YANG JUNGHOON (US)
KAUFFMAN KEVIN (US)
BARNES THOMAS (US)
WESSELHOEFT ROBERT ALEXANDER (US)
BECKER AMY M (US)
MOTZ GREGORY (US)
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WHAT IS CLAIMED IS 1. An ionizable lipid, wherein the ionizable lipid is represented by Formula (13c-1) or is a pharmaceutically acceptable salt thereof: Formula (13c-1) wherein: n* is an integer from 1 to 7; Ra is hydrogen or hydroxyl; Rb is hydrogen or C1-C6 alkyl; LA and LB are each independently linear C1-C12 alkyl; ZA and ZB are each independently a direct bond or a linking group selected from -C(O)O-, -O(CO)-, and -O(CO)O-; and RA and RB are independently linear or branched C1-C20 alkyl or C2-C20 alkenyl. 2. An ionizable lipid, wherein the ionizable lipid is represented by Formula (13c-2) or is a pharmaceutically acceptable salt thereof: Formula (13c-2) wherein: n is an integer from 1 to 7; Ra is hydrogen or hydroxyl; LA and LB are each independently linear C1-C12 alkyl; ZA and ZB are each independently a direct bond or a linking group selected from -C(O)O-, -O(CO)-, and -O(CO)O-; and RA and RB are independently linear or branched C1-C20 alkyl or C2-C20 alkenyl. 3. The ionizable lipid of claim 1, wherein Rb is C1-C6 alkyl. 4. The ionizable lipid of claim 1 or 3, wherein Rb is methyl or ethyl. 5. The ionizable lipid of any one of claims 1-4, wherein RB is linear or branched C1-C20 alkyl. 6. The ionizable lipid of claim 5, wherein RB is branched C1-C20 alkyl. 7. The ionizable lipid of claim 5, wherein RB is linear C1-C20 alkyl. 8. The ionizable lipid of any one of claims 1-7, wherein RA is linear or branched C1-C20 alkyl. 9. The ionizable lipid of claim 8, wherein RA is branched C1-C20 alkyl. 10. The ionizable lipid of claim 8, wherein RA is linear C1-C20 alkyl. 11. The ionizable lipid of any one of claims 1-4, wherein both RA and RB are branched C1-C20 alkyl or C2-C20 alkenyl. 12. The ionizable lipid of claim 11, wherein both RA and RB are branched C1-C20 alkyl. 13. The ionizable lipid of claim 11, wherein both RA and RB are branched C2-C20 alkenyl. 14. The ionizable lipid of any one of claims 1-13, wherein Ra is hydrogen. 15. The ionizable lipid of any one of claims 1-13, wherein Ra is hydroxyl. 16. The ionizable lipid of any one of claims 1-15, wherein ZA is selected from -C(O)O-, -OC(O)-, and -OC(O)O-, and ZB is a direct bond. 17. The ionizable lipid of any one of claims 1-15, wherein ZB is selected from -C(O)O-, -OC(O)-, and -OC(O)O-, and ZA is a direct bond. 18. The ionizable lipid of claim 1 or 2, wherein the lipid is of Formula (13d-2) Formula (13d-2) wherein: q and q’ are each independently an integer from 1 to 12, r and r’ are each independently an integer from 0 to 6, R8A is H or R10A, R8B is H or R10B, and R9A, R9B, R10A, and R10A are each independently unsubstituted linear C1-C12 alkyl or unsubstituted linear C2-C12-alkenyl. 19. The ionizable lipid of claim 18, wherein R8B is R10B. 20. The ionizable lipid of claim 19, wherein R10B and R9B are different. 21. The ionizable lipid of claim 20, wherein R8A is R10A. 22. The ionizable lipid of claim 21, wherein R10A and R9A are different. 23. The ionizable lipid of claim 22, wherein R10A and R9A are different unsubstituted linear C1-C12 alkyl. 24. The ionizable lipid of claim 22, wherein R10A and R9A are different unsubstituted linear C2-C12 alkenyl. 25. The ionizable lipid of claim 18, wherein R9A, R9B, R10A, and R10A are each independently unsubstituted linear C4-C8 alkyl. 26. The ionizable lipid of claim 18, wherein q is an integer from 3 to 6. 27. The ionizable lipid of claim 18, wherein q’ is an integer from 1 to 12. 28. The ionizable lipid of claim 26, wherein q’ is an integer from 3 to 6. 29. The ionizable lipid of claim 18, wherein r is an integer from 1 to 6. 30. The ionizable lipid of claim 18, wherein r is 0. 31. The ionizable lipid of claim 18, wherein r’ is an integer from 1 to 6. 32. The ionizable lipid of claim 18, wherein r’ is 0. 33. The ionizable lipid of claim 18, wherein Ra is hydrogen. 34. The ionizable lipid of claim 33, wherein ZA is selected from -C(O)O-, -OC(O)-, and -OC(O)O-, and ZB is a direct bond. 35. The ionizable lipid of claim 33, wherein ZB is selected from -C(O)O-, -OC(O)-, and -OC(O)O-, and ZA is a direct bond. 36. The ionizable lipid of claim 18, wherein Ra is hydroxyl. 37. The ionizable lipid of claim 36, wherein ZA is selected from -C(O)O-, -OC(O)-, and -OC(O)O-, and ZB is a direct bond. 38. The ionizable lipid of claim 36, wherein ZB is selected from -C(O)O-, -OC(O)-, and -OC(O)O-, and ZA is a direct bond. 39. The ionizable lipid of any one of claims 18-33, wherein ZA is -C(O)O-. 40. The ionizable lipid of any one of claims 18-33, wherein ZA is -OC(O)-. 41. The ionizable lipid of any one of claims 18-33, wherein ZA is -OC(O)O-. 42. The ionizable lipid of any one of claims 18-33, wherein ZA is a direct bond. 43. The ionizable lipid of any one of claims 18-33, wherein ZB is -C(O)O-. 44. The ionizable lipid of any one of claims 18-33, wherein ZB is -OC(O)-. 45. The ionizable lipid of any one of claims 18-33, wherein ZB is -OC(O)O-. 46. The ionizable lipid of any one of claims 18-33, wherein ZB is a direct bond. 47. The ionizable lipid of claims 1-2 and 18, wherein the ionizable lipid is selected from , , OH N OH O O OH , , , , , , , , , , , and , or a pharmaceutically acceptable salt thereof. 48. A pharmaceutical composition comprising the ionizable lipid of any one of claims 1-47. 49. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition further comprises a RNA polynucleotide. 50. The pharmaceutical composition of claim 49, wherein the RNA polynucleotide is a linear RNA polynucleotide. 51. The pharmaceutical composition of claim 49, wherein the RNA polynucleotide is a circular RNA polynucleotide. 52. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition further comprises: a helper lipid, wherein the helper lipid is DOPE or DSPC, cholesterol, and a PEG-lipid, wherein the PEG-lipid is DSPE-PEG(2000) or DMG-PEG(2000). 53. A method of treating or preventing a disease, disorder, or condition, comprising administering an effective amount of a pharmaceutical composition of claim 48 further comprising a drug. 54. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 48 further comprising a drug. |
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
24 25 26 27
28 29 30 31 32 33 34 35 36 37 38 39
40 41 42 43 44
45 46 47 48 49 O O O OH O O O N O OH O
50 51 52 53 O OH O N OH 54 55 OH OH O N O O O 56
57 58 59 OH O N OH O 60 61
62 63 64 HO O O OH O N O 65
66 67 68 69 70 71 72 73 74 O O O O HO N OH HO 75 76 77 78 79 O O O OH O O O N OH OH 80
81 82 83 O OH N O HO O O 84 O N OH O HO O O
85 86 87 88 OH O N OH O HO O O 89 OH O OH N O HO O O 90 OH O N OH O HO O O 91 92
93 O OH N O HO O O 94 95 OH O OH N O HO O O 96
97 98 99 100
101 102 103 104
105 106 107 108
109 110 111 112
113 114 115 116
117 118 119 120
121 122 123 124 125
126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142
143 144 145 O OH O N OH O O 146 O OH O N OH O O 147 148 149 150 151 152 153 154
155 156 157 158 O OH O O N OH O O O 159 160
161 O O O N OH O O O OH 162 163 O OH O O N OH O O OH 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 [0459] In some embodiments, an ionizable lipid has one of the structures set forth in Table 10f below, or is a pharmaceutically acceptable salt thereof. Table 10f Ionizable Structure lipid number 1
7 8 9 10 11
12 13 14 15 16
17 18 19 20
21 22 23 24 25
26 27 28 29 30
31 32 33 34 35
36 37 38 [0460] In some embodiments, an ionizable lipid has one of the structures set forth in Table 10g below, or is a pharmaceutically acceptable salt thereof. Table 10g Number Structure 1
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 [0461] In some embodiments, an ionizable lipid is as described in international patent application PCT/US2020/038678. [0462] In some embodiments, the ionizable lipid is represented by Formula (14*): Formula (14*) or a pharmaceutically acceptable salt thereof, wherein L 1 is C2-C11 alkylene, C4-C10-alkenylene, or C4-C10-alkynylene; X 1 is OR 1 , SR 1 , or N(R 1 ) 2 , where R 1 is independently H or unsubstituted C 1 -C 6 alkyl; and R2 and R3 are each independently a linear or branched C1-C30 alkyl, C2-C30 alkenyl, or C1-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl. [0463] In some embodiments, the ionizable lipid is represented by Formula (14): Formula (14) or a pharmaceutically acceptable salt thereof, wherein L 1 is C 2 -C 11 alkylene, C 4 -C 10 -alkenylene, or C 4 -C 10 -alkynylene; X 1 is OR 1 , SR 1 , or N(R 1 )2, where R 1 is independently H or unsubstituted C1-C6 alkyl; and R 2 and R 3 are each independently C 6 -C 30 -alkyl, C 6 -C 30 -alkenyl, or C 6 -C 30 -alkynyl. [0464] In some embodiments, X 1 is OR 1 . In some embodiments, X 1 is OH. In some embodiments, X 1 is SR 1 . In some embodiments, X 1 is SH. In some embodiments, X 1 is N(R 1 )2. In some embodiments, X 1 is NH2. [0465] In some embodiments, L 1 is C2-C10 alkylene. In some embodiments, L 1 is unsubstituted C2- C10 alkylene. In some embodiments, L 1 is C4-C10 alkenylene. In some embodiments, L 1 is unsubstituted C4-C10 alkenylene. In some embodiments, L 1 is C4-C10 alkynylene. In some embodiments, L 1 is unsubstituted C4-C10 alkynylene. [0466] In some embodiments of Formula (14), a lipid has a structure according to Formula (14-2), Formula (14-2) or a pharmaceutically acceptable salt thereof, wherein n is an integer of 2-10. [0467] In some embodiments, n is 2, 3, 4, or 5. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. [0468] In some embodiments of Formula (14*) or Formula (14-2), R2 and R3 are independently a linear or branched C1-C20 alkyl, C2-C20 alkenyl, or C1-C20 heteroalkyl, optionally substituted by one or more substituents each independently selected from linear or branched C1-C20 alkoxy, linear or branched C1- C20 alkyloxycarbonyl, linear or branched C1-C20 alkylcarbonyloxy, linear or branched C1-C20 alkylcarbonate, linear or branched C 2 -C 20 alkenyloxycarbonyl, linear or branched C 2 -C 20 alkenylcarbonyloxy, linear or branched C 2 -C 20 alkenylcarbonate, linear or branched C 2 -C 20 alkynyloxycarbonyl, linear or branched C2-C20 alkynylcarbonyloxy, and linear or branched C2-C20 alkynylcarbonate. [0469] In certain embodiments of Formula (14*) or Formula (14-2), one or each of R 2 and R 3 is unsubstituted C 6 -C 30 -alkyl, unsubstituted C 6 -C 30 -alkenyl, or unsubstituted C 6 -C 30 -alkynyl. In certain embodiments, each of R 2 and R 3 is unsubstituted C6-C30-alkyl. In certain embodiments, each of R 2 and R 3 is unsubstituted C6-C30-alkenyl. In certain embodiments, each of R 2 and R 3 is unsubstituted C6-C30- alkynyl. [0470] In some embodiments of Formula (14*), the alkyloxycarbonyl substituent is of the formula - C(O)OR 6 , wherein R 6 is unsubstituted C6-C30 alkyl or C6-C30 alkenyl. In some embodiments of Formula (14*) or Formula (14-2), at least one of R2 and R3 is substituted with an alkylcarbonyloxy. In some embodiments, the alkylcarbonyloxy is of the formula -OC(O)R 6 , wherein R 6 is unsubstituted C6-C30 alkyl or C6-C30 alkenyl. In some embodiments, at least one of R2 and R3 is substituted with an alkylcarbonate. In some embodiments, the alkylcarbonate is of the formula -O(CO)OR 6 , wherein R 6 is unsubstituted C 6 -C 30 alkyl or C 6 -C 30 alkenyl. In some embodiments, R 2 and R 3 are independently C 1 - C 12 alkyl substituted by -O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 , wherein R 6 is unsubstituted C 6 -C 30 alkyl or C6-C30 alkenyl. In some embodiments, R2 and R3 are each C1-C12 alkyl substituted by -O(CO)R 6 . In some embodiments, R2 and R3 are each C1-C12 alkyl substituted by -C(O)OR 6 . In some embodiments, R2 and R3 are each C1-C12 alkyl substituted by -O(CO)OR 6 . In some embodiments R2 is -C(O)OR 6 or -O(CO)R 6 and R 3 is -O(CO)OR 6 . In some embodiments, R 2 is -O(CO)OR 6 and R 3 is -C(O)OR 6 or - O(CO)R 6 . [0471] In some embodiments of Formula (14*) or Formula (14-2), at least one of R 2 and R 3 is selected from the following formulae: (i) -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), (ii) -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and (iii) -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), wherein: q is 1 to 12, r is 0 to 6, R 8 is H or R 10 , and R 9 and R 10 are independently unsubstituted linear C 1 -C 12 alkyl or unsubstituted linear C 2 -C 12 - alkenyl. [0472] In some embodiments of Formula (14*) or Formula (14-2), each of R 2 and R 3 is independently selected from one of the following formulae: (i) -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), (ii) -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and (iii) -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), wherein: q is 1 to 12, r is 0 to 6, R 8 is H or R 10 , and R 9 and R 10 are independently unsubstituted linear C1-C12 alkyl or unsubstituted linear C2-C12- alkenyl. [0473] In some embodiments, of any one of formulae (i)-(iii), q is 1 to 6. In some embodiments of any one of formulae (i)-(iii), q is 0. In some embodiments, of any one of formulae (i)-(iii), q is 1. In some embodiments of any one of formulae (i)-(iii), q is 2. In some embodiments, of any one of formulae (i)- (iii), q is 3 to 12. In some embodiments, of any one of formulae (i)-(iii), q is 3 to 6. [0474] In some embodiments of any one of formulae (i)-(iii), r is 0. In some embodiments of any one of formulae (i)-(iii), r is 1 to 6. In some embodiments of any one of formulae (i)-(iii), r is 1. In some embodiments of any one of formulae (i)-(iii), r is 2. In some embodiments of any one of formulae (i)- (iii), r is 3. In some embodiments of any one of formulae (i)-(iii), r is 4. [0475] In some embodiments of formulae (i)-(iii), R 8 is H. In some embodiments of formulae (i)-(iii), R 8 is R 10 . In some embodiments of formulae (i)-(iii), R 9 and R 10 are different. In some embodiments of formulae (i)-(iii), R 9 and R 10 are the same. [0476] In some embodiments of formulae (i)-(iii), R 8 is H, and R 9 is unsubstituted linear C1-C12 alkyl or unsubstituted linear C1-C12-alkenyl. In some embodiments of formulae (i)-(iii), R 8 is H, and R 9 is unsubstituted linear C 2 -C 12 alkyl. In some embodiments of formulae (i)-(iii), R 8 is H, and R 9 is unsubstituted linear C 2 -C 8 alkyl. In some embodiments of formulae (i)-(iii), R 8 is H, and R 9 is unsubstituted linear C4-C8 alkyl. In some embodiments of formulae (i)-(iii), R 8 is H, and R 9 is unsubstituted linear C5-C8 alkyl. In some embodiments of formulae (i)-(iii), R 8 is H, and R 9 is unsubstituted linear C6-C8 alkyl. [0477] In some embodiments of formulae (i)-(iii), R 8 and R 9 are each independently unsubstituted linear C1-C12 alkyl or unsubstituted linear C1-C12-alkenyl. In some embodiments of formulae (i)-(iii), R 8 and R 9 are each independently unsubstituted linear C2-C12 alkyl. In some embodiments of formulae (i)-(iii), R 8 and R 9 are each independently unsubstituted linear C2-C8 alkyl. In some embodiments of formulae (i)-(iii), R 8 and R 9 are each independently unsubstituted linear C 4 -C 8 alkyl. In some embodiments of formulae (i)-(iii), R 8 and R 9 are each independently unsubstituted linear C 6 -C 8 alkyl. [0478] In some embodiments of Formula (14*) or Formula (14-2), at least one of R2 and R3 is - (CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In some embodiments at least one of R 2 and R 3 is -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In some embodiments at least one of R 2 and R 3 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In some embodiments of Formula (14*) or Formula (14-2), at least one of R 2 and R3 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), where q is 3 to 12 (e.g., 6 to 12), r is 1 to 6 (e.g., 1, 2 or 3), and R 8 and R 9 are each independently unsubstituted linear C4-C8 alkyl. [0479] In certain embodiments of Formula (14*) or Formula (14-2), at least one of R 2 and R 3 is - (CH 2 ) q C(O)O(CH 2 ) r CH(R 8 )(R 9 ) or -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In other embodiments, at least one of R2 and R3 is -(CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ) or -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In some embodiments, at least one of R2 and R3 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ) or -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. [0480] In other embodiments of Formula (14*) or Formula (14-2), at least one of R2 and R3 is - (CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ) where q, r, R 8 and R 9 are as defined above. In some embodiments, at least one of R2 and R3 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ) or -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. [0481] In certain embodiments of Formula (14*) or Formula (14-2), R 2 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), and R3 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In certain embodiments, R2 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), and R3 is - (CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In certain embodiments, R is -(CH 2 ) q C(O)O(CH 2 ) r CH(R 8 )(R 9 ), and R 3 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. [0482] In certain embodiments of Formula (14*) or Formula (14-2), R2 is -(CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ), and R3 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In certain embodiments, R2 is -(CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ), and R3 is - (CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In certain embodiments, R 2 is -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and R 3 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. [0483] In certain embodiments of Formula (14*) or Formula (14-2), R 2 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), and R 3 is -(CH 2 ) q C(O)O(CH 2 ) r CH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In certain embodiments, R 2 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), and R 3 is - (CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. In certain embodiments, R2 is -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), and R3 is -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), where q, r, R 8 and R 9 are as defined above. [0484] In certain embodiments of Formula (14*) or Formula (14-2), one or each of R 2 and R 3 is unsubstituted C6-C22-alkyl, or one or each of R 2 and R 3 is unsubstituted C6-C22-alkenyl. In certain embodiments, each of R 2 and R 3 is unsubstituted C6-C22-alkyl. In certain embodiments, each of R2 and R3 is unsubstituted C6-C22-alkenyl. [0485] In certain embodiments, one or each of R 2 and R 3 is -C 6 H 13 , -C 7 H 15 , -C 8 H 17 , -C 9 H 19 , -C 10 H 21 , - C11H23, -C12H25, -Cl3H27, -Cl4H29, -Cl5H3l, -Cl6H33, -Cl7H35, -C18H37, -C19H39, -C20H41, -C21H43, -C22H45, - C23H47, -C24H49, -C25H51. [0486] In certain embodiments, one or each of R 2 and R 3 is -(CH 2 ) 4 CH=CH 2 , -(CH 2 ) 5 CH=CH 2 , -(CH2)6CH=CH2, -(CH2)7CH=CH2, -(CH2)8SCH=CH2, -(CH2)9CH=CH2, -(CH2)10CH=CH2, -(CH 2 ) 11 CH=CH 2 , -(CH 2 ) 12 CH=CH 2 , -(CH 2 ) 13 CH=CH 2 , -(CH 2 ) 14 CH=CH 2 , -(CH 2 ) 15 CH=CH 2 , - (CH2)16CH=CH2, -(CH2)17CH=CH2, -(CH2)18CH=CH2, -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)6CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH 2 ) 7 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH 3 , -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, -(CH 2 ) 3 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH=CHCH 2 CH 3 , -(CH2)IICH=CH(CH2)7CH3, or -(CH2)2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH3. [0487] In certain embodiments, one or each of R2 and R3 is C6-C12 alkyl substituted by -O(CO)R 6 or - C(O)OR 6 , wherein R 6 is unsubstituted C 6 -C 14 alkyl. In certain embodiments, R 6 is unsubstituted linear C 6 -C 14 alkyl. In certain embodiments, R 6 is unsubstituted branched C 6 -C 14 alkyl. [0488] In certain embodiments, one or each of R2 and R3 is (CH2)7C(O)O(CH2)2CH(C5H11)2 or (CH2)8C(O)O(CH2)2CH(C5H11)2. In certain embodiments, one or each of R 2 and R 3 is , , , , . , , , , , or . [0489] In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . In certain embodiments, one or each of R 2 and R 3 is . [0490] In some embodiments, an ionizable lipid is described in Table 10h.
Table 10h
[0491] Some embodiments of the present disclosure provide nucleic acid-lipid nanoparticle compositions comprising one or more of the novel cationic lipids described herein as structures listed in the tables, that provide increased activity of the nucleic acid and improved tolerability of the compositions in vivo. Other Ionizable Lipids [0492] In some embodiments, one or more (e.g., two or more, or three or more) ionizable lipids are utilized in the transfer vehicles of this disclosure. In some embodiments, the transfer vehicle includes a first ionizable lipid (e.g., as described herein, such as a lipid of Formula (13*) or (14*)), and one or more additional ionizable lipids. [0493] Lipids of interest, including ionizable lipids that can be used in combination with a first ionizable lipid as described herein, such as by being incorporated into the transfer vehicles of this disclosure, include, but are not limited to, lipids as described in: international application PCT/US2018/058555, international application PCT/US2020/038678, US publication US2019/0314524, WO2019/152848, international application PCT/US2010/061058, international application PCT/US2017/028981, WO2015/095340, WO2014/136086, US2019/0321489, WO2010/053572, U.S. provisional patent application 61/617,468, international patent application PCT/US2019/025246, US patent publications 2017/0190661 and 2017/0114010, US publication 20190314284, WO2015/095340, WO2019/152557, WO2019/152848, international application PCT/US2019/015913, US patent 9,708,628, US patent 9,765,022; Wang et al., ACS Synthetic Biology, 1, 403-07 (2012); WO 2008/042973, US Patent 8,071,082, the disclosures of which are incorporated herein by reference in their entirety. [0494] In some embodiments, tail groups as used in the lipids may be as described in. WO2015/095340, WO2019/152557, and WO2019/152848, the disclosures of which are incorporated herein by reference in their entirety. [0495] The lipid-like compounds can be prepared by methods well known the art. See [0496] In some embodiments, the ionizable lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride or “DOTMA” is used. (Felgner et al. Proc. Nat’l Acad. Sci. 84, 7413 (1987); U.S. Pat. No.4,897,355). DOTMA can be formulated with an ionizable lipid (e.g., as described herein), and/or can be combined with a neutral lipid, dioleoylphosphatidylethanolamine or “DOPE” or other cationic or non-cationic lipids into a lipid nanoparticle. [0497] Other suitable lipids include, for example, ionizable cationic lipids, such as, e.g., (15Z,18Z)- N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15 ,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)t etracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)t etracosa-5,15,18- trien-1-amine (HGT5002), C12-200 (described in WO 2010/053572), 2-(2,2-di((9Z,12Z)-octadeca- 9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLinKC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), 2-(2,2-di((9Z,2Z)-octadeca-9,12- dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA), (3S,10R,13R,17R)-10,13- dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro- 1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl- 6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-am ine (HGT5000), (15Z,18Z)-N,N- dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15, 18-trien-1-amine (HGT5001), (15Z,18 Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracos a-5,15,18-trien-1-amine (HGT5002), 5-carboxyspermylglycine-dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) (Behr et al. Proc. Nat.’l Acad. Sci.86, 6982 (1989); U.S. Pat. No. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane (DODAP), 1,2-Dioleoyl-3-Trimethylammonium-Propane or (DOTAP). Contemplated ionizable lipids also include 1,2-distcaryloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N- dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis, cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy l-1-(cis,cis-9′,1-2′- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2- N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (DLinDAP), 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylamninopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane (DLin-K-XTC2-DMA) or GL67, or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). The use of cholesterol-based ionizable lipids to formulate the transfer vehicles (e.g., lipid nanoparticles) is also contemplated by the present disclosure. Such cholesterol- based ionizable lipids can be used, either alone or in combination with other lipids. Suitable cholesterol- based ionizable lipids include, for example, DC-Cholesterol (N,N-dimethyl-N- ethylcarboxamidocholesterol), and 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al., Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335). [0498] Also contemplated are cationic lipids such as dialkylamino-based, imidazole-based, and guanidinium-based lipids. For example, also contemplated is the use of the ionizable lipid (3S,10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17- tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate (ICE), as disclosed in International Application No. PCT/US2010/058457, incorporated herein by reference. [0499] Also contemplated are ionizable lipids such as the dialkylamino-based, imidazole-based, and guanidinium-based lipids. For example, certain embodiments are directed to a composition comprising one or more imidazole-based ionizable lipids, for example, the imidazole cholesterol ester or “ICE” lipid, (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate. [0500] Without wishing to be bound by a particular theory, it is believed that the fusogenicity of the imidazole-based cationic lipid ICE is related to the endosomal disruption which is facilitated by the imidazole group, which has a lower pKa relative to traditional ionizable lipids. The endosomal disruption in turn promotes osmotic swelling and the disruption of the liposomal membrane, followed by the transfection or intracellular release of the nucleic acid(s) contents loaded therein into the target cell. [0501] The imidazole-based ionizable lipids are also characterized by their reduced toxicity relative to other ionizable lipids. [0502] In certain embodiments, transfer vehicle compositions for the delivery of circular RNA comprise an amine lipid. In certain embodiments, an ionizable lipid is an amine lipid. In some embodiments, an amine lipid is described in international patent application PCT/US2018/053569. [0503] In some embodiments, the amine lipid is Lipid E, which is (9Z, 12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)car bonyl)oxy)methyl)propyl octadeca-9, 12-dienoate. [0504] In certain embodiments, an amine lipid is an analog of Lipid E. In certain embodiments, a Lipid E analog is an acetal analog of Lipid E. In particular transfer vehicle compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In additional embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is chosen from a C4, C5, C6, C7, C9, C10, C11 and C12 acetal analog. [0505] Amine lipids and other biodegradable lipids suitable for use in the transfer vehicles, e.g., lipid nanoparticles, described herein are biodegradable in vivo. The amine lipids described herein have low toxicity (e.g., are tolerated in animal models without adverse effect in amounts of greater than or equal to 10 mg/kg). In certain embodiments, transfer vehicles composing an amine lipid include those where at least 75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. [0506] Biodegradable lipids include, for example, the biodegradable lipids of WO2017/173054, WO2015/095340 , and WO2014/136086. [0507] Lipid clearance may be measured by methods known by persons of skill in the art. See, for example, Maier, M.A., et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther.2013, 21(8), 1570-78. [0508] Transfer vehicle compositions comprising an amine lipid can lead to an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example the rate at which a lipid is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example the rate at which an circRNA is cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which transfer vehicles are cleared from the blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which transfer vehicles are cleared from a tissue, such as liver tissue or spleen tissue. In certain embodiments, a high rate of clearance leads to a safety profile with no substantial adverse effects. The amine lipids and biodegradable lipids may reduce transfer vehicle accumulation in circulation and in tissues. In some embodiments, a reduction in transfer vehicle accumulation in circulation and in tissues leads to a safety profile with no substantial adverse effects. [0509] Lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipid, such as an amine lipid, may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood, where pH is approximately 7.35, the lipid, such as an amine lipid, may not be protonated and thus bear no charge. [0510] The ability of a lipid to bear a charge is related to its intrinsic pKa. In some embodiments, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.1 to about 7.4. For example, the amine lipids of the present disclosure may each, independently, have a pKa in the range of from about 5.8 to about 6.5 . Lipids with a pKa ranging from about 5.1 to about 7.4 are effective for delivery of cargo in vivo, e.g.,to the liver. Further, it has been found that lipids with a pKa ranging from about 5.3 to about 6.4 are effective for delivery in vivo, e.g.,into tumors. See, e.g., WO2014/136086. [0511] A lipid of the present disclosure may have an —S—S— (disulfide) bond. [0512] Lipid-like compounds of this disclosure can be prepared using suitable starting materials through synthetic route known in the art. The method can include an additional step(s) to add or remove suitable protecting groups in order to ultimately allow synthesis of the lipid-like compounds. In addition, various synthetic steps can be performed in an alternate sequence or order to give the desired material. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable lipid-like compounds are known in the art, including, for example, R. Larock, Comprehensive Organic Transformations (2nd Ed., VCH Publishers 1999); P. G. M. Wuts and T. W. Greene, Greene’s Protective Groups in Organic Synthesis (4th Ed., John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser’ s Reagents for Organic Synthesis (John Wiley and Sons 1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd ed., John Wiley and Sons 2009) and subsequent editions thereof. Certain lipid-like compounds may contain a non-aromatic double bond and one or more asymmetric centers. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- isomeric forms. All such isomeric forms are contemplated. [0513] Preparation methods for the above compounds and compositions are described herein below and/or known in the art. [0514] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include, e.g., hydroxyl, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxyl include, e.g., trialkylsilyl or diarylalkylsilyl (for example, t- butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include, e.g., t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include, e.g., -C(O)-R’’ (where R’’ is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include, e.g., alkyl, aryl, or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in, e.g., Green, T. W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin, or a 2- chlorotrityl-chloride resin. [0515] It will also be appreciated by those skilled in the art, although such protected derivatives of compounds of this disclosure may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form compounds of the disclosure which are pharmacologically active. Such derivatives may therefore be described as prodrugs. All prodrugs of compounds of this disclosure are included within the scope of the disclosure. [0516] Furthermore, all compounds of the disclosure which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of the disclosure can also be converted to their free base or acid form by standard techniques. [0517] It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make other compounds of Formula (1) not specifically illustrated herein by using the appropriate starting materials and modifying the parameters of the synthesis. In general, starting materials may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this disclosure. [0518] As mentioned above, these lipid-like compounds are useful for delivery of pharmaceutical agents. They can be preliminarily screened for their efficacy in delivering pharmaceutical agents by an in vitro assay and then confirmed by animal experiments and clinic trials. Other methods will also be apparent to those of ordinary skill in the art. [0519] Not to be bound by any theory, the lipid-like compounds of this disclosure can facilitate delivery of pharmaceutical agents by forming complexes, e.g., nanocomplexes and microparticles. The hydrophilic head of such a lipid-like compound, positively or negatively charged, binds to a moiety of a pharmaceutical agent that is oppositely charged and its hydrophobic moiety binds to a hydrophobic moiety of the pharmaceutical agent. Either binding can be covalent or non-covalent. [0520] The above described complexes can be prepared using procedures described in publications such as Wang et al., ACS Synthetic Biology, 1, 403-07 (2012). Generally, they are obtained by incubating a lipid-like compound and a pharmaceutical agent in a buffer such as a sodium acetate buffer or a phosphate buffered saline ("PBS"). [0521] In certain embodiments, the selected hydrophilic functional group or moiety may alter or otherwise impart properties to the compound or to the transfer vehicle of which such compound is a component (e.g., by improving the transfection efficiencies of a lipid nanoparticle of which the compound is a component). For example, the incorporation of guanidinium as a hydrophilic head-group in the compounds described herein may promote the fusogenicity of such compounds (or of the transfer vehicle of which such compounds are a component) with the cell membrane of one or more target cells, thereby enhancing, for example, the transfection efficiencies of such compounds. It has been hypothesized that the nitrogen from the hydrophilic guanidinium moiety forms a six-membered ring transition state which grants stability to the interaction and thus allows for cellular uptake of encapsulated materials. (Wender, et al., Adv. Drug Del. Rev. (2008) 60: 452-472.) Similarly, the incorporation of one or more amino groups or moieties into the described compounds (e.g., as a head- group) may further promote disruption of the endosomal/lysosomal membrane of the target cell by exploiting the fusogenicity of such amino groups. This is based not only on the pKa of the amino group of the composition, but also on the ability of the amino group to undergo a hexagonal phase transition and fuse with the target cell surface, i.e. the vesicle membrane. (Koltover, et al. Science (1998) 281: 78-81.) The result is believed to promote the disruption of the vesicle membrane and release of the lipid nanoparticle contents into the target cell. [0522] Similarly, in certain embodiments the incorporation of, for example, imidazole as a hydrophilic head-group in the compounds described herein may serve to promote endosomal or lysosomal release of, for example, contents that are encapsulated in a transfer vehicle (e.g., lipid nanoparticle) of the disclosure. Such enhanced release may be achieved by one or both of a proton-sponge mediated disruption mechanism and/or an enhanced fusogenicity mechanism. The proton-sponge mechanism is based on the ability of a compound, and in particular a functional moiety or group of the compound, to buffer the acidification of the endosome. This may be manipulated or otherwise controlled by the pKa of the compound or of one or more of the functional groups comprising such compound (e.g., imidazole). Accordingly, in certain embodiments the fusogenicity of, for example, the imidazole-based compounds described herein (e.g., HGT4001 and HGT4004) are related to the endosomal disruption properties, which are facilitated by such imidazole groups, which have a lower pKa relative to other traditional ionizable lipids. Such endosomal disruption properties in turn promote osmotic swelling and the disruption of the liposomal membrane, followed by the transfection or intracellular release of the polynucleotide materials loaded or encapsulated therein into the target cell. This phenomenon can be applicable to a variety of compounds with desirable pKa profiles in addition to an imidazole moiety. Such embodiments also include multi-nitrogen based functionalities such as polyamines, poly-peptide (histidine), and nitrogen-based dendritic structures. [0523] Exemplary ionizable and/or cationic lipids are described in International PCT patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004 143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/129709, WO2010/144740 , WO20 12/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO2010/088537, WO2010/054401, WO2010/054406 , WO2010/054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126803, WO2006/007712, WO2011/038160, WO2005/121348, WO2011/066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823, WO2015/095346, and WO2013/086354, and US patent publications US2016/0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US2016/0376224, US2017/0119904, US2012/0149894, US2015/0057373, US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203446, US2018/0005363, US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US2013/0123338, US2015/0064242, US2006/0051405, US2013/0065939, US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US2018/0028664, US2016/0317458, and US2013/0195920, the contents of all of which are incorporated herein by reference in their entirety. International patent application WO 2019/131770 is also incorporated herein by reference in its entirety. B. PEG LIPIDS [0524] The use and inclusion of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1- [Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) in the liposomal and pharmaceutical compositions described herein is contemplated, preferably in combination with one or more of the compounds and lipids described herein. Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, the PEG-modified lipid employed in the compositions and methods of the disclosure is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (2000 MW PEG) “DMG-PEG2000.” The addition of PEG-modified lipids to the lipid delivery vehicle may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-polynucleotide composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No.5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivatized lipids of the present disclosure may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in a liposomal lipid nanoparticle. [0525] In an embodiment, a PEG-modified lipid is described in International Pat. Appl. No. PCT/US2019/015913 or PCT/US2020/046407, which are incorporated herein by reference in their entirety. In an embodiment, a transfer vehicle comprises one or more PEG-modified lipids. [0526] Non-limiting examples of PEG-modified lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. In some further embodiments, a PEG-modified lipid may be, e,g,, PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE. [0527] In some still further embodiments, the PEG-modified lipid includes, but is not limited to 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG- DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c- DMA). [0528] In some still further embodiments, the PEG-modified lipid is DSPE-PEG, DMG-PEG, PEG- DAG, PEG-S-DAG, PEG-PE, PEG-S-DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1. In some embodiments, the PEG-modified lipid is DSPE- PEG(2000). [0529] In some embodiments, the PEG-modified lipid comprises a PEG moiety comprising 10-70 (e.g., 30-60) oxyethylene (−O−CH2−CH2−) units or portions thereof. In some embodiments, the PEG- modified lipid comprises (OCH 2 CH 2 ) v –OR w , and v is an integer from 0 and 70 (inclusive) (e.g., an integer from 30 and 60), w is hydrogen or alkyl. [0530] In various embodiments, a PEG-modified lipid may also be referred to as “PEGylated lipid” or “PEG-lipid.” [0531] In one embodiment, the PEG-lipid is selected from a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. [0532] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C 14 to about C 22 , such as from about C 14 to about C 16 . In some embodiments, a PEG moiety, for example a mPEG-NH 2 , has a size of about 1000, about 2000, about 5000, about 10,000, about 15,000 or about 20,000 daltons. In one embodiment, the PEG-lipid is PEG2k-DMG. [0533] In one embodiment, the lipid nanoparticles described herein can comprise a lipid modified with a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG- DSPE. [0534] PEG-lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Pat. Publ. No. WO2015/130584 A2, which are incorporated herein by reference in their entirety. [0535] In various embodiments, lipids (e.g., PEG-lipids), described herein may be synthesized as described International Pat. Publ. No. PCT/US2016/000129, which is incorporated by reference in its entirety. [0536] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. [0537] In some embodiments the PEG-modified lipids are a modified form of PEG-DMG. PEG-DMG has the following structure: . [0538] In some embodiments the PEG-modified lipids are a modified form of PEG-C18, or PEG-1. PEG-1 has the following structure . [0539] In one embodiment, PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an –OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present disclosure. [0540] In some embodiments, the PEG lipid is a compound of Formula (P1): (P1) or a salt or isomer thereof, wherein: r is an integer from 1 and 100; R is C10-40 alkyl, C10-40 alkenyl, or C10-40 alkynyl; and optionally one or more methylene groups of R are independently replaced with C3-10 carbocyclylene, 4 to 10 membered heterocyclylene, C6- 10 arylene, 4 to 10 membered heteroarylene, –N(R N )–, –O–, –S–, –C(O)–,–C(O)N(R N )–, –NR N C(O)–, –NR N C(O)N(R N )–, –C(O)O–, –OC(O)–, –OC(O)O– ,–OC(O)N(R N )–, –NR N C(O)O–, –C(O)S–, – SC(O)–, –C(=NR N )–, –C(=NR N )N(R N )–, –NR N C(=NR N )–, –NR N C(=NR N )N(R N )– ,–C(S)–, – C(S)N(R N )–, –NR N C(S)–, –NR N C(S)N(R N )–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O) 2 –, – S(O)2O–, –OS(O)2O–, –N(R N )S(O)–, –S(O)N(R N )–, –N(R N )S(O)N(R N )–, –OS(O)N(R N )–, – N(R N )S(O)O–, –S(O)2–, –N(R N )S(O)2–, –S(O)2N(R N )–, –N(R N )S(O)2N(R N )–, –OS(O)2N(R N )–, or – N(R N )S(O)2O–; and each instance of R N is independently hydrogen, C1-6 alkyl, or a nitrogen protecting group. [0541] For example, R is C17 alkyl. For example, the PEG lipid is a compound of Formula (P1-a): . or a salt or isomer thereof, wherein r is an integer from 1 and 100. [0542] For example, the PEG lipid is a compound of the following formula: . C. HELPER LIPIDS [0543] In some embodiments, the transfer vehicle (e.g., LNP) described herein comprises one or more non-cationic helper lipids. In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is a phospholipid substitute or replacement. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. [0544] A phospholipid moiety can be selected, for example, from the non-limiting group of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. [0545] A fatty acid moiety can be selected, for example, from the non-limiting group of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. [0546] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. [0547] In some embodiments, the helper lipid is a 1,2-distearoyl-177-glycero-3-phosphocholine (DSPC) analog, a DSPC substitute, oleic acid, or an oleic acid analog. [0548] In some embodiments, a helper lipid is a non-phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a DSPC substitute. [0549] In some embodiments, a helper lipid is described in PCT/US2018/053569. Helper lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Such helper lipids are preferably used in combination with one or more of the compounds and lipids described herein. Examples of helper lipids include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoylsn-glycero-3- phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2- palmitoyl phosphatidylcholine (MPPC), 1-paimitoyl-2-myristoyl phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), paimitoyioieoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanol amine (DOPE) dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC) or dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC). Helper lipids function to stabilize and improve processing of the transfer vehicles. Such helper lipids are preferably used in combination with other excipients, for example, one or more of the ionizable lipids described herein. In some embodiments, when used in combination with an ionizable lipid, the helper lipid may comprise a molar ratio of 5% to about 90%, or about 10% to about 70% of the total lipid present in the lipid nanoparticle. D. STRUCTURAL LIPIDS [0550] In an embodiment, a structural lipid is described in international patent application PCT/US2019/015913. [0551] The transfer vehicles described herein comprise one or more structural lipids. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. [0552] In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha- tocopherol. [0553] The transfer vehicles described herein comprise one or more structural lipids. Incorporation of structural lipids in a transfer vehicle, e.g., a lipid nanoparticle, may help mitigate aggregation of other lipids in the particle. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. [0554] In some embodiments, the structural lipid is a sterol. Structural lipids can include, but are not limited to, sterols (e.g., phytosterols or zoosterols). [0555] In certain embodiments, the structural lipid is a steroid. For example, sterols can include, but are not limited to, cholesterol, β-sitosterol, fecosterol, ergosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol. [0556] In some embodiments, a transfer vehicle includes an effective amount of an immune cell delivery potentiating lipid, e.g., a cholesterol analog or an amino lipid or combination thereof, that, when present in a transfer vehicle, e.g., an lipid nanoparticle, may function by enhancing cellular association and/or uptake, internalization, intracellular trafficking and/or processing, and/or endosomal escape and/or may enhance recognition by and/or binding to immune cells, relative to a transfer vehicle lacking the immune cell delivery potentiating lipid. Accordingly, while not intending to be bound by any particular mechanism or theory, in one embodiment, a structural lipid or other immune cell delivery potentiating lipid of the disclosure binds to C1q or promotes the binding of a transfer vehicle comprising such lipid to C1q. Thus, for in vitro use of the transfer vehicles of the disclosure for delivery of a nucleic acid molecule to an immune cell, culture conditions that include C1q are used (e.g., use of culture media that includes serum or addition of exogenous C1q to serum-free media). For in vivo use of the transfer vehicles of the disclosure, the requirement for C1q is supplied by endogenous C1q. [0557] In certain embodiments, the structural lipid is cholesterol or an analog of cholesterol. In some embodiments, the structural lipid is a lipid in Table 16: Table 16
E. LIPID NANOPARTICLE (LNP) FORMULATIONS [0558] The formation of a lipid nanoparticle (LNP) described herein may be accomplished by any methods known in the art. For example, as described in U.S. Pat. Pub. No. US2012/0178702 A1, which is incorporated herein by reference in its entirety. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety). [0559] In one embodiment, the LNP formulation may be prepared by, e.g., the methods described in International Pat. Pub. No. WO 2011/127255 or WO 2008/103276, the contents of each of which are herein incorporated by reference in their entirety. [0560] In one embodiment, LNP formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be a composition selected from Formulae 1-60 of U.S. Pat. Pub. No. US2005/0222064 A1, the content of which is herein incorporated by reference in its entirety. [0561] In one embodiment, the lipid nanoparticle may be formulated by the methods described in U.S. Pat. Pub. No. US2013/0156845 A1, and International Pat. Pub. No. WO2013/093648 A2 or WO2012/024526 A2, each of which is herein incorporated by reference in its entirety. [0562] In one embodiment, the lipid nanoparticles described herein may be made in a sterile environment by the system and/or methods described in U.S. Pat. Pub. No. US2013/0164400 A1, which is incorporated herein by reference in its entirety. [0563] In one embodiment, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle described in U.S. Pat. No.8,492,359, which is incorporated herein by reference in its entirety. [0564] A nanoparticle composition may optionally comprise one or more coatings. .For example, a nanoparticle composition may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density. [0565] In some embodiments, the lipid nanoparticles described herein may be synthesized using methods comprising microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to, a slit interdigitial micromixer including, but not limited to, those manufactured by Precision Nanosystems (Vancouver, BC, Canada), Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al. (2012) Langmuir. 28:3633-40; Belliveau, N.M. et al. Mol. Ther. Nucleic. Acids. (2012) 1:e37; Chen, D. et al. J. Am. Chem. Soc. (2012) 134(16):6948-51; each of which is herein incorporated by reference in its entirety). [0566] In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Pat. Pub. Nos. US2004/0262223 A1 and US2012/0276209 A1, each of which is incorporated herein by reference in their entirety. [0567] In one embodiment, the lipid nanoparticles may be formulated using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM)from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany). In one embodiment, the lipid nanoparticles are created using microfluidic technology (see, Whitesides (2006) Nature. 442: 368-373; and Abraham et al. (2002) Science. 295: 647-651; each of which is herein incorporated by reference in its entirety). As a non- limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g., Abraham et al. (2002) Science.295: 647651; which is herein incorporated by reference in its entirety). [0568] In one embodiment, the circRNA of the present disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA), Dolomite Microfluidics (Royston, UK), or Precision Nanosystems (Van Couver, BC, Canada). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism. [0569] In one embodiment, the lipid nanoparticles may have a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm. In one embodiment, the lipid nanoparticles may have a diameter from about 10 to 500 nm. In one embodiment, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm. Each possibility represents a separate embodiment of the present disclosure. [0570] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm. [0571] In some embodiments, the lipid nanoparticles described herein can have a diameter from below 0.1 µm to up to 1 mm such as, but not limited to, less than 0.1 µm, less than 1.0 µm, less than 5 µm, less than 10 µm, less than 15 µm, less than 20 µm, less than 25 µm, less than 30 µm, less than 35 µm, less than 40 µm, less than 50 µm, less than 55 µm, less than 60 µm, less than 65 µm, less than 70 µm, less than 75 µm, less than 80 µm, less than 85 µm, less than 90 µm, less than 95 µm, less than 100 µm, less than 125 µm, less than 150 µm, less than 175 µm, less than 200 µm, less than 225 µm, less than 250 µm, less than 275 µm, less than 300 µm, less than 325 µm, less than 350 µm, less than 375 µm, less than 400 µm, less than 425 µm, less than 450 µm, less than 475 µm, less than 500 µm, less than 525 µm, less than 550 µm, less than 575 µm, less than 600 µm, less than 625 µm, less than 650 µm, less than 675 µm, less than 700 µm, less than 725 µm, less than 750 µm, less than 775 µm, less than 800 µm, less than 825 µm, less than 850 µm, less than 875 µm, less than 900 µm, less than 925 µm, less than 950 µm, less than 975 µm. [0572] In another embodiment, LNPs may have a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from about 70 to about 90 nm. Each possibility represents a separate embodiment of the present disclosure. [0573] A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition may be from about 0.10 to about 0.20. Each possibility represents a separate embodiment of the present disclosure. [0574] The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition may be from about -20 mV to about +20 mV, from about -20 mV to about +15 mV, from about -20 mV to about +10 mV, from about -20 mV to about +5 mV, from about -20 mV to about 0 mV, from about -20 mV to about -5 mV, from about -20 mV to about -10 mV, from about -20 mV to about -15 mV from about -20 mV to about +20 mV, from about -20 mV to about +15 mV, from about -20 mV to about +10 mV, from about -20 mV to about +5 mV, from about -20 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. Each possibility represents a separate embodiment of the present disclosure. [0575] The efficiency of encapsulation of a therapeutic agent describes the amount of therapeutic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic agent in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic agent (e.g., nucleic acids) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic agent may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%. Each possibility represents a separate embodiment of the present disclosure. In some embodiments, the lipid nanoparticle has a polydiversity value of less than 0.4. In some embodiments, the lipid nanoparticle has a net neutral charge at a neutral pH. In some embodiments, the lipid nanoparticle has a mean diameter of 50-200nm. [0576] The properties of a lipid nanoparticle formulation may be influenced by factors including, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the selection of the non-cationic lipid component, the degree of noncationic lipid saturation, the selection of the structural lipid component, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. As described herein, the purity of a PEG lipid component is also important to an LNP’s properties and performance. F. METHODS FOR LIPID NANOPARTICLES (LNP) [0577] In one embodiment, a lipid nanoparticle formulation may be prepared by the methods described in International Publication Nos. WO2011127255 or WO2008103276, each of which is herein incorporated by reference in their entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. WO2019131770, which is herein incorporated by reference in its entirety. [0578] In some embodiments, circular RNA is formulated according to a process described in US patent application 15/809,680. In some embodiments, the present disclosure provides a process of encapsulating circular RNA in transfer vehicles comprising the steps of forming lipids into pre-formed transfer vehicles (i.e. formed in the absence of RNA) and then combining the pre-formed transfer vehicles with RNA. In some embodiments, the novel formulation process results in an RNA formulation with higher potency (peptide or protein expression) and higher efficacy (improvement of a biologically relevant endpoint) both in vitro and in vivo with potentially better tolerability as compared to the same RNA formulation prepared without the step of preforming the lipid nanoparticles (e.g., combining the lipids directly with the RNA). [0579] For certain cationic lipid nanoparticle formulations of RNA, in order to achieve high encapsulation of RNA, the RNA in buffer (e.g., citrate buffer) has to be heated. In those processes or methods, the heating is required to occur before the formulation process (i.e. heating the separate components) as heating post-formulation (post-formation of nanoparticles) does not increase the encapsulation efficiency of the RNA in the lipid nanoparticles. In contrast, in some embodiments of the novel processes of the present disclosure, the order of heating of RNA does not appear to affect the RNA encapsulation percentage. In some embodiments, no heating (i.e. maintaining at ambient temperature) of one or more of the solutions comprising the pre-formed lipid nanoparticles, the solution comprising the RNA and the mixed solution comprising the lipid nanoparticle encapsulated RNA is required to occur before or after the formulation process. [0580] RNA may be provided in a solution to be mixed with a lipid solution such that the RNA may be encapsulated in lipid nanoparticles. A suitable RNA solution may be any aqueous solution containing RNA to be encapsulated at various concentrations. For example, a suitable RNA solution may contain an RNA at a concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, a suitable RNA solution may contain an RNA at a concentration in a range from about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01- 0.8 mg/ml, 0.01-0.7 mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml. [0581] Typically, a suitable RNA solution may also contain a buffering agent and/or salt. Generally, buffering agents can include HEPES, Tris, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate or sodium phosphate. In some embodiments, suitable concentration of the buffering agent may be in a range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. [0582] Exemplary salts can include sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, suitable concentration of salts in an RNA solution may be in a range from about 1 mM to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM. [0583] In some embodiments, a suitable RNA solution may have a pH in a range from about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5. [0584] Various methods may be used to prepare an RNA solution suitable for the present disclosure. In some embodiments, RNA may be directly dissolved in a buffer solution described herein. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. [0585] According to the present disclosure, a lipid solution contains a mixture of lipids suitable to form transfer vehicles for encapsulation of RNA. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e.100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide. [0586] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration in a range from about 0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0- 60 mg/ml, 1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml, 1.0-10 mg/ml, 1.0- 9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or 1.0-5 mg/ml. G. LIPOSOMES [0587] In certain embodiments, liposomes or other lipid bilayer vesicles are described herein and may be used as a component or as the whole transfer vehicle to facilitate or enhance the delivery and release of circular RAN to one or more target cells. Liposomes are usually characterized by having an interior space sequestered from an outer medium by a membrane of one or more bilayers forming a microscopic sack or vesicle. Bilayer membranes of liposomes are typically formed by lipids, i.e. amphiphilic molecules of synthetic or natural origin that comprise spatially separated hydrophobic or hydrophilic domains (Lasic, D, and Papahadjopoulos, D., eds. Medical Applications of Liposomes. Elsevier, Amsterdam, 1998). [0588] In some embodiments, the circular RNA is encapsulated, or the liposome can be prepared using various methods, including but not limited to mechanical dispersion, solvent dispersion, and or detergent removal. Each of these methods include the steps of drying the lipids from organic solvents, dispersing the lipid in aqueous media, resizing the liposomes and purifying the/liposome suspension (Gomez et al., ACS Omega.2019.4(6): 10866-10876). Various other methods of liposome preparation can be found in Akbarzadeh et al., Nanoscale Res Lett. 2013; 8(1): 102. In some embodiments, the circular RNA may be loaded passively (i.e. the circular RNA is encapsulated during liposome formation) or actively (i.e. after liposome formation). [0589] In some embodiments, the liposome described herein may comprise one or more bilayers. In certain embodiments, the liposome may comprise a multilamellar vesicle or a unilamellar vesicle. [0590] In certain embodiments, the liposome as described herein comprises of naturally derived or engineered phospholipids. In some embodiments, the liposomes may further comprise PEG-lipids that aid with stability of the overall liposome structure. Other improvements, including but not limited to corticosteroid and other steroids may be used to help with maintaining structure and stability of the liposome. H. DENDRIMER [0591] In certain embodiments, the transfer vehicle for transporting the circular RNA comprises a dendrimer. Use of “dendrimer” describes the architectural motif of the transfer vehicle. In some embodiments, the dendrimer includes but is not limited to containing an interior core and one or more layers (i.e. generations) that extend or attach out from the interior core. In some of the embodiments, the generations may contain one or more branching points and an exterior surface of terminal groups that attach to the outermost generation. The branching points, in certain embodiments, may be mostly monodispersed and contain symmetric branching units built around the interior core. In some embodiments, the interior core [0592] Synthesis of the dendrimer may comprise the divergent method, convergent growth, hypercore and branched monomer growth, double exponential growth, lego chemistry, click chemistry and other methods as available in the art (Mendes L. et al., Molecules.2017.22 (9): 1401 further describes these methods). I. POLYMER-BASED DELIVERY [0593] In certain embodiments, as described herein, the transfer vehicle for the circular RNA polynucleotide comprises a polymer nanoparticle. In some embodiments, the polymer nanoparticle includes nanocapsules and nanospheres. Nanocapsules, in some embodiments, are composed of an oily core surrounded by a polymeric shell. In some embodiments, the circular RNA is contained within the core and the polymeric shell controls the release of the circular RNA. On the other hand, nanospheres comprise a continuous polymeric network in which the circular RNA is retained or absorbed onto the surface. In some embodiments, cationic polymers is used to encapsulate the circular RNA due to the favorable electrostatic interaction of the cations to the negatively charged nucleic acids and cell membrane. [0594] The polymer nanoparticle may be prepared by various methods. In some embodiments, the polymer nanoparticle may be prepared by nanoprecipitation, emulsion techniques, solvent evaporation, solvent diffusion, reverse salting-out or other methods available in the art. J. POLYMER-LIPID HYBRIDS [0595] In certain embodiments, as described herein, the transfer vehicle for the circular RNA polynucleotide comprises a polymer-lipid hybrid nanoparticle (LPHNP). In some embodiments, the LPHNP comprises a polymer core enveloped within a lipid bilayer. In some embodiments, the polymer core encapsulates the circular RNA polynucleotide. In some embodiments, the LPHNP further comprises an outer lipid bilayer. In certain embodiments this outer lipid bilayer comprises a PEG-lipid, helper lipid, cholesterol or other molecule as known in the art to help with stability in a lipid-based nanoparticle. The lipid bilayer closest to the polymer core mitigates the loss of the entrapped circular RNA during LPHNP formation and protects from degradation of the polymer core by preventing diffusion of water from outside of the transfer vehicle into the polymer core (Mukherjee et al., In J. Nanomedicine.2019; 14: 1937-1952). [0596] There are various methods of developing and formulating a LPHNP. In certain embodiments, the LPHNP is developed using a one-step or a two-step method available in the art. In some embodiments, the one-step method for forming an LPHNP is through nanoprecipitation or emulsification-solvent evaporation. In certain embodiments, the two-step method includes nanoprecipitation, emulsification-solvent evaporation, high-pressure homogenization, or other method available in the art. K. PEPTIDE-BASED DELIVERY [0597] In certain embodiments, the circular RNA can be transported using a peptide-based delivery mechanism. In some embodiments, the peptide-based delivery mechanism comprises a lipoprotein. Based on the size of the drug to be delivered, the lipoprotein may be either a low-density (LDL) or high- density lipoprotein (HDL). As seen in US8734853B2, high-density lipoproteins are capable of transporting a nucleic acid in vivo and in vitro. [0598] In particular embodiments, the lipid component includes cholesterol. In more particular embodiments, the lipid component includes a combination of cholesterol and cholesterol oleate. [0599] The HDL-nucleic acid particle can be of any size, but in particular embodiments the particle has a molecular size of from about 100 Angstroms to about 500 Angstroms. In more particular embodiments, the particle has a molecular size of from about 100 Angstroms to about 300 Angstroms. The size may be dependent on the size of the nucleic acid component incorporated into the particle. [0600] The HDL-nucleic acid particle can have a broad range in molecular weight. The weight is dependent on the size of the nucleic acid incorporated into the particle. For example, in some embodiments, the particle has a molecular weight of from about 100,000 Daltons to about 1,000,000 Daltons. In more particular embodiments, the particle has a molecular weight of from about 100,000 Daltons to about 500,000 Daltons. In specific embodiments, the particle has a molecular weight of from about 100,000 Daltons to about 300,000 Daltons. [0601] The HDL-nucleic acid particles of the present disclosure can be made by different methods. For example, a nucleic acid (e.g., siRNA) may be neutralized by combining the nucleic acid with peptides or polypeptides composed of contiguous positively-charged amino acids. For example, amino acid sequences may include 2 or more contiguous lysine residues. The positive charge of the amino acid sequences neutralizes the negatively charged nucleic acid molecule. The nucleic acid can then be encapsulated in an HDL particle using a method as described in Lacko et al. (2002). L. CARBOHYDRATE CARRIER [0602] In certain embodiments, the circular RNA polynucleotide can be transported using a carbohydrate carrier or a sugar-nanocapsule. In certain embodiments, the carbohydrate carrier comprises a sugar-decorated nanoparticle, peptide- and saccharide-conjugated dendrimer, nanoparticles based on polysaccharides, and other carbohydrate-based carriers available in the art. As described herein, the incorporation of carbohydrate molecules may be through synthetic means. [0603] In some embodiments, the carbohydrate carrier comprises polysaccharides. These polysaccharides may be made from the microbial cell wall of the target cell. For example, carbohydrate carriers comprise of mannan carbohydrates have been shown to successfully deliver mRNA (Son et al., Nano Lett.2020.20(3): 1499-1509). M. GLYCAN-DECORATED NANOPARTICLES/GLYCONANOPARTICLES [0604] In certain embodiments, as provided herein, the transfer vehicle for the circular RNA is a glyconanoparticle (GlycoNP). As known in the art, glyconanoparticles comprise a core comprising gold, iron oxide, semiconductor nanoparticles or a combination thereof. In some embodiments, the glyconanoparticle is functionalized using carbohydrates. In certain embodiments, the glyconanoparticle comprises a carbon nanotube or graphene. In one embodiment the glyconanoparticle comprises a polysaccharide-based GlycoNP (e.g., chitosan-based GlycoNP). In certain embodiments, the glyconanoparticle is a glycodendrimer. 7. COMBINATIONS OF PROTEINS AND IRES [0605] In certain embodiments, as provided herein, the payload encoded by the circular RNA polynucleotide may be optimized through use of a specific internal ribosome entry sites (IRES) within the translation initiation element (TIE). In some embodiments, IRES specificity within a circular RNA can significantly enhance expression of specific proteins encoded within the coding element. 8. TARGETING A. TARGETING METHODS [0606] The present disclosure also contemplates the discriminatory targeting of target cells and tissues by both passive and active targeting means. The phenomenon of passive targeting exploits the natural distributions patterns of a transfer vehicle in vivo without relying upon the use of additional excipients or means to enhance recognition of the transfer vehicle by target cells. For example, transfer vehicles which are subject to phagocytosis by the cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen, and accordingly may provide a means to passively direct the delivery of the compositions to such target cells. [0607] Alternatively, the present disclosure contemplates active targeting, which involves the use of targeting moieties that may be bound (either covalently or non-covalently) to the transfer vehicle to encourage localization of such transfer vehicle at certain target cells or target tissues. For example, targeting may be mediated by the inclusion of one or more endogenous targeting moieties in or on the transfer vehicle to encourage distribution to the target cells or tissues. Recognition of the targeting moiety by the target tissues actively facilitates tissue distribution and cellular uptake of the transfer vehicle and/or its contents in the target cells and tissues (e.g., the inclusion of an apolipoprotein-E targeting ligand in or on the transfer vehicle encourages recognition and binding of the transfer vehicle to endogenous low density lipoprotein receptors expressed by hepatocytes). As provided herein, the composition can comprise a moiety capable of enhancing affinity of the composition to the target cell. Targeting moieties may be linked to the outer bilayer of the lipid particle during formulation or post- formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S. patent application Ser. No.08/835,281, and 60/083,294, which are incorporated herein by reference) and other features useful for in vivo and/or intracellular delivery. In other some embodiments, the compositions of the present disclosure demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest. Contemplated therefore are compositions which comprise one or more moieties (e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents for the target cells or tissues. Suitable moieties may optionally be bound or linked to the surface of the transfer vehicle. In some embodiments, the targeting moiety may span the surface of a transfer vehicle or be encapsulated within the transfer vehicle. Suitable moieties and are selected based upon their physical, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features). Cell-specific target sites and their corresponding targeting ligand can vary widely. Suitable targeting moieties are selected such that the unique characteristics of a target cell are exploited, thus allowing the composition to discriminate between target and non-target cells. For example, compositions of the disclosure may include surface markers (e.g., apolipoprotein-B or apolipoprotein- E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers). As an example, the use of galactose as a targeting moiety would be expected to direct the compositions of the present disclosure to parenchymal hepatocytes, or alternatively the use of mannose containing sugar residues as a targeting ligand would be expected to direct the compositions of the present disclosure to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor present in hepatocytes). (See Hillery A M, et al. “Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists” (2002) Taylor & Francis, Inc.) The presentation of such targeting moieties that have been conjugated to moieties present in the transfer vehicle (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions of the present disclosure in target cells and tissues. Examples of suitable targeting moieties include one or more peptides, proteins, aptamers, vitamins and oligonucleotides. [0608] In particular embodiments, a transfer vehicle comprises a targeting moiety. In some embodiments, the targeting moiety mediates receptor-mediated endocytosis selectively into a specific population of cells. In some embodiments, the targeting moiety is capable of binding to a T cell antigen. In some embodiments, the targeting moiety is capable of binding to a NK, NKT, or macrophage antigen. In some embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, PD-1, 4-1BB, and CD2. In some embodiments, the targeting moiety is an single chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, heavy chain variable region, light chain variable region or fragment thereof. In some embodiments, the targeting moiety is selected from T-cell receptor motif antibodies, T-cell α chain antibodies, T-cell β chain antibodies, T- cell γ chain antibodies, T-cell δ chain antibodies, CCR7 antibodies, CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8 antibodies, CD11b antibodies, CD11c antibodies, CD16 antibodies, CD19 antibodies, CD20 antibodies, CD21 antibodies, CD22 antibodies, CD25 antibodies, CD28 antibodies, CD34 antibodies, CD35 antibodies, CD40 antibodies, CD45RA antibodies, CD45RO antibodies, CD52 antibodies, CD56 antibodies, CD62L antibodies, CD68 antibodies, CD80 antibodies, CD95 antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, CD137 (4-1BB) antibodies, CD163 antibodies, F4/80 antibodies, IL-4Rα antibodies, Sca-1 antibodies, CTLA-4 antibodies, GITR antibodies GARP antibodies, LAP antibodies, granzyme B antibodies, LFA-1 antibodies, transferrin receptor antibodies, and fragments thereof. In some embodiments, the targeting moiety is a small molecule binder of an ectoenzyme on lymphocytes. Small molecule binders of ectoenzymes include A2A inhibitors CD73 inhibitors, CD39 or adesines receptors A2aR and A2bR. Potential small molecules include AB928. [0609] In some embodiments, transfer vehicles are formulated and/or targeted as described in Shobaki N, Sato Y, Harashima H. Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting. Int J Nanomedicine. 2018;13:8395–8410. Published 2018 Dec 10. In some embodiments, a transfer vehicle is made up of 3 lipid types. In some embodiments, a transfer vehicle is made up of 4 lipid types. In some embodiments, a transfer vehicle is made up of 5 lipid types. In some embodiments, a transfer vehicle is made up of 6 lipid types. B. TARGET CELLS [0610] Where it is desired to deliver a nucleic acid to an immune cell, the immune cell represents the target cell. In some embodiments, the compositions of the disclosure transfect the target cells on a discriminatory basis (i.e., do not transfect non-target cells). The compositions of the disclosure may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, T cells, B cells, macrophages, and dentritic cells. [0611] In some embodiments, the target cells are deficient in a protein or enzyme of interest. For example, where it is desired to deliver a nucleic acid to a hepatocyte, the hepatocyte represents the target cell. In some embodiments, the compositions of the disclosure transfect the target cells on a discriminatory basis (i.e., do not transfect non-target cells). The compositions of the disclosure may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells. [0612] The compositions of the disclosure may be prepared to preferentially distribute to target cells such as in the heart, lungs, kidneys, liver, and spleen. In some embodiments, the compositions of the disclosure distribute into the cells of the liver or spleen to facilitate the delivery and the subsequent expression of the circRNA comprised therein by the cells of the liver (e.g., hepatocytes) or the cells of spleen (e.g., immune cells). The targeted cells may function as a biological “reservoir” or “depot” capable of producing, and systemically excreting a functional protein or enzyme. Accordingly, in one embodiment of the disclosure the transfer vehicle may target hepatocytes or immune cells and/or preferentially distribute to the cells of the liver or spleen upon delivery. In an embodiment, following transfection of the target hepatocytes or immune cells, the circRNA loaded in the vehicle are translated and a functional protein product is produced, excreted and systemically distributed. In other embodiments, cells other than hepatocytes (e.g., lung, spleen, heart, ocular, or cells of the central nervous system) can serve as a depot location for protein production. [0613] In one embodiment, the compositions of the disclosure facilitate a subject’s endogenous production of one or more functional proteins and/or enzymes. In an embodiment of the present disclosure, the transfer vehicles comprise circRNA which encode a deficient protein or enzyme. Upon distribution of such compositions to the target tissues and the subsequent transfection of such target cells, the exogenous circRNA loaded into the transfer vehicle (e.g., a lipid nanoparticle) may be translated in vivo to produce a functional protein or enzyme encoded by the exogenously administered circRNA (e.g., a protein or enzyme in which the subject is deficient). Accordingly, the compositions of the present disclosure exploit a subject’s ability to translate exogenously- or recombinantly-prepared circRNA to produce an endogenously-translated protein or enzyme, and thereby produce (and where applicable excrete) a functional protein or enzyme. The expressed or translated proteins or enzymes may also be characterized by the in vivo inclusion of native post-translational modifications which may often be absent in recombinantly-prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme. [0614] The administration of circRNA encoding a deficient protein or enzyme avoids the need to deliver the nucleic acids to specific organelles within a target cell. Rather, upon transfection of a target cell and delivery of the nucleic acids to the cytoplasm of the target cell, the circRNA contents of a transfer vehicle may be translated and a functional protein or enzyme expressed. [0615] In some embodiments, a circular RNA comprises one or more miRNA binding sites. In some embodiments, a circular RNA comprises one or more miRNA binding sites recognized by miRNA present in one or more non-target cells or non-target cell types (e.g., Kupffer cells or hepatic cells) and not present in one or more target cells or target cell types (e.g., hepatocytes or T cells). In some embodiments, a circular RNA comprises one or more miRNA binding sites recognized by miRNA present in an increased concentration in one or more non-target cells or non-target cell types (e.g., Kupffer cells or hepatic cells) compared to one or more target cells or target cell types (e.g., hepatocytes or T cells). miRNAs are thought to function by pairing with complementary sequences within RNA molecules, resulting in gene silencing. [0616] In some embodiments, the compositions of the disclosure transfect or distribute to target cells on a discriminatory basis (i.e. do not transfect non-target cells). The compositions of the disclosure may also be prepared to preferentially target a variety of target cells, which include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells. 9. PHARMACEUTICAL COMPOSITIONS [0617] In certain embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising a therapeutic agent provided herein. In some embodiments, the therapeutic agent is a circular RNA polynucleotide provided herein. In some embodiments the therapeutic agent is a vector provided herein. In some embodiments, the therapeutic agent is a cell comprising a circular RNA or vector provided herein (e.g., a human cell, such as a human T cell). In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the compositions provided herein comprise a therapeutic agent provided herein in combination with other pharmaceutically active agents or drugs, such as anti-inflammatory drugs or antibodies capable of targeting B cell antigens, e.g., anti-CD20 antibodies, e.g., rituximab. [0618] With respect to pharmaceutical compositions, the pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active agent(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the therapeutic agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0619] The choice of carrier will be determined in part by the particular therapeutic agent, as well as by the particular method used to administer the therapeutic agent. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions provided herein. [0620] In certain embodiments, the pharmaceutical composition comprises a preservative. In certain embodiments, suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. [0621] In some embodiments, the pharmaceutical composition comprises a buffering agent. In some embodiments, suitable buffering agents may include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents optionally may be used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. [0622] In some embodiments, the concentration of therapeutic agent in the pharmaceutical composition can vary, e.g., less than about 1%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50% or more by weight, and can be selected primarily by fluid volumes, and viscosities, in accordance with the particular mode of administration selected. [0623] The following formulations for oral, aerosol, parenteral (e.g., subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and intrathecal), and topical administration are merely exemplary and are in no way limiting. More than one route can be used to administer the therapeutic agents provided herein, and in certain instances, a particular route can provide a more immediate and more effective response than another route. [0624] Formulations suitable for oral administration can comprise or consist of (a) liquid solutions, such as an effective amount of the therapeutic agent dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard or soft shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and other pharmacologically compatible excipients. Lozenge forms can comprise the therapeutic agent with a flavorant, usually sucrose, acacia or tragacanth. Pastilles can comprise the therapeutic agent with an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to, such excipients as are known in the art. [0625] Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In some embodiments, the therapeutic agents provided herein can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol or hexadecyl alcohol, a glycol such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0626] Oils, which can be used in parenteral formulations in some embodiments, include petroleum, animal oils, vegetable oils, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. [0627] Suitable soaps for use in certain embodiments of parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides. and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alky, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof. [0628] In some embodiments, the parenteral formulations will contain, for example, from about 0.5% to about 25% by weight of the therapeutic agent in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having, for example, a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range, for example, from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol, sorbitan, fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules or vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. [0629] In certain embodiments, injectable formulations are provided herein. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed, pages 622-630 (1986)). [0630] In some embodiments, topical formulations are provided herein. Topical formulations, including those that are useful for transdermal drug release, are suitable in the context of certain embodiments provided herein for application to skin. In some embodiments, the therapeutic agent alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. [0631] In certain embodiments, the therapeutic agents provided herein can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes can serve to target the therapeutic agents to a particular tissue. Liposomes also can be used to increase the half-life of the therapeutic agents. Many methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369. [0632] In some embodiments, the therapeutic agents provided herein are formulated in time-released, delayed release, or sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to, cause sensitization of the site to be treated. Such systems can avoid repeated administrations of the therapeutic agent, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain composition embodiments provided herein. In one embodiment, the compositions of the disclosure are formulated such that they are suitable for extended-release of the circRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present disclosure are administered to a subject twice a day, daily or every other day. In an embodiment, the compositions of the present disclosure are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, every four weeks, once a month, every six weeks, every eight weeks, every three months, every four months, every six months, every eight months, every nine months or annually. [0633] In some embodiments, a protein encoded by an inventive polynucleotide is produced by a target cell for sustained amounts of time. For example, the protein may be produced for more than one hour, more than four, more than six, more than 12, more than 24, more than 48 hours, or more than 72 hours after administration. In some embodiments the polypeptide is expressed at a peak level about six hours after administration. In some embodiments the expression of the polypeptide is sustained at least at a therapeutic level. In some embodiments, the polypeptide is expressed at least at a therapeutic level for more than one, more than four, more than six, more than 12, more than 24, more than 48, or more than 72 hours after administration. In some embodiments, the polypeptide is detectable at a therapeutic level in patient tissue (e.g., liver or lung). In some embodiments, the level of detectable polypeptide is from continuous expression from the circRNA composition over periods of time of more than one, more than four, more than six, more than 12, more than 24, more than 48, or more than 72 hours after administration. [0634] In certain embodiments, a protein encoded by an inventive polynucleotide is produced at levels above normal physiological levels. The level of protein may be increased as compared to a control. In some embodiments, the control is the baseline physiological level of the polypeptide in a normal individual or in a population of normal individuals. In other embodiments, the control is the baseline physiological level of the polypeptide in an individual having a deficiency in the relevant protein or polypeptide or in a population of individuals having a deficiency in the relevant protein or polypeptide. In some embodiments, the control can be the normal level of the relevant protein or polypeptide in the individual to whom the composition is administered. In other embodiments, the control is the expression level of the polypeptide upon other therapeutic intervention, e.g., upon direct injection of the corresponding polypeptide, at one or more comparable time points. [0635] In certain embodiments, the levels of a protein encoded by an inventive polynucleotide are detectable at 3 days, 4 days, 5 days, or 1 week or more after administration. Increased levels of protein may be observed in a tissue (e.g., liver or lung). [0636] In some embodiments, the method yields a sustained circulation half-life of a protein encoded by an inventive polynucleotide. For example, the protein may be detected for hours or days longer than the half-life observed via subcutaneous injection of the protein or mRNA encoding the protein. In some embodiments, the half-life of the protein is 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week or more. [0637] Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems: wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Patents 4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patents 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. [0638] In some embodiments, the therapeutic agent can be conjugated either directly or indirectly through a linking moiety to a targeting moiety. Methods for conjugating therapeutic agents to targeting moieties is known in the art. See, for instance, Wadwa et al., J, Drug Targeting 3:111 (1995) and U.S. Patent 5,087,616. [0639] In some embodiments, the therapeutic agents provided herein are formulated into a depot form, such that the manner in which the therapeutic agent is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent 4,450,150). Depot forms of therapeutic agents can be, for example, an implantable composition comprising the therapeutic agents and a porous or non-porous material, such as a polymer, wherein the therapeutic agents are encapsulated by or diffused throughout the material and/or degradation of the non-porous material. The depot is then implanted into the desired location within the body and the therapeutic agents are released from the implant at a predetermined rate. 10. THERAPEUTIC METHODS [0640] In certain aspects, provided herein is a method of treating and/or preventing a condition, e.g., an autoimmune disorder or cancer. [0641] In certain embodiments, the therapeutic agents provided herein are co-administered with one or more additional therapeutic agents (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the therapeutic agent provided herein can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the therapeutic agent provided herein and the one or more additional therapeutic agents can be administered simultaneously. [0642] In some embodiments, the subject is a mammal. In some embodiments, the mammal referred to herein can be any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, or mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs), or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human. 11. ADDITIONAL EMBODIMENTS [0643] Aspects of this disclosure are set forth in the following clauses: [644] Clause 1. A pharmaceutical composition comprising: a. an RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid represented by Formula (13*): Formula (13*) wherein: n * is 1 to 7; R a is hydrogen or hydroxyl; R b is hydrogen or C1-C6 alkyl; R 1 and R 2 are each independently a linear or branched C 1 -C 30 alkyl, C 2 -C 30 alkenyl, or C 1 -C 30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; with the proviso that the ionizable lipid is not or . [645] Clause 2. The pharmaceutical composition of clause 1, wherein R b is C1-C6 alkyl. [646] Clause 3. The pharmaceutical composition of clause 1, wherein R b is H and the ionizable lipid is represented by Formula (13): , wherein n is 1 to 7. [647] Clause 4. The pharmaceutical composition of clause 3, wherein n is 1 to 4. [648] Clause 5. The pharmaceutical composition of any one of clauses 1 to 4, wherein Ra is hydrogen. [649] Clause 6. The pharmaceutical composition of clause 5, wherein the ionizable lipid is of Formula (13a-1), Formula (13a-2), or Formula (13a-3): Formula (13a-1) Formula (13a-2) Formula (13a-3). [650] Clause 7. The pharmaceutical composition of any one of clauses 1 to 4, wherein R a is hydroxyl. [651] Clause 8. The pharmaceutical composition of clause 7, wherein the ionizable lipid is represented by Formula (13b-1), Formula (13b-2), or Formula (13b-3): Formula (13b-1) Formula (13b-2) Formula (13b-3). [652] Clause 9. The pharmaceutical composition of clause 7, wherein the ionizable lipid is represented by Formula (13b-4), Formula (13b-5), Formula (13b-6), Formula (13b-7), Formula (13b- 8), or Formula (13b-9): Formula (13b-4) Formula (13b-5) Formula (13b-6) Formula (13b-7) Formula (13b-8) Formula (13b-9). [653] Clause 10. The pharmaceutical composition of any one of clauses 1-9, wherein R1 and R2 are independently a linear or branched C1-C20 alkyl, C2-C20 alkenyl, or C1-C20 heteroalkyl, optionally substituted by one or more substituents selected from C 1 -C 20 alkoxy, C 1 -C 20 alkyloxycarbonyl, C 1 -C 20 alkylcarbonyloxy, C1-C20 alkylcarbonate, C2-C20 alkenyloxycarbonyl, C2-C20 alkenylcarbonyloxy, C2- C20 alkenylcarbonate, C2-C20 alkynyloxycarbonyl, C2-C20 alkynylcarbonyloxy, and C2-C20 alkynylcarbonate. [654] Clause 11. The pharmaceutical composition of any one of clauses 1-10, wherein at least one of R1 and R2 is an unsubstituted, linear or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl. [655] Clause 12. The pharmaceutical composition of any one of clauses 1-11, wherein at least one of R 1 and R 2 is a linear C 1 -C 12 alkyl substituted by -O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 , wherein each R 6 is independently linear or branched C1-C20 alkyl or C2-C20 alkenyl. [656] Clause 13. The pharmaceutical composition of clause 12, wherein R 1 and R 2 are each independently a linear C 1 -C 12 alkyl substituted by -O(CO)R 6 , -C(O)OR 6 , or -O(CO)OR 6 , wherein each R 6 is independently linear or branched C 1 -C 20 alkyl or C 2 -C 20 alkenyl. [657] Clause 14. The pharmaceutical composition of any one of clauses 1-13, wherein the at least one of R 1 and R 2 is selected from: -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), wherein: q is 1 to 12, r is 0 to 6, R 8 is H or R 10 , and R 9 and R 11 are independently unsubstituted linear C 1 -C 12 alkyl or unsubstituted linear C 2 -C 12 - alkenyl. [658] Clause 15. The pharmaceutical composition of clause 14, wherein R1 and R2 are independently selected from: -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and -(CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ), wherein: q is 1 to 12, r is 0 to 6, R 8 is H or R 10 , and R 9 and R 10 are independently unsubstituted linear C1-C12 alkyl or unsubstituted linear C2-C12- alkenyl. [659] Clause 16. The pharmaceutical composition of clause 14, wherein R 1 is unsubstituted, linear or branched C 6 -C 30 alkyl. [660] Clause 17. The pharmaceutical composition of clause 14 or 15, wherein R1 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ). [661] Clause 18. The pharmaceutical composition of clause 14 or 15, wherein R1 is -(CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ). [662] Clause 19. The pharmaceutical composition of clause 14 or 15, wherein R 1 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ). [663] Clause 20. The pharmaceutical composition of any one of clauses 16-19, wherein R2 is unsubstituted, linear or branched C6-C30 alkyl. [664] Clause 21. The pharmaceutical composition of any one of clauses 16-19, wherein R 2 is -(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ). [665] Clause 22. The pharmaceutical composition of any one of clauses 16-19, wherein R 2 is -(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ). [666] Clause 23. The pharmaceutical composition of any one of clauses 16-19, wherein R2 is -(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ). [667] Clause 24. The pharmaceutical composition of any one of clauses 14 to 23, wherein q is 1 to 6. [668] Clause 25. The pharmaceutical composition of any one of clauses 14 to 23, wherein q is 3 to 6. [669] Clause 26. The pharmaceutical composition of any one of clauses 14 to 25, wherein r is 0. [670] Clause 27. The pharmaceutical composition of any one of clauses 14 to 25, wherein r is 1 to 6. [671] Clause 28. The pharmaceutical composition of any one of clauses 14 to 25, wherein r is 1. [672] Clause 29. The pharmaceutical composition of any one of clauses 14 to 25, wherein r is 2. [673] Clause 30. The pharmaceutical composition of any one of clauses 14 to 29, wherein R 8 is H. [674] Clause 31. The pharmaceutical composition of any one of clauses 14 to 29, wherein R 8 is R 10 . [675] Clause 32. The pharmaceutical composition of clause 31, wherein R 9 and R 10 are independently unsubstituted linear C 1 -C 12 alkyl. [676] Clause 33. The pharmaceutical composition of clause 32, wherein R 9 and R 10 are independently unsubstituted linear C4-C8 alkyl. [677] Clause 34. The pharmaceutical composition of clause 33, wherein R 9 and R 10 are independently unsubstituted linear C6-C8 alkyl. [678] Clause 35. The pharmaceutical composition of any one of clauses 1-13, wherein R 1 and R 2 are each independently selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and . [679] Clause 36. The pharmaceutical composition of any one of clauses 1-15, wherein R1 and R2 are the same. [680] Clause 37. The pharmaceutical composition of any one of clauses 1-15, wherein R1 and R2 are different. [681] Clause 38. The pharmaceutical composition of any one of clauses 1-14, wherein the ionizable lipid is selected from , , , , , , , , O OH O N OH O O and . [682] Clause 39. The pharmaceutical composition of any one of clauses 1-14, wherein the ionizable lipid is selected from and . [683] Clause 40. The pharmaceutical composition of any one of clauses 1-14, wherein the ionizable lipid is selected from Table 10e. [684] Clause 41. The pharmaceutical composition of any one of clauses 1-40, wherein the RNA polynucleotide is a linear or circular RNA polynucleotide. [685] Clause 42. The pharmaceutical composition of any one of clauses 1-40, wherein the RNA polynucleotide is a circular RNA polynucleotide. [0686] Clause 43. A pharmaceutical composition comprising: an RNA polynucleotide, wherein the RNA polynucleotide is a circular RNA polynucleotide, and a transfer vehicle comprising an ionizable lipid selected from HO O N OH O O O or . [0687] Clause 44. The pharmaceutical composition of any one of clauses 1-43, wherein the RNA polynucleotide is encapsulated in the transfer vehicle. [0688] Clause 45. The pharmaceutical composition of any one of clauses 1-44, wherein the RNA polynucleotide is encapsulated in the transfer vehicle with an encapsulation efficiency of at least 80%. [0689] Clause 46. The pharmaceutical composition of any one of clauses 1-45, wherein the RNA comprises a first expression sequence. [0690] Clause 47. The pharmaceutical composition of clause 46, wherein the first expression sequence encodes a therapeutic protein. [0691] Clause 48. The pharmaceutical composition of clause 46, wherein the first expression sequence encodes a cytokine or a functional fragment thereof. [0692] Clause 49. The pharmaceutical composition of clause 47, wherein the first expression sequence encodes a transcription factor. [0693] Clause 50. The pharmaceutical composition of clause 47, wherein the first expression sequence encodes an immune checkpoint inhibitor. [0694] Clause 51. The pharmaceutical composition of clause 47, wherein the first expression sequence encodes a chimeric antigen receptor (CAR). [0695] Clause 52. The pharmaceutical composition of any one of clauses 1-51, wherein the RNA polynucleotide further comprises a second expression sequence. [0696] Clause 53. The pharmaceutical composition of clause 52, wherein the RNA polynucleotide further comprises an internal ribosome entry site (IRES). [0697] Clause 54. The pharmaceutical composition of clause 53, wherein the first and second expression sequences are separated by a ribosomal skipping element or a nucleotide sequence encoding a protease cleavage site. [0698] Clause 55. The pharmaceutical composition of any one of clauses 52 or 54, wherein the first expression sequence encodes a first T-cell receptor (TCR) chain, and the second expression sequence encodes a second TCR chain. [0699] Clause 56. The pharmaceutical composition of any one of clauses 1-55, wherein the RNA polynucleotide comprises one or more microRNA binding sites. [0700] Clause 57. The pharmaceutical composition of clause 56, wherein the microRNA binding site is recognized by a microRNA expressed in the liver. [0701] Clause 58. The pharmaceutical composition of clause 56 or 57, wherein the microRNA binding site is recognized by miR-122. [0702] Clause 59. The pharmaceutical composition of any one of clauses 1-58, wherein the RNA polynucleotide comprises a first IRES associated with greater protein expression in a human immune cell than in a reference human cell. [0703] Clause 60. The pharmaceutical composition of clause 59, wherein the human immune cell is a T cell, an NK cell, an NKT cell, a macrophage, or a neutrophil. [0704] Clause 61. The pharmaceutical composition of clause 59 or 60, wherein the reference human cell is a hepatic cell. [0705] Clause 62. The pharmaceutical composition of any one of clauses 1-61, wherein the RNA polynucleotide comprises, in the following order: a 5’ enhanced exon element, a core functional element, and a 3’ enhanced exon element. [0706] Clause 63. The pharmaceutical composition of any one of clauses 1-62, further comprising a post-splicing intron fragment. [0707] Clause 64. The pharmaceutical composition of clause 62 or 63, wherein the 5’ enhanced exon element comprises a 3’ exon fragment. [0708] Clause 65. The pharmaceutical composition of any one of clauses 62-64, wherein the 5’ enhanced exon element comprises a 5’ internal duplex region located downstream to the 3’ exon fragment. [0709] Clause 66. The pharmaceutical composition of any one of clauses 62-65, wherein the 5’ enhanced exon element comprises a 5’ internal spacer located downstream to the 3’ exon fragment. [0710] Clause 67. The pharmaceutical composition of clause 66, wherein the 5’ internal spacer has a length of about 10 to about 60 nucleotides. [0711] Clause 68. The pharmaceutical composition of clause 66 or 67, wherein the 5’ internal spacer comprises a polyA or polyA-C sequence. [0712] Clause 69. The pharmaceutical composition of clause 68, wherein the polyA or polyA-C sequence comprises a length of about 10-50 nucleotides. [0713] Clause 70. The pharmaceutical composition of any one of clauses 62-69, wherein the core functional element comprises a translation initiation element (TIE). [0714] Clause 71. The pharmaceutical composition of any one of clauses 70, wherein the translation initiation element (TIE) comprises an untranslated region (UTR) or fragment thereof. [0715] Clause 72. The pharmaceutical composition of clause 71, wherein the UTR or fragment thereof comprises a viral internal ribosome entry site (IRES) or eukaryotic IRES. [0716] Clause 73. The pharmaceutical composition of clause 72, wherein the IRES is selected from Table 17, or is a functional fragment or variant thereof. [0717] Clause 74. The pharmaceutical composition of clause 72 or 73, wherein the IRES has a sequence in whole or in part from a Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c- IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Apodemus Agrarius Picornavirus, Caprine Kobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or an aptamer to eIF4G. [0718] Clause 75. The pharmaceutical composition of any one of clauses 70-74, wherein the translation initiation element (TIE) comprises an aptamer complex. [0719] Clause 76. The pharmaceutical composition of clause 70, wherein the aptamer complex comprises at least two aptamers. [0720] Clause 77. The pharmaceutical composition of any one of clauses 62-76, wherein the core functional element comprises a coding region. [0721] Clause 78. The pharmaceutical composition of clause 77, wherein the coding region encodes for a therapeutic protein. [0722] Clause 79. The pharmaceutical composition of clause 78, wherein the therapeutic protein is a chimeric antigen receptor (CAR), a cytokine, a transcription factor, a T cell receptor (TCR), B-cell receptor (BCR), ligand, immune cell activation or inhibitory receptor, recombinant fusion protein, chimeric mutant protein, or fusion protein or a functional fragment thereof. [0723] Clause 80. The pharmaceutical composition of clause 79, wherein the therapeutic protein is an antigen. [0724] Clause 81. The pharmaceutical composition of clause 80, wherein the antigen is a viral polypeptide from an adenovirus; Herpes simplex, type 1; Herpes simplex, type 2; encephalitis virus, papillomavirus, Varicella-zoster virus; Epstein-barr virus; Human cytomegalovirus; Human herpes virus, type 8; Human papillomavirus; BK virus; JC virus; Smallpox; polio virus; Hepatitis B virus; Human bocavirus; Parvovirus B19; Human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; Severe acute respiratory syndrome virus; Hepatitis C virus; Yellow Fever virus; Dengue virus; West Nile virus; Rubella virus; Hepatitis E virus; Human Immunodeficiency virus (HIV); Influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; Measles virus; Mumps virus; Parainfluenza virus; Respiratory syncytial virus; Human metapneumo virus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D; Rotavirus; Orbivirus; Coltivirus; Banna virus; Human Enterovirus; Hanta virus; West Nile virus; Middle East Respiratory Syndrome Corona Virus; Japanese encephalitis virus; Vesicular exanthernavirus; SARS-CoV-2; Eastern equine encephalitis, or a combination of any two or more of the foregoing. [0725] Clause 82. The pharmaceutical composition of any one of clauses 62-81, wherein the core functional element comprises a stop codon or a stop cassette. [0726] Clause 83. The pharmaceutical composition of any one of clause 62-81, wherein the core functional element comprises a noncoding region. [0727] Clause 84. The pharmaceutical composition of any one of clause 62-81, wherein the core functional element comprises an accessory or modulatory element. [0728] Clause 85. The pharmaceutical composition of clause 84, wherein the accessory or modulatory element comprises a miRNA binding site or a fragment thereof, a restriction site or a fragment thereof, a RNA editing motif or a fragment thereof, a zip code element or a fragment thereof, a RNA trafficking element or fragment thereof, or a combination thereof. [0729] Clause 86. The pharmaceutical composition of clause 84, wherein the accessory or modulatory element comprises a binding domain to an IRES transacting factor (ITAF). [0730] Clause 87. The pharmaceutical composition of any one of clauses 62-86, wherein the 3’ enhanced exon element comprises a 5’ exon fragment. [0731] Clause 88. The pharmaceutical composition of clauses 87, wherein the 3’ enhanced exon element comprises a 3’ internal spacer located upstream to the 5’ exon fragment. [0732] Clause 89. The pharmaceutical composition of clause 88, wherein the 3’ internal spacer is a polyA or polyA-C sequence. [0733] Clause 90. The pharmaceutical composition of clause 88 or 89, wherein the 3’ internal spacer has a length of about 10 to about 60 nucleotides. [0734] Clause 91. The pharmaceutical composition of any one of clauses 87-90, wherein the 3’ enhanced exon element comprises a 3’ internal duplex element located upstream to the 5’ exon fragment. [0735] Clause 92. The pharmaceutical composition of any one of clauses 1-91, wherein the RNA polynucleotide is made via circularization of a RNA polynucleotide comprising, in the following order: a 5’ enhanced intron element, a 5’ enhanced exon element, a core functional element, a 3’ enhanced exon element, and a 3’ enhanced intron element. [0736] Clause 93. The pharmaceutical composition of clause 92, wherein the 5’ enhanced intron element comprises a 3’ intron fragment. [0737] Clause 94. The pharmaceutical composition of clause 93, wherein the 3’ intron fragment comprises a first or a first and second nucleotide of a 3’ group I intron splice site dinucleotide. [0738] Clause 95. The pharmaceutical composition of clause 92 or 93, wherein the 5’ enhanced intron element comprises a 5’ affinity tag located upstream to the 3’ intron fragment. [0739] Clause 96. The pharmaceutical composition of any one of clauses 93-95, wherein the 5’ enhanced intron element comprises a 5’ external spacer located upstream to the 3’ intron fragment. [0740] Clause 97. The pharmaceutical composition of any one of clauses 92-96, wherein the 5’ enhanced intron element comprises a leading untranslated sequence located at the 5’ end of said 5’ enhanced intron element. [0741] Clause 98. The pharmaceutical composition of any one of clauses 92-97, wherein the 3’ enhanced intron element comprises a 5’ intron fragment. [0742] Clause 99. The pharmaceutical composition of any one of clauses 92-98, wherein the 3’ enhanced intron element comprises a 3’ external spacer located downstream to the 5’ intron fragment. [0743] Clause 100. The pharmaceutical composition of any one of clauses 92-99, wherein the 3’ enhanced intron element comprises a 3’ affinity tag located downstream to the 5’ intron fragment. [0744] Clause 101. The pharmaceutical composition of any one of clauses 92-100, wherein the 3’ enhanced intron element comprises a 3’ terminal untranslated sequence at the 3’ end of the said 5’ enhanced intron element. [0745] Clause 102. The pharmaceutical composition of any one of clauses 92-101, wherein the 5’ enhanced intron element comprises a 5’ external duplex region upstream to the 3’ intron fragment, and the 3’ enhanced intron element comprises a 3’ external duplex region downstream to the 5’ intron fragment. [0746] Clause 103. The pharmaceutical composition of clause 102, wherein the 5’ external duplex region and the 3’ external duplex region are the same. [0747] Clause 104. The pharmaceutical composition of clause 102, wherein the 5’ external duplex region and the 3’ external duplex region are different. [0748] Clause 105. The pharmaceutical composition of any one of clauses 94-104, wherein the group I intron comprises in part or in whole from a bacterial phage, viral vector, organelle genome, or a nuclear rDNA gene. [0749] Clause 106. The pharmaceutical composition of clause 105, wherein the nuclear rDNA gene comprises a nuclear rDNA gene derived from a fungi, plant, or algae, or a fragment thereof. [0750] Clause 107. The pharmaceutical composition of any one of clauses 1-106, wherein the RNA polynucleotide contains at least about 80%, at least about 90%, at least about 95%, or at least about 99% naturally occurring nucleotides. [0751] Clause 108. The pharmaceutical composition of any one of clauses 1-107, wherein the RNA polynucleotide consists of naturally occurring nucleotides. [0752] Clause 109. The pharmaceutical composition of any one of clauses 62-108, wherein the expression sequence is codon optimized. [0753] Clause 110. The pharmaceutical composition of any one of clauses 1-109, wherein the RNA polynucleotide is optimized to lack at least one microRNA binding site present in an equivalent pre- optimized polynucleotide. [0754] Clause 111. The pharmaceutical composition of any one of clauses 1-110, wherein the RNA polynucleotide is optimized to lack at least one microRNA binding site capable of binding to a microRNA present in a cell within which the RNA polynucleotide is expressed. [0755] Clause 112. The pharmaceutical composition of any one of clauses 1-111, wherein the RNA polynucleotide is optimized to lack at least one endonuclease susceptible site present in an equivalent pre-optimized polynucleotide. [0756] Clause 113. The pharmaceutical composition of any one of clauses 1-112, wherein the RNA polynucleotide is optimized to lack at least one endonuclease susceptible site capable of being cleaved by an endonuclease present in a cell within which the endonuclease is expressed. [0757] Clause 114. The pharmaceutical composition of any one of clauses 1-113, wherein the RNA polynucleotide is optimized to lack at least one RNA editing susceptible site present in an equivalent pre-optimized polynucleotide. [0758] Clause 115. The pharmaceutical composition of any one of clauses 1-114, wherein the RNA polynucleotide is from about 100nt to about 10,000nt in length. [0759] Clause 116. The pharmaceutical composition of any one of clauses 1-115, wherein the RNA polynucleotide is from about 100nt to about 15,000nt in length. [0760] Clause 117. The pharmaceutical composition of any one of clauses 1-116, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the circular RNA polynucleotide is more compact than a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. [0761] Clause 118. The pharmaceutical composition of any one of clauses 1-117, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the composition has a duration of therapeutic effect in a human cell greater than or equal to that of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. [0762] Clause 119. The pharmaceutical composition of clause 118, wherein the reference linear RNA polynucleotide is a linear, unmodified or nucleoside-modified, fully-processed mRNA comprising a cap1 structure and a polyA tail at least 80nt in length. [0763] Clause 120. The pharmaceutical composition of any one of clauses 1-119, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the composition has a duration of therapeutic effect in vivo in humans greater than that of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. [0764] Clause 121. The pharmaceutical composition of any one of clauses 1-120, wherein the composition has a duration of therapeutic effect in vivo in humans of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 hours. [0765] Clause 122. The pharmaceutical composition of any one of clauses 1-121, wherein the composition has a functional half-life in a human cell greater than or equal to that of a pre-determined threshold value. [0766] Clause 123. The pharmaceutical composition of any one of clauses 1-122, wherein the composition has a functional half-life in vivo in humans greater than that of a pre-determined threshold value. [0767] Clause 124. The pharmaceutical composition of clause 122 or 123, wherein the functional half- life is determined by a functional protein assay. [0768] Clause 125. The pharmaceutical composition of clause 124, wherein the functional protein assay is an in vitro luciferase assay. [0769] Clause 126. The pharmaceutical composition of clause 124, wherein the functional protein assay comprises measuring levels of protein encoded by the expression sequence of the RNA polynucleotide in a patient serum or tissue sample. [0770] Clause 127. The pharmaceutical composition of any one of clauses 122-126, wherein the pre- determined threshold value is the functional half-life of a reference linear RNA polynucleotide comprising the same expression sequence as the RNA polynucleotide. [0771] Clause 128. The pharmaceutical composition of any one of clauses 1-127, wherein the composition has a functional half-life of at least about 20 hours. [0772] Clause 129. The pharmaceutic composition of any one of clauses 1-128, further comprising a structural lipid and a PEG-modified lipid. [0773] Clause 130. The pharmaceutical composition of any one of clause 129, wherein the structural lipid binds to C1q and/or promotes the binding of the transfer vehicle comprising said lipid to C1q compared to a control transfer vehicle lacking the structural lipid and/or increases uptake of C1q-bound transfer vehicle into an immune cell compared to a control transfer vehicle lacking the structural lipid. [0774] Clause 131. The pharmaceutical composition of any one of clause 125-130, wherein the immune cell is a T cell, an NK cell, an NKT cell, a macrophage, or a neutrophil. [0775] Clause 132. The pharmaceutical composition of any one of clauses 129-131, wherein the structural lipid is cholesterol. [0776] Clause 133. The pharmaceutical composition of clause 130, wherein the structural lipid is beta- sitosterol. [0777] Clause 134. The pharmaceutical composition of clause 130, wherein the structural lipid is not beta-sitosterol. [0778] Clause 135. The pharmaceutical composition of any one of clauses 129-134, wherein the PEG- modified lipid is DSPE-PEG, DMG-PEG, PEG-DAG, PEG-S-DAG, PEG-PE, PEG-S-DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1. [0779] Clause 136. The pharmaceutical composition of clause 135, wherein the PEG-modified lipid is DSPE-PEG(2000). [0780] Clause 137. The pharmaceutical composition of any one of clauses 1-136, further comprising a helper lipid. [0781] Clause 138. The pharmaceutical composition of clause 137, wherein the helper lipid is DSPC or DOPE. [0782] Clause 139. The pharmaceutical composition of any one of clauses 1-138, further comprising DSPC, cholesterol, and DMG-PEG(2000). [0783] Clause 140. The pharmaceutical composition of any one of clauses 130-139, wherein the transfer vehicle comprises about 0.5% to about 4% PEG-modified lipids by molar ratio. [0784] Clause 141. The pharmaceutical composition of any one of clauses 130-140, wherein the transfer vehicle comprises about 1% to about 2% PEG-modified lipids by molar ratio. [0785] Clause 142. The pharmaceutical composition of any one of clauses 1-141, wherein the transfer vehicle comprises: an ionizable lipid selected from: , , and ,or a mixture thereof, a helper lipid selected from DOPE or DSPC, cholesterol, and a PEG-lipid selected from DSPE- PEG(2000) or DMG-PEG(2000). [0786] Clause 143. The pharmaceutical composition of clause 142, wherein the molar ratio of ionizable lipid:helper lipid:cholesterol:PEG-lipid is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0787] Clause 144. The pharmaceutical composition of clause 142, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0788] Clause 145. The pharmaceutical composition of clause 145, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0789] Clause 146. The pharmaceutical composition of clause 145, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is 62:4:33:1. [0790] Clause 147. The pharmaceutical composition of clause 142, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is 53:5:41:1. [0791] Clause 148. The pharmaceutical composition of clause 142, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0792] Clause 149. The pharmaceutical composition of clause 148, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 50:10:38.5:1.5. [0793] Clause 150. The pharmaceutical composition of clause 148, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 41:12:45:2. [0794] Clause 151. The pharmaceutical composition of clause 148, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 45:9:44:2. [0795] Clause 152. The pharmaceutical composition of clause 142, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid: DSPC:cholesterol:DSPE-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0796] Clause 153. The pharmaceutical composition of clause 142, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid is C14-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:C14-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0797] Clause 154. The pharmaceutical composition of clause 142, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DMG-PEG(2000), wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. [0798] Clause 155. The pharmaceutical composition of any one of clauses 1-154, having a lipid to phosphate (IL:P) molar ratio of about 3 to about 9. [0799] Clause 156. The pharmaceutical composition of any one of clauses 1-155, having a lipid to phosphate (IL:P) molar ratio of about 3, about 4, about 4.5, about 5, about 5.5, about 5.7, about 6, about 6.2, about 6.5, or about 7. [0800] Clause 157. The pharmaceutical composition of any one of clauses 1-156, wherein the transfer vehicle is formulated for endosomal release of the RNA polynucleotide. [0801] Clause 158. The pharmaceutical composition of any one of clauses 1-157, wherein the transfer vehicle is capable of binding to APOE. [0802] Clause 159. The pharmaceutical composition of any one of clauses 1-158, wherein the transfer vehicle interacts with apolipoprotein E (APOE) less than an equivalent transfer vehicle loaded with a reference linear RNA having the same expression sequence as the RNA polynucleotide. [0803] Clause 160. The pharmaceutical composition of any one of clauses 1-159, wherein the exterior surface of the transfer vehicle is substantially free of APOE binding sites. [0804] Clause 161. The pharmaceutical composition of any one of clauses 1-160, wherein the transfer vehicle has a diameter of less than about 120 nm. [0805] Clause 162. The pharmaceutical composition of any one of clauses 1-161, wherein the transfer vehicle does not form aggregates with a diameter of more than 300 nm. [0806] Clause 163. The pharmaceutical composition of any one of clauses 1-162, wherein the transfer vehicle has an in vivo half-life of less than about 30 hours. [0807] Clause 164. The pharmaceutical composition of any one of clauses 1-163, wherein the transfer vehicle is capable of low density lipoprotein receptor (LDLR) dependent uptake into a cell. [0808] Clause 165. The pharmaceutical composition of any one of clauses 1-164, wherein the transfer vehicle is capable of LDLR independent uptake into a cell. [0809] Clause 166. The pharmaceutical composition of any one of clauses 1-165, wherein the pharmaceutical composition is substantially free of linear RNA. [0810] Clause 167. The pharmaceutical composition of any one of clauses 1-166, further comprising a targeting moiety operably connected to the transfer vehicle. [0811] Clause 168. The pharmaceutical composition of clause 167, wherein the targeting moiety specifically binds an immune cell antigen or indirectly. [0812] Clause 169. The pharmaceutical composition of clause 168, wherein the immune cell antigen is a T cell antigen. [0813] Clause 170. The pharmaceutical composition of clause 169, wherein the T cell antigen is selected from CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2 integrin, and C1qR. [0814] Clause 171. The pharmaceutical composition of clause 170, further comprising an adapter molecule comprising a transfer vehicle binding moiety and a cell binding moiety, wherein the targeting moiety specifically binds the transfer vehicle binding moiety and the cell binding moiety specifically binds a target cell antigen. [0815] Clause 172. The pharmaceutical composition of clause 171, wherein the target cell antigen is an immune cell antigen. [0816] Clause 173. The pharmaceutical composition of clause 172, wherein the immune cell antigen is a T cell antigen, an NK cell, an NKT cell, a macrophage, or a neutrophil. [0817] Clause 174. The pharmaceutical composition of clause 173, wherein the T cell antigen is selected from CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2 integrin, CD25, CD39, CD73, A2a Receptor, A2b Receptor, and C1qR. [0818] Clause 175. The pharmaceutical composition of clause 168 or 171, wherein the immune cell antigen is a macrophage antigen. [0819] Clause 176. The pharmaceutical composition of clause 175, wherein the macrophage antigen is selected from mannose receptor, CD206, and C1q. [0820] Clause 177. The pharmaceutical composition of any one of clauses 167-176, wherein the targeting moiety is a small molecule. [0821] Clause 178. The pharmaceutical composition of clause 177, wherein the small molecule is mannose, a lectin, acivicin, biotin, or digoxigenin. [0822] Clause 179. The pharmaceutical composition of clause 177, wherein the small molecule binds to an ectoenzyme on an immune cell, wherein the ectoenzyme is selected from CD38, CD73, adenosine 2a receptor, and adenosine 2b receptor. [0823] Clause 180. The pharmaceutical composition of any one of clauses 167-176, wherein the targeting moiety is a single chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, small molecule ligand such as folate, arginylglycylaspartic acid (RGD), or phenol-soluble modulin alpha 1 peptide (PSMA1), heavy chain variable region, light chain variable region or fragment thereof. [0824] Clause 181. The pharmaceutical composition of any one of clauses 1-180, wherein the ionizable lipid has a half-life in a cell membrane less than about 2 weeks. [0825] Clause 182. The pharmaceutical composition of any one of clauses 1-181, wherein the ionizable lipid has a half-life in a cell membrane less than about 1 week. [0826] Clause 183. The pharmaceutical composition of any one of clauses 1-182, wherein the ionizable lipid has a half-life in a cell membrane less than about 30 hours. [0827] Clause 184. The pharmaceutical composition of any one of clauses 1-183, wherein the ionizable lipid has a half-life in a cell membrane less than the functional half-life of the RNA polynucleotide. [0828] Clause 185. A method of treating or preventing a disease, disorder, or condition, comprising administering an effective amount of a pharmaceutical composition of any one of clauses 1-184. [0829] Clause 186. The method of clause 185, wherein the disease, disorder, or condition is associated with aberrant expression, activity, or localization of a polypeptide selected from ASCII Tables L and M. [0830] Clause 187. The method of clause 185 or 186, wherein the RNA polynucleotide encodes a therapeutic protein. [0831] Clause 188. The method of clause 187, wherein therapeutic protein expression in the spleen is higher than therapeutic protein expression in the liver. [0832] Clause 189. The method of clause 188, wherein therapeutic protein expression in the spleen is at least about 2.9x therapeutic protein expression in the liver. [0833] Clause 190. The method of clause 188, wherein the therapeutic protein is not expressed at functional levels in the liver. [0834] Clause 191. The method of clause 188, wherein the therapeutic protein is not expressed at detectable levels in the liver. [0835] Clause 192. The method of clause 188, wherein therapeutic protein expression in the spleen is at least about 50% of total therapeutic protein expression. [0836] Clause 193. The method of clause 188, wherein therapeutic protein expression in the spleen is at least about 63% of total therapeutic protein expression. [0837] Clause 194. A pharmaceutical composition of any one of clauses 1-184, wherein the transfer vehicle comprises a nanoparticle, and optionally, a targeting moiety operably connected to the nanoparticle. [0838] Clause 195. The pharmaceutical composition of clause 194, wherein the nanoparticle is a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymer nanoparticle, or a biodegradable polymer nanoparticle. [0839] Clause 196. The pharmaceutical composition of clause 194 or 195, comprising a targeting moiety, wherein the targeting moiety mediates receptor-mediated endocytosis or direct fusion selectively into cells of a selected cell population or tissue in the absence of cell isolation or purification. [0840] Clause 197. The pharmaceutical composition of any one of clauses 194-196, wherein the targeting moiety is a scfv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof. [0841] Clause 198. The pharmaceutical composition of any one of clauses 194-197, wherein less than 1%, by weight, of the polynucleotides in the composition are double stranded RNA, DNA splints, or triphosphorylated RNA. [0842] Clause 199. The pharmaceutical composition of any one of clauses 194-198, wherein less than 1%, by weight, of the polynucleotides and proteins in the pharmaceutical composition are double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes. [0843] Clause 200. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of clauses 194-199. [0844] Clause 201. The method of clause 200, wherein the targeting moiety is a scfv, nanobody, peptide, minibody, heavy chain variable region, light chain variable region, an extracellular domain of a TCR, or a fragment thereof. [0845] Clause 202. The method of clause 200 or 201, wherein the nanoparticle comprises one or more cationic lipids, ionizable lipids, or poly β-amino esters. [0846] Clause 203. The method of any one of clauses 200-202, wherein the nanoparticle comprises one or more non-cationic lipids. [0847] Clause 204. The method of any one of clauses 200-203, wherein the nanoparticle comprises one or more PEG-modified lipids, polyglutamic acid lipids, or Hyaluronic acid lipids. [0848] Clause 205. The method of any one of clauses 200-204, wherein the nanoparticle comprises cholesterol. [0849] Clause 206. The method of any one of clauses 200-205, wherein the nanoparticle comprises arachidonic acid or oleic acid. [0850] Clause 207. The method of any one of clauses 200-206, wherein the pharmaceutical composition comprises a targeting moiety, wherein the targeting moiety mediates receptor-mediated endocytosis selectively into cells of a selected cell population in the absence of cell selection or purification. [0851] Clause 208. The method of any one of clauses 200-207, wherein the nanoparticle comprises more than one circular RNA polynucleotide. [0852] Clause 209. A DNA vector encoding the RNA polynucleotide of any one of clauses 92-106. [0853] Clause 210. The DNA vector of clause 209, further comprising a transcription regulatory sequence. [0854] Clause 211. The DNA vector of clause 210, wherein the transcription regulatory sequence comprises a promoter and/or an enhancer. [0855] Clause 212. The DNA vector of clause 211, wherein the promoter comprises a T7 promoter. [0856] Clause 213. The DNA vector of any one of clauses 209-212, wherein the DNA vector comprises a circular DNA. [0857] Clause 214. The DNA vector of any one of clauses 209-213, wherein the DNA vector comprises a linear DNA. [0858] Clause 215. A prokaryotic cell comprising the DNA vector according to any one of clauses 209-214. [0859] Clause 216. A eukaryotic cell comprising the RNA polynucleotide according to any one of clauses 1-215. [0860] Clause 217. The eukaryotic cell of clause 217, wherein the eukaryotic cell is a human cell. [0861] Clause 218. A method of producing a circular RNA polynucleotide, the method comprising incubating the RNA polynucleotide of any one of clauses 92-106 under suitable conditions for circularization. [0862] Clause 219. A method of producing a circular RNA polynucleotide, the method comprising incubating DNA of any one of clauses 209-214 under suitable conditions for transcription. [0863] Clause 220. The method of clause 219, wherein the DNA is transcribed in vitro. [0864] Clause 221. The method of clause 219, wherein the suitable conditions comprises adenosine triphosphate (ATP), guanine triphosphate (GTP), cytosine triphosphate (CTP), uridine triphosphate (UTP), and an RNA polymerase. [0865] Clause 222. The method of clause 219, wherein the suitable conditions further comprises guanine monophosphate (GMP). [0866] Clause 223. The method of clause 222, wherein the ratio of GMP concentration to GTP concentration is within the range of about 3:1 to about 15:1, optionally about 4:1, 5:1, or 6:1. [0867] Clause 224. A method of producing a circular RNA polynucleotide, the method comprising culturing the prokaryotic cell of clause 215 under suitable conditions for transcribing the DNA in the cell. [0868] Clause 225. The method of any one of clauses 218-224, further comprising purifying a circular RNA polynucleotide. [0869] Clause 226. The method of clause 225, wherein the circular RNA polynucleotide is purified by negative selection using an affinity oligonucleotide that hybridizes with the first or second spacer conjugated to a solid surface. [0870] Clause 227. The method of clause 226, wherein the first or second spacer comprises a polyA sequence, and wherein the affinity oligonucleotide is a deoxythymine oligonucleotide. [0871] The disclosure is further described by the following non-limiting exemplary embodiments: Enbodiment 1. A pharmaceutical composition comprising: a. an RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid represented by Formula (13): Formula (13) wherein: n is an integer from 1 to 4; R a is hydrogen or hydroxyl; R1 and R2 are each independently a linear or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; with the proviso that the ionizable lipid is not or . Enbodiment 2. The pharmaceutical composition of embodiment 1, wherein Ra is hydrogen. Enbodiment 3. The pharmaceutical composition of embodiment 2, wherein the ionizable lipid is represented by Formula (13a-1), Formula (13a-2), or Formula (13a-3): Formula (13a-1) Formula (13a-2) Formula (13a-3). Enbodiment 4. The pharmaceutical composition of embodiment 1, wherein R a is hydroxyl. Enbodiment 5. The pharmaceutical composition of embodiment 4, wherein the ionizable lipid is represented by Formula (13b-1), Formula (13b-2), or Formula (13b-3): Formula (13b-1) Formula (13b-2) Formula (13b-3). Enbodiment 6. The pharmaceutical composition of embodiment 4, wherein the ionizable lipid is represented by Formula (13b-4), Formula (13b-5), Formula (13b-6), Formula (13b-7), Formula (13b- 8), or Formula (13b-9): Formula (13b-4) Formula (13b-5) Formula (13b-6) Formula (13b-7) Formula (13b-8) Formula (13b-9). Enbodiment 7. The pharmaceutical composition of any one of embodiments 1-6, wherein R1 and R 2 are each independently selected from: , , , , , , , , , , , , , , , , , , O , O , , , , , , , , , , , , , , , and . Enbodiment 8. The pharmaceutical composition of any one of embodiments 1-7, wherein R1 and R2 are the same. Enbodiment 9. The pharmaceutical composition of any one of embodiments 1-7, wherein R 1 and R 2 are different. Enbodiment 10. The pharmaceutical composition of any one of embodiments 1-9, wherein the ionizable lipid is selected from: , , , , , , O O N OH HO O , O , and . Enbodiment 11. The pharmaceutical composition of any one of embodiments 1-9, wherein the ionizable lipid is selected from: and . Enbodiment 12. The pharmaceutical composition of any one of embodiments 1-9, wherein the ionizable lipid is selected from Table 10e. Enbodiment 13. The pharmaceutical composition of any one of embodiments 1-12, wherein the RNA polynucleotide is a linear or circular RNA polynucleotide. Enbodiment 14. The pharmaceutical composition of any one of embodiments 1-13, wherein the RNA polynucleotide is a circular RNA polynucleotide. Enbodiment 15. A pharmaceutical composition comprising: a. an RNA polynucleotide, wherein the RNA polynucleotide is a circular RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid selected from or . Enbodiment 16. The pharmaceutical composition of any one of embodiments 1-15, wherein the RNA polynucleotide is encapsulated in the transfer vehicle. Enbodiment 17. The pharmaceutical composition of any one of embodiments 1-16, wherein the RNA polynucleotide is encapsulated in the transfer vehicle with an encapsulation efficiency of at least 80%. Enbodiment 18. The pharmaceutical composition of any one of embodiments 1-14, wherein the RNA comprises a first expression sequence. Enbodiment 19. The pharmaceutical composition of embodiment 18, wherein the first expression sequence encodes a therapeutic protein. Enbodiment 20. The pharmaceutical composition of embodiment 19, wherein the first expression sequence encodes a cytokine or a functional fragment thereof. Enbodiment 21. The pharmaceutical composition of embodiment 19, wherein the first expression sequence encodes a transcription factor. Enbodiment 22. The pharmaceutical composition of embodiment 19, wherein the first expression sequence encodes an immune checkpoint inhibitor. Enbodiment 23. The pharmaceutical composition of embodiment 19, wherein the first expression sequence encodes a chimeric antigen receptor (CAR). Enbodiment 24. The pharmaceutical composition of any one of embodiments 1-23, wherein the RNA polynucleotide further comprises a second expression sequence. Enbodiment 25. The pharmaceutical composition of embodiment 24, wherein the RNA polynucleotide further comprises an internal ribosome entry site (IRES). Enbodiment 26. The pharmaceutical composition of embodiment 25, wherein the first and second expression sequences are separated by a ribosomal skipping element or a nucleotide sequence encoding a protease cleavage site. Enbodiment 27. The pharmaceutical composition of any one of embodiments 24 or 26, wherein the first expression sequence encodes a first T-cell receptor (TCR) chain, and the second expression sequence encodes a second TCR chain. Enbodiment 28. The pharmaceutical composition of any one of embodiments 1-27, wherein the RNA polynucleotide comprises one or more microRNA binding sites. Enbodiment 29. The pharmaceutical composition of embodiment 28, wherein the microRNA binding site is recognized by a microRNA expressed in the liver. Enbodiment 30. The pharmaceutical composition of embodiment 28 or 29, wherein the microRNA binding site is recognized by miR-122. Enbodiment 31. The pharmaceutical composition of any one of embodiments 1-30, wherein the RNA polynucleotide comprises a first IRES associated with greater protein expression in a human immune cell than in a reference human cell. Enbodiment 32. The pharmaceutical composition of embodiment 31, wherein the human immune cell is a T cell, an NK cell, an NKT cell, a macrophage, or a neutrophil. Enbodiment 33. The pharmaceutical composition of embodiment 31 or 32, wherein the reference human cell is a hepatic cell. Enbodiment 34. The pharmaceutical composition of any one of embodiments 1-33, wherein the RNA polynucleotide comprises, in the following order: a. a 5’ enhanced exon element, b. a core functional element, and c. a 3’ enhanced exon element. Enbodiment 35. The pharmaceutical composition of any one of embodiments 1-34, further comprising a post-splicing intron fragment. Enbodiment 36. The pharmaceutical composition of embodiment 34 or 35, wherein the 5’ enhanced exon element comprises a 3’ exon fragment. Enbodiment 37. The pharmaceutical composition of any one of embodiments 34-36, wherein the 5’ enhanced exon element comprises a 5’ internal duplex region located downstream to the 3’ exon fragment. Enbodiment 38. The pharmaceutical composition of any one of embodiments 34-37, wherein the 5’ enhanced exon element comprises a 5’ internal spacer located downstream to the 3’ exon fragment. Enbodiment 39. The pharmaceutical composition of embodiment 38, wherein the 5’ internal spacer has a length of about 10 to about 60 nucleotides. Enbodiment 40. The pharmaceutical composition of embodiment 38 or 39, wherein the 5’ internal spacer comprises a polyA or polyA-C sequence. Enbodiment 41. The pharmaceutical composition of embodiment 40, wherein the polyA or polyA- C sequence comprises a length of about 10-50 nucleotides. Enbodiment 42. The pharmaceutical composition of any one of embodiments 34-41, wherein the core functional element comprises a translation initiation element (TIE). Enbodiment 43. The pharmaceutical composition of any one of embodiments 42, wherein the translation initiation element (TIE) comprises an untranslated region (UTR) or fragment thereof. Enbodiment 44. The pharmaceutical composition of embodiment 43, wherein the UTR or fragment thereof comprises a viral internal ribosome entry site (IRES) or eukaryotic IRES. Enbodiment 45. The pharmaceutical composition of embodiment 44, wherein the IRES is selected from Table 17, or is a functional fragment or variant thereof. Enbodiment 46. The pharmaceutical composition of embodiment 44 or 45, wherein the IRES has a sequence in whole or in part from a Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Apodemus Agrarius Picornavirus, Caprine Kobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or an aptamer to eIF4G. Enbodiment 47. The pharmaceutical composition of any one of embodiments 42-46, wherein the translation initiation element (TIE) comprises an aptamer complex. Enbodiment 48. The pharmaceutical composition of embodiment 42, wherein the aptamer complex comprises at least two aptamers. Enbodiment 49. The pharmaceutical composition of any one of embodiments 34-48, wherein the core functional element comprises a coding region. Enbodiment 50. The pharmaceutical composition of embodiment 49, wherein the coding region encodes for a therapeutic protein. Enbodiment 51. The pharmaceutical composition of embodiment 50, wherein the therapeutic protein is a chimeric antigen receptor (CAR), a cytokine, a transcription factor, a T cell receptor (TCR), B-cell receptor (BCR), ligand, immune cell activation or inhibitory receptor, recombinant fusion protein, chimeric mutant protein, or fusion protein or a functional fragment thereof. Enbodiment 52. The pharmaceutical composition of embodiment 51, wherein the therapeutic protein is an antigen. Enbodiment 53. The pharmaceutical composition of embodiment 52, wherein the antigen is a viral polypeptide from an adenovirus; Herpes simplex, type 1; Herpes simplex, type 2; encephalitis virus, papillomavirus, Varicella-zoster virus; Epstein-barr virus; Human cytomegalovirus; Human herpes virus, type 8; Human papillomavirus; BK virus; JC virus; Smallpox; polio virus; Hepatitis B virus; Human bocavirus; Parvovirus B19; Human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; Severe acute respiratory syndrome virus; Hepatitis C virus; Yellow Fever virus; Dengue virus; West Nile virus; Rubella virus; Hepatitis E virus; Human Immunodeficiency virus (HIV); Influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; Measles virus; Mumps virus; Parainfluenza virus; Respiratory syncytial virus; Human metapneumo virus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D; Rotavirus; Orbivirus; Coltivirus; Banna virus; Human Enterovirus; Hanta virus; West Nile virus; Middle East Respiratory Syndrome Corona Virus; Japanese encephalitis virus; Vesicular exanthernavirus; SARS-CoV-2; Eastern equine encephalitis, or a combination of any two or more of the foregoing. Enbodiment 54. The pharmaceutical composition of any one of embodiments 34-53, wherein the core functional element comprises a stop codon or a stop cassette. Enbodiment 55. The pharmaceutical composition of any one of embodiment 34-53, wherein the core functional element comprises a noncoding region. Enbodiment 56. The pharmaceutical composition of any one of embodiment 34-53, wherein the core functional element comprises an accessory or modulatory element. Enbodiment 57. The pharmaceutical composition of embodiment 56, wherein the accessory or modulatory element comprises a miRNA binding site or a fragment thereof, a restriction site or a fragment thereof, a RNA editing motif or a fragment thereof, a zip code element or a fragment thereof, a RNA trafficking element or fragment thereof, or a combination thereof. Enbodiment 58. The pharmaceutical composition of embodiment 56, wherein the accessory or modulatory element comprises a binding domain to an IRES transacting factor (ITAF). Enbodiment 59. The pharmaceutical composition of any one of embodiments 34-58, wherein the 3’ enhanced exon element comprises a 5’ exon fragment. Enbodiment 60. The pharmaceutical composition of embodiments 59, wherein the 3’ enhanced exon element comprises a 3’ internal spacer located upstream to the 5’ exon fragment. Enbodiment 61. The pharmaceutical composition of embodiment 60, wherein the 3’ internal spacer is a polyA or polyA-C sequence. Enbodiment 62. The pharmaceutical composition of embodiment 60 or 61, wherein the 3’ internal spacer has a length of about 10 to about 60 nucleotides. Enbodiment 63. The pharmaceutical composition of any one of embodiments 59-62, wherein the 3’ enhanced exon element comprises a 3’ internal duplex element located upstream to the 5’ exon fragment. Enbodiment 64. The pharmaceutical composition of any one of embodiments 1-63, wherein the RNA polynucleotide is made via circularization of a RNA polynucleotide comprising, in the following order: a. a 5’ enhanced intron element, b. a 5’ enhanced exon element, c. a core functional element, d. a 3’ enhanced exon element, and e. a 3’ enhanced intron element. Enbodiment 65. The pharmaceutical composition of embodiment 64, wherein the 5’ enhanced intron element comprises a 3’ intron fragment. Enbodiment 66. The pharmaceutical composition of embodiment 65, wherein the 3’ intron fragment comprises a first or a first and second nucleotide of a 3’ group I intron splice site dinucleotide. Enbodiment 67. The pharmaceutical composition of embodiment 64 or 65, wherein the 5’ enhanced intron element comprises a 5’ affinity tag located upstream to the 3’ intron fragment. Enbodiment 68. The pharmaceutical composition of any one of embodiments 65-67, wherein the 5’ enhanced intron element comprises a 5’ external spacer located upstream to the 3’ intron fragment. Enbodiment 69. The pharmaceutical composition of any one of embodiments 64-68, wherein the 5’ enhanced intron element comprises a leading untranslated sequence located at the 5’ end of said 5’ enhanced intron element. Enbodiment 70. The pharmaceutical composition of any one of embodiments 64-69, wherein the 3’ enhanced intron element comprises a 5’ intron fragment. Enbodiment 71. The pharmaceutical composition of any one of embodiments 64-70, wherein the 3’ enhanced intron element comprises a 3’ external spacer located downstream to the 5’ intron fragment. Enbodiment 72. The pharmaceutical composition of any one of embodiments 64-71, wherein the 3’ enhanced intron element comprises a 3’ affinity tag located downstream to the 5’ intron fragment. Enbodiment 73. The pharmaceutical composition of any one of embodiments 64-72, wherein the 3’ enhanced intron element comprises a 3’ terminal untranslated sequence at the 3’ end of the said 5’ enhanced intron element. Enbodiment 74. The pharmaceutical composition of any one of embodiments 64-73, wherein the 5’ enhanced intron element comprises a 5’ external duplex region upstream to the 3’ intron fragment, and the 3’ enhanced intron element comprises a 3’ external duplex region downstream to the 5’ intron fragment. Enbodiment 75. The pharmaceutical composition of embodiment 74, wherein the 5’ external duplex region and the 3’ external duplex region are the same. Enbodiment 76. The pharmaceutical composition of embodiment 74, wherein the 5’ external duplex region and the 3’ external duplex region are different. Enbodiment 77. The pharmaceutical composition of any one of embodiments 66-76, wherein the group I intron comprises in part or in whole from a bacterial phage, viral vector, organelle genome, or a nuclear rDNA gene. Enbodiment 78. The pharmaceutical composition of embodiment 77, wherein the nuclear rDNA gene comprises a nuclear rDNA gene derived from a fungi, plant, or algae, or a fragment thereof. Enbodiment 79. The pharmaceutical composition of any one of embodiments 1-78, wherein the RNA polynucleotide contains at least about 80%, at least about 90%, at least about 95%, or at least about 99% naturally occurring nucleotides. Enbodiment 80. The pharmaceutical composition of any one of embodiments 1-79, wherein the RNA polynucleotide consists of naturally occurring nucleotides. Enbodiment 81. The pharmaceutical composition of any one of embodiments 34-80, wherein the expression sequence is codon optimized. Enbodiment 82. The pharmaceutical composition of any one of embodiments 1-81, wherein the RNA polynucleotide is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide. Enbodiment 83. The pharmaceutical composition of any one of embodiments 1-82, wherein the RNA polynucleotide is optimized to lack at least one microRNA binding site capable of binding to a microRNA present in a cell within which the RNA polynucleotide is expressed. Enbodiment 84. The pharmaceutical composition of any one of embodiments 1-83, wherein the RNA polynucleotide is optimized to lack at least one endonuclease susceptible site present in an equivalent pre-optimized polynucleotide. Enbodiment 85. The pharmaceutical composition of any one of embodiments 1-84, wherein the RNA polynucleotide is optimized to lack at least one endonuclease susceptible site capable of being cleaved by an endonuclease present in a cell within which the endonuclease is expressed. Enbodiment 86. The pharmaceutical composition of any one of embodiments 1-85, wherein the RNA polynucleotide is optimized to lack at least one RNA editing susceptible site present in an equivalent pre-optimized polynucleotide. Enbodiment 87. The pharmaceutical composition of any one of embodiments 1-86, wherein the RNA polynucleotide is from about 100nt to about 10,000nt in length. Enbodiment 88. The pharmaceutical composition of any one of embodiments 1-87, wherein the RNA polynucleotide is from about 100nt to about 15,000nt in length. Enbodiment 89. The pharmaceutical composition of any one of embodiments 1-88, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the circular RNA polynucleotide is more compact than a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. Enbodiment 90. The pharmaceutical composition of any one of embodiments 1-89, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the composition has a duration of therapeutic effect in a human cell greater than or equal to that of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. Enbodiment 91. The pharmaceutical composition of embodiment 90, wherein the reference linear RNA polynucleotide is a linear, unmodified or nucleoside-modified, fully-processed mRNA comprising a cap1 structure and a polyA tail at least 80nt in length. Enbodiment 92. The pharmaceutical composition of any one of embodiments 1-91, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the composition has a duration of therapeutic effect in vivo in humans greater than that of a composition comprising a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide. Enbodiment 93. The pharmaceutical composition of any one of embodiments 1-92, wherein the composition has a duration of therapeutic effect in vivo in humans of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 hours. Enbodiment 94. The pharmaceutical composition of any one of embodiments 1-93, wherein the composition has a functional half-life in a human cell greater than or equal to that of a pre-determined threshold value. Enbodiment 95. The pharmaceutical composition of any one of embodiments 1-94, wherein the composition has a functional half-life in vivo in humans greater than that of a pre-determined threshold value. Enbodiment 96. The pharmaceutical composition of embodiment 94 or 95, wherein the functional half-life is determined by a functional protein assay. Enbodiment 97. The pharmaceutical composition of embodiment 96, wherein the functional protein assay is an in vitro luciferase assay. Enbodiment 98. The pharmaceutical composition of embodiment 96, wherein the functional protein assay comprises measuring levels of protein encoded by the expression sequence of the RNA polynucleotide in a patient serum or tissue sample. Enbodiment 99. The pharmaceutical composition of any one of embodiments 94-98, wherein the pre-determined threshold value is the functional half-life of a reference linear RNA polynucleotide comprising the same expression sequence as the RNA polynucleotide. Enbodiment 100. The pharmaceutical composition of any one of embodiments 1-99, wherein the composition has a functional half-life of at least about 20 hours. Enbodiment 101. The pharmaceutic composition of any one of embodiments 1-100, further comprising a structural lipid and a PEG-modified lipid. Enbodiment 102. The pharmaceutical composition of any one of embodiment 101, wherein the structural lipid binds to C1q and/or promotes the binding of the transfer vehicle comprising said lipid to C1q compared to a control transfer vehicle lacking the structural lipid and/or increases uptake of C1q-bound transfer vehicle into an immune cell compared to a control transfer vehicle lacking the structural lipid. Enbodiment 103. The pharmaceutical composition of any one of embodiment 97-102, wherein the immune cell is a T cell, an NK cell, an NKT cell, a macrophage, or a neutrophil. Enbodiment 104. The pharmaceutical composition of any one of embodiments 101-103, wherein the structural lipid is cholesterol. Enbodiment 105. The pharmaceutical composition of embodiment 102, wherein the structural lipid is beta-sitosterol. Enbodiment 106. The pharmaceutical composition of embodiment 102, wherein the structural lipid is not beta-sitosterol. Enbodiment 107. The pharmaceutical composition of any one of embodiments 101-106, wherein the PEG-modified lipid is DSPE-PEG, DMG-PEG, PEG-DAG, PEG-S-DAG, PEG-PE, PEG-S- DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1. Enbodiment 108. The pharmaceutical composition of embodiment 107, wherein the PEG-modified lipid is DSPE-PEG(2000). Enbodiment 109. The pharmaceutical composition of any one of embodiments 1-108, further comprising a helper lipid. Enbodiment 110. The pharmaceutical composition of embodiment 109, wherein the helper lipid is DSPC or DOPE. Enbodiment 111. The pharmaceutical composition of any one of embodiments 1-110, further comprising DSPC, cholesterol, and DMG-PEG(2000). Enbodiment 112. The pharmaceutical composition of any one of embodiments 102-111, wherein the transfer vehicle comprises about 0.5% to about 4% PEG-modified lipids by molar ratio. Enbodiment 113. The pharmaceutical composition of any one of embodiments 102-112, wherein the transfer vehicle comprises about 1% to about 2% PEG-modified lipids by molar ratio. Enbodiment 114. The pharmaceutical composition of any one of embodiments 1-113, wherein the transfer vehicle comprises: a. an ionizable lipid selected from: OH N OH O O , , and , or a mixture thereof, b. a helper lipid selected from DOPE or DSPC, c. cholesterol, and d. a PEG-lipid selected from DSPE-PEG(2000) or DMG-PEG(2000). Enbodiment 115. The pharmaceutical composition of embodiment 114, wherein the molar ratio of ionizable lipid:helper lipid:cholesterol:PEG-lipid is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 116. The pharmaceutical composition of embodiment 114, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 117. The pharmaceutical composition of embodiment 117, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 118. The pharmaceutical composition of embodiment 117, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is 62:4:33:1. Enbodiment 119. The pharmaceutical composition of embodiment 114, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is 53:5:41:1. Enbodiment 120. The pharmaceutical composition of embodiment 114, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 121. The pharmaceutical composition of embodiment 120, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 50:10:38.5:1.5. Enbodiment 122. The pharmaceutical composition of embodiment 120, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 41:12:45:2. Enbodiment 123. The pharmaceutical composition of embodiment 120, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is 45:9:44:2. Enbodiment 124. The pharmaceutical composition of embodiment 114, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid: DSPC:cholesterol:DSPE-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 125. The pharmaceutical composition of embodiment 114, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid is C14-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:C14-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 126. The pharmaceutical composition of embodiment 114, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DMG-PEG(2000), wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is 45:9:44:2, 50:10:38.5:1.5, 41:12:45:2, 62:4:33:1, or 53:5:41:1. Enbodiment 127. The pharmaceutical composition of any one of embodiments 1-126, having a lipid to phosphate (IL:P) molar ratio of about 3 to about 9. Enbodiment 128. The pharmaceutical composition of any one of embodiments 1-127, having a lipid to phosphate (IL:P) molar ratio of about 3, about 4, about 4.5, about 5, about 5.5, about 5.7, about 6, about 6.2, about 6.5, or about 7. Enbodiment 129. The pharmaceutical composition of any one of embodiments 1-128, wherein the transfer vehicle is formulated for endosomal release of the RNA polynucleotide. Enbodiment 130. The pharmaceutical composition of any one of embodiments 1-129, wherein the transfer vehicle is capable of binding to APOE. Enbodiment 131. The pharmaceutical composition of any one of embodiments 1-130, wherein the transfer vehicle interacts with apolipoprotein E (APOE) less than an equivalent transfer vehicle loaded with a reference linear RNA having the same expression sequence as the RNA polynucleotide. Enbodiment 132. The pharmaceutical composition of any one of embodiments 1-131, wherein the exterior surface of the transfer vehicle is substantially free of APOE binding sites. Enbodiment 133. The pharmaceutical composition of any one of embodiments 1-132, wherein the transfer vehicle has a diameter of less than about 120 nm. Enbodiment 134. The pharmaceutical composition of any one of embodiments 1-133, wherein the transfer vehicle does not form aggregates with a diameter of more than 300 nm. Enbodiment 135. The pharmaceutical composition of any one of embodiments 1-134, wherein the transfer vehicle has an in vivo half-life of less than about 30 hours. Enbodiment 136. The pharmaceutical composition of any one of embodiments 1-135, wherein the transfer vehicle is capable of low density lipoprotein receptor (LDLR) dependent uptake into a cell. Enbodiment 137. The pharmaceutical composition of any one of embodiments 1-136, wherein the transfer vehicle is capable of LDLR independent uptake into a cell. Enbodiment 138. The pharmaceutical composition of any one of embodiments 1-137, wherein the pharmaceutical composition is substantially free of linear RNA. Enbodiment 139. The pharmaceutical composition of any one of embodiments 1-138, further comprising a targeting moiety operably connected to the transfer vehicle. Enbodiment 140. The pharmaceutical composition of embodiment 139, wherein the targeting moiety specifically binds an immune cell antigen or indirectly. Enbodiment 141. The pharmaceutical composition of embodiment 140, wherein the immune cell antigen is a T cell antigen. Enbodiment 142. The pharmaceutical composition of embodiment 141, wherein the T cell antigen is selected from CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2 integrin, and C1qR. Enbodiment 143. The pharmaceutical composition of embodiment 142, further comprising an adapter molecule comprising a transfer vehicle binding moiety and a cell binding moiety, wherein the targeting moiety specifically binds the transfer vehicle binding moiety and the cell binding moiety specifically binds a target cell antigen. Enbodiment 144. The pharmaceutical composition of embodiment 143, wherein the target cell antigen is an immune cell antigen. Enbodiment 145. The pharmaceutical composition of embodiment 144, wherein the immune cell antigen is a T cell antigen, an NK cell, an NKT cell, a macrophage, or a neutrophil. Enbodiment 146. The pharmaceutical composition of embodiment 145, wherein the T cell antigen is selected from CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2 integrin, CD25, CD39, CD73, A2a Receptor, A2b Receptor, and C1qR. Enbodiment 147. The pharmaceutical composition of embodiment 140 or 143, wherein the immune cell antigen is a macrophage antigen. Enbodiment 148. The pharmaceutical composition of embodiment 147, wherein the macrophage antigen is selected from mannose receptor, CD206, and C1q. Enbodiment 149. The pharmaceutical composition of any one of embodiments 139-148, wherein the targeting moiety is a small molecule. Enbodiment 150. The pharmaceutical composition of embodiment 149, wherein the small molecule is mannose, a lectin, acivicin, biotin, or digoxigenin. Enbodiment 151. The pharmaceutical composition of embodiment 149, wherein the small molecule binds to an ectoenzyme on an immune cell, wherein the ectoenzyme is selected from CD38, CD73, adenosine 2a receptor, and adenosine 2b receptor. Enbodiment 152. The pharmaceutical composition of any one of embodiments 139-148, wherein the targeting moiety is a single chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, small molecule ligand such as folate, arginylglycylaspartic acid (RGD), or phenol-soluble modulin alpha 1 peptide (PSMA1), heavy chain variable region, light chain variable region or fragment thereof. Enbodiment 153. The pharmaceutical composition of any one of embodiments 1-152, wherein the ionizable lipid has a half-life in a cell membrane less than about 2 weeks. Enbodiment 154. The pharmaceutical composition of any one of embodiments 1-153, wherein the ionizable lipid has a half-life in a cell membrane less than about 1 week. Enbodiment 155. The pharmaceutical composition of any one of embodiments 1-154, wherein the ionizable lipid has a half-life in a cell membrane less than about 30 hours. Enbodiment 156. The pharmaceutical composition of any one of embodiments 1-155, wherein the ionizable lipid has a half-life in a cell membrane less than the functional half-life of the RNA polynucleotide. Enbodiment 157. A method of treating or preventing a disease, disorder, or condition, comprising administering an effective amount of a pharmaceutical composition of any one of embodiments 1- 156. Enbodiment 158. The method of embodiment 157, wherein the disease, disorder, or condition is associated with aberrant expression, activity, or localization of a polypeptide selected from ASCII Tables L and M. Enbodiment 159. The method of embodiment 157 or 158, wherein the RNA polynucleotide encodes a therapeutic protein. Enbodiment 160. The method of embodiment 159, wherein therapeutic protein expression in the spleen is higher than therapeutic protein expression in the liver. Enbodiment 161. The method of embodiment 160, wherein therapeutic protein expression in the spleen is at least about 2.9x therapeutic protein expression in the liver. Enbodiment 162. The method of embodiment 160, wherein the therapeutic protein is not expressed at functional levels in the liver. Enbodiment 163. The method of embodiment 160, wherein the therapeutic protein is not expressed at detectable levels in the liver. Enbodiment 164. The method of embodiment 160, wherein therapeutic protein expression in the spleen is at least about 50% of total therapeutic protein expression. Enbodiment 165. The method of embodiment 160, wherein therapeutic protein expression in the spleen is at least about 63% of total therapeutic protein expression. Enbodiment 166. A pharmaceutical composition of any one of embodiments 1-156, wherein the transfer vehicle comprises a nanoparticle, and optionally, a targeting moiety operably connected to the nanoparticle. Enbodiment 167. The pharmaceutical composition of embodiment 166, wherein the nanoparticle is a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymer nanoparticle, or a biodegradable polymer nanoparticle. Enbodiment 168. The pharmaceutical composition of embodiment 166 or 167, comprising a targeting moiety, wherein the targeting moiety mediates receptor-mediated endocytosis or direct fusion selectively into cells of a selected cell population or tissue in the absence of cell isolation or purification. Enbodiment 169. The pharmaceutical composition of any one of embodiments 166-168, wherein the targeting moiety is a scfv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof. Enbodiment 170. The pharmaceutical composition of any one of embodiments 166-169, wherein less than 1%, by weight, of the polynucleotides in the composition are double stranded RNA, DNA splints, or triphosphorylated RNA. Enbodiment 171. The pharmaceutical composition of any one of embodiments 166-170, wherein less than 1%, by weight, of the polynucleotides and proteins in the pharmaceutical composition are double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes. Enbodiment 172. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 166-171. Enbodiment 173. The method of embodiment 172, wherein the targeting moiety is a scfv, nanobody, peptide, minibody, heavy chain variable region, light chain variable region, an extracellular domain of a TCR, or a fragment thereof. Enbodiment 174. The method of embodiment 172 or 173, wherein the nanoparticle comprises one or more cationic lipids, ionizable lipids, or poly β-amino esters. Enbodiment 175. The method of any one of embodiments 172-174, wherein the nanoparticle comprises one or more non-cationic lipids. Enbodiment 176. The method of any one of embodiments 172-175, wherein the nanoparticle comprises one or more PEG-modified lipids, polyglutamic acid lipids, or Hyaluronic acid lipids. Enbodiment 177. The method of any one of embodiments 172-176, wherein the nanoparticle comprises cholesterol. Enbodiment 178. The method of any one of embodiments 172-177, wherein the nanoparticle comprises arachidonic acid or oleic acid. Enbodiment 179. The method of any one of embodiments 172-178, wherein the pharmaceutical composition comprises a targeting moiety, wherein the targeting moiety mediates receptor-mediated endocytosis selectively into cells of a selected cell population in the absence of cell selection or purification. Enbodiment 180. The method of any one of embodiments 172-179, wherein the nanoparticle comprises more than one circular RNA polynucleotide. Enbodiment 181. A DNA vector encoding the RNA polynucleotide of any one of embodiments 64-78. Enbodiment 182. The DNA vector of embodiment 181, further comprising a transcription regulatory sequence. Enbodiment 183. The DNA vector of embodiment 182, wherein the transcription regulatory sequence comprises a promoter and/or an enhancer. Enbodiment 184. The DNA vector of embodiment 183, wherein the promoter comprises a T7 promoter. Enbodiment 185. The DNA vector of any one of embodiments 181-184, wherein the DNA vector comprises a circular DNA. Enbodiment 186. The DNA vector of any one of embodiments 181-185, wherein the DNA vector comprises a linear DNA. Enbodiment 187. A prokaryotic cell comprising the DNA vector according to any one of embodiments 181-186. Enbodiment 188. A eukaryotic cell comprising the RNA polynucleotide according to any one of embodiments 1-187. Enbodiment 189. The eukaryotic cell of embodiment 189, wherein the eukaryotic cell is a human cell. Enbodiment 190. A method of producing a circular RNA polynucleotide, the method comprising incubating the RNA polynucleotide of any one of embodiments 64-78 under suitable conditions for circularization. Enbodiment 191. A method of producing a circular RNA polynucleotide, the method comprising incubating DNA of any one of embodiments 181-186 under suitable conditions for transcription. Enbodiment 192. The method of embodiment 191, wherein the DNA is transcribed in vitro. Enbodiment 193. The method of embodiment 191, wherein the suitable conditions comprises adenosine triphosphate (ATP), guanine triphosphate (GTP), cytosine triphosphate (CTP), uridine triphosphate (UTP), and an RNA polymerase. Enbodiment 194. The method of embodiment 191, wherein the suitable conditions further comprises guanine monophosphate (GMP). Enbodiment 195. The method of embodiment 194, wherein the ratio of GMP concentration to GTP concentration is within the range of about 3:1 to about 15:1, optionally about 4:1, 5:1, or 6:1. Enbodiment 196. A method of producing a circular RNA polynucleotide, the method comprising culturing the prokaryotic cell of embodiment 187 under suitable conditions for transcribing the DNA in the cell. Enbodiment 197. The method of any one of embodiments 190-196, further comprising purifying a circular RNA polynucleotide. Enbodiment 198. The method of embodiment 197, wherein the circular RNA polynucleotide is purified by negative selection using an affinity oligonucleotide that hybridizes with the first or second spacer conjugated to a solid surface. Enbodiment 199. The method of embodiment 198, wherein the first or second spacer comprises a polyA sequence, and wherein the affinity oligonucleotide is a deoxythymine oligonucleotide. [0872] Additional aspects of this disclosure are set forth in the following clauses: [0873] Clause 1. An ionizable lipid represented by Formula (13*): Formula (13*) or a pharmaceutically acceptable salt thereof, wherein: n * is an integer from 1 to 7; R a is hydrogen or hydroxyl; R b is hydrogen or C 1 -C 6 alkyl; R1 and R2 are each independently a linear or branched C1-C30 alkyl, C2-C30 alkenyl, or C1-C30 heteroalkyl, optionally substituted by one or more substituents selected from oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, alkylcarbonyloxy, alkylcarbonate, alkenyloxycarbonyl, alkenylcarbonyloxy, alkenylcarbonate, alkynyloxycarbonyl, alkynylcarbonyloxy, alkynylcarbonate, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; with the proviso that the ionizable lipid is not or . [0874] Clause 2. The ionizable lipid of clause 1, wherein R b is C 1 -C 6 alkyl. [0875] Clause 3. The ionizable lipid of clause 1, wherein R b is H and the ionizable lipid is represented by Formula (13): Formula (13) wherein n is an integer from 1 to 7. [0876] Clause 4. The ionizable lipid of clause 3, wherein n is 1, 2, 3, or 4. [0877] Clause 5. The ionizable lipid of any one of clauses 1-4, wherein R a is hydrogen. [0878] Clause 6. The ionizable lipid of clause 2, wherein the ionizable lipid is of Formula (13a-1), Formula (13a-2), or Formula (13a-3): Formula (13a-1) Formula (13a-2) Formula (13a-3). [0879] Clause 7. The ionizable lipid of any one of clauses 1-4, wherein R a is hydroxyl. [0880] Clause 8. The ionizable lipid of clause 7, wherein the ionizable lipid is represented by Formula (13b-1), Formula (13b-2), or Formula (13b-3): Formula (13b-1) Formula (13b-2) Formula (13b-3). [0881] Clause 9. The ionizable lipid of clause 7, wherein the ionizable lipid is represented by Formula (13b-4), Formula (13b-5), Formula (13b-6), Formula (13b-7), Formula (13b-8), or Formula (13b-9): Formula (13b-4) Formula (13b-5) Formula (13b-6) Formula (13b-7) Formula (13b-8) Formula (13b-9). [0882] Clause 10. The ionizable lipid of any one of clauses 1-9, wherein R 1 and R 2 are each independently a linear or branched C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 1 -C 20 heteroalkyl, optionally substituted by one or more substituents selected from C 1 -C 20 alkoxy, C 1 -C 20 alkyloxycarbonyl, C 1 -C 20 alkylcarbonyloxy, C1-C20 alkylcarbonate, C2-C20 alkenyloxycarbonyl, C2-C20 alkenylcarbonyloxy, C2- C20 alkenylcarbonate, C2-C20 alkynyloxycarbonyl, C2-C20 alkynylcarbonyloxy, and C2-C20 alkynylcarbonate. [0883] Clause 11. The ionizable lipid of any one of clauses 1-10, wherein at least one of R1 and R2 is an unsubstituted, linear or branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl. [0884] Clause 12. The compound of any one of clauses 1-11, wherein R 1 is a branched C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 1 -C 20 heteroalkyl, optionally substituted by one or more substituents selected from C 1 -C 20 alkoxy, C 1 -C 20 alkyloxycarbonyl, C 1 -C 20 alkylcarbonyloxy, C 1 -C 20 alkylcarbonate, C 2 -C 20 alkenyloxycarbonyl, C2-C20 alkenylcarbonyloxy, C2-C20 alkenylcarbonate, C2-C20 alkynyloxycarbonyl, C2-C20 alkynylcarbonyloxy, and C2-C20 alkynylcarbonate. [0885] Clause 13. The compound of any one of clauses 1-12, wherein R 1 and R 2 are each a branched C1-C20 alkyl, C2-C20 alkenyl, or C1-C20 heteroalkyl, optionally substituted by one or more substituents selected from C1-C20 alkoxy, C1-C20 alkyloxycarbonyl, C1-C20 alkylcarbonyloxy, C1-C20 alkylcarbonate, C2-C20 alkenyloxycarbonyl, C2-C20 alkenylcarbonyloxy, C2-C20 alkenylcarbonate, C2-C20 alkynyloxycarbonyl, C 2 -C 20 alkynylcarbonyloxy, and C 2 -C 20 alkynylcarbonate. [0886] Clause 14. The ionizable lipid of any one of clauses 1-13, wherein R1 is an unsubstituted branched C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 heteroalkyl. [0887] Clause 15. The ionizable lipid of any one of clauses 1-14, wherein R 1 and R 2 are each an unsubstituted branched C 6 -C 30 alkyl, C 6 -C 30 alkenyl, or C 6 -C 30 heteroalkyl. [0888] Clause 16. The ionizable lipid of any one of clauses 1-15, wherein at least one of R1 and R2 is a linear C 1 -C 12 alkyl substituted by –O(CO)R 6 , –C(O)OR 6 , or –O(CO)OR 6 , wherein each R 6 is independently linear or branched C 1 -C 20 alkyl or C 2 -C 20 alkenyl. [0889] Clause 17. The ionizable lipid of clause 16, wherein R1 and R2 are each independently a linear C 1 -C 12 alkyl substituted by–OC(O)R 6 ,–C(O)OR 6 , or–OC(O)OR 6 , wherein each R 6 is independently linear or branched C 1 -C 20 alkyl or C 2 -C 20 alkenyl. [0890] Clause 18. The ionizable lipid of clause 16 or 17, wherein R 6 is branched C1-C20 alkyl or C2- C 20 alkenyl. [0891] Clause 19. The ionizable lipid of any one of clauses 1-18, wherein the at least one of R1 and R2 is selected from: –(CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ), –(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and –(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), wherein: q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 , and R 9 and R 10 are independently unsubstituted linear C 1 -C 12 alkyl or unsubstituted linear C 2 -C 12 - alkenyl. [0892] Clause 20. The ionizable lipid of clause 19, wherein R1 and R2 are each independently selected from: –(CH 2 ) q C(O)O(CH 2 ) r CH(R 8 )(R 9 ), –(CH 2 ) q OC(O)(CH 2 ) r CH(R 8 )(R 9 ), and –(CH 2 ) q OC(O)O(CH 2 ) r CH(R 8 )(R 9 ), wherein: q is an integer from 0 to 12, r is an integer from 0 to 6, R 8 is H or R 10 , and R 9 and R 10 are independently unsubstituted linear C1-C12 alkyl or unsubstituted linear C2-C12- alkenyl. [0893] Clause 21. The ionizable lipid of clause 19 or 20, wherein R 8 and R 9 are different. [0894] Clause 22. The ionizable lipid of clause 19, wherein R1 is unsubstituted, linear or branched C6- C 30 alkyl. [0895] Clause 23. The ionizable lipid of any one of clauses 19-21, wherein R1 is – (CH2)qC(O)O(CH2)rCH(R 8 )(R 9 ). [0896] Clause 24. The ionizable lipid of any one of clauses 19-21, wherein R1 is – (CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ). [0897] Clause 25. The ionizable lipid of any one of clauses 19-21, wherein R 1 is – (CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ). [0898] Clause 26. The ionizable lipid of any one of clauses 22-25, wherein R 2 is unsubstituted, linear or branched C 6 -C 30 alkyl. [0899] Clause 27. The ionizable lipid of any one of clauses 22-25, wherein R2 is – (CH 2 ) q C(O)O(CH 2 ) r CH(R 8 )(R 9 ). [0900] Clause 28. The ionizable lipid of any one of clauses 22-25, wherein R2 is – (CH2)qOC(O)(CH2)rCH(R 8 )(R 9 ). [0901] Clause 29. The ionizable lipid of any one of clauses 22-25, wherein R2 is – (CH2)qOC(O)O(CH2)rCH(R 8 )(R 9 ). [0902] Clause 30. The ionizable lipid of any one of clauses 19-29, wherein q is an integer from 1 to 6. [0903] Clause 31. The ionizable lipid of any one of clauses 19-29, wherein q is 3, 4, 5, or 6. [0904] Clause 32. The ionizable lipid of any one of clauses 19-31, wherein r is 0. [0905] Clause 33. The ionizable lipid of any one of clauses 19-31, wherein r is an integer from 1 to 6. [0906] Clause 34. The ionizable lipid of any one of clauses 19-31, wherein r is 1. [0907] Clause 35. The ionizable lipid of any one of clauses 19-31, wherein r is 2. [0908] Clause 36. The ionizable lipid of any one of clauses 19-35, wherein R 8 is H. [0909] Clause 37. The ionizable lipid of any one of clauses 19-35, wherein R 8 is R 10 . [0910] Clause 38. The ionizable lipid of clause 37, wherein R9 and R10 are each independently unsubstituted linear C1-C12 alkyl. [0911] Clause 39. The ionizable lipid of clause 38, wherein R9 and R10 are each independently unsubstituted linear C4-C8 alkyl. [0912] Clause 40. The ionizable lipid of clause 39, wherein R9 and R10 are each independently unsubstituted linear C6-C8 alkyl. [0913] Clause 41. The ionizable lipid of any one of clauses 37-40, wherein R9 and R10 are different. [0914] Clause 42. The ionizable lipid of any one of clauses 1-9, wherein R1 and R2 are each – (CH2)m–L–R’, wherein: [0915] m is an integer from 0 to 10; [0916] L is absent (i.e., a direct bond), –C(H)(RL)–*, –OC(O)–*, or –C(O)O–*, wherein “–*” indicates the attachment point to R’; [0917] R’ is selected from: C1-C30 alkyl, C2-C30 alkenyl, C1-C30 alkoxy, 2-30-membered heteroalkylene, and 3-12-membered heterocyclyl, wherein 2-30-membered heteroalkylene is optionally substituted with one or more R’’, and 3-12-membered heterocyclyl is optionally substituted with one or more C1-C30 alkyl; [0918] RL is selected from: C1-C30 alkyl, C2-C30 alkenyl, C1-C30 alkoxy, 2-30-membered heteroalkylene, wherein 3-12-membered heteroalkylene is optionally substituted one or more with R’’, [0919] R’’ is each independently selected from: oxo, C1-C30 alkoxy, –C(O)-C1-C30 alkyl, –C(O)- C1-C30 alkoxy, and –C(O)-C1-C30 alkylene-C(O)-C1-C30 alkoxy. [0920] Clause 43. The ionizable lipid of any one of clauses 1-19 and 42, wherein R1 and R2 are each independently selected from: [0921] , , [0922] , , , , , , , , , , , , , , , , O , O , , , , , , , , , , , , , , , , , , , , , , and . [0923] Clause 44. The ionizable lipid of any one of clauses 1-19 and 42-43, wherein R1 and R2 are the same. [0924] Clause 45. The ionizable lipid of any one of clauses 1-19 and 42-43, wherein R1 and R2 are different. [0925] Clause 46. The ionizable lipid of clause 1, wherein the lipid is of Formula (13c-1) or Formula (13c-2): Formula (13c-1) Formula (13c-2) wherein: n* and n are each an integer from 1 to 7; R a is hydrogen or hydroxyl, R b is hydrogen or C 1 -C 6 alkyl, L A and L B are each independently linear C1-C12 alkyl; Z A and Z B are optional linking groups each independently selected from -C(O)O-, -O(CO)-, and -O(CO)O-; and R A and R B are independently linear or branched C1-C20 alkyl or C2-C20 alkenyl. [0926] Clause 47. The ionizable lipid of clause 46, wherein R B is branched C1-C20 alkyl or C2-C20 alkenyl. [0927] Clause 48. The ionizable lipid of clause 47, wherein R A is branched C 1 -C 20 alkyl or C 2 -C 20 alkenyl. [0928] Clause 49. The ionizable lipid of clause 46, wherein the lipid is of Formula (13d-2) Formula (13d-2) wherein: q and q’ are each independently an integer from 1 to 12, r and r’ are each independently an integer from 0 to 6, R 8A is H or R 10A , R 8B is H or R 10B , and R 9A , R 9B , R 10A , and R 10A are each independently unsubstituted linear C 1 -C 12 alkyl or unsubstituted linear C2-C12-alkenyl. [0929] Clause 50. The ionizable lipid of clause 49, wherein R 8B is R 10B . [0930] Clause 51. The ionizable lipid of clause 50, wherein R 10B and R 9B are different. [0931] Clause 52. The ionizable lipid of clause 51, wherein R 8A is R 10A . [0932] Clause 53. The ionizable lipid of clause 52, wherein R 10A and R 9A are different. [0933] Clause 54. The ionizable lipid of any one of clauses 1-53, wherein the ionizable lipid is selected from , , , , , , O O N OH HO O , O , and . [0934] Clause 55. The ionizable lipid of any one of clauses 1-53, wherein the ionizable lipid is selected from: and . [0935] Clause 56. The ionizable lipid of any one of clauses 1-53, wherein the ionizable lipid is selected from Table 10e. [0936] Clause 57. A pharmaceutical composition comprising a transfer vehicle, wherein the transfer vehicle comprises an ionizable lipid of any one of clauses 1-56. [0937] Clause 58. The pharmaceutical composition of clause 57, wherein the pharmaceutical composition further comprises a RNA polynucleotide. [0938] Clause 59. The pharmaceutical composition of clause 58, wherein the RNA polynucleotide is a linear or circular RNA polynucleotide. [0939] Clause 60. The pharmaceutical composition of clause 58 or 59, wherein the RNA polynucleotide is a circular RNA polynucleotide. [0940] Clause 61. A pharmaceutical composition comprising: a. an RNA polynucleotide, wherein the RNA polynucleotide is a circular RNA polynucleotide, and b. a transfer vehicle comprising an ionizable lipid selected from or . [0941] Clause 62. The pharmaceutical composition of any one of clauses 57-61, wherein the transfer vehicle comprises a nanoparticle, such as a lipid nanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, a biodegradable lipid nanoparticle, a polymer nanoparticle, or a biodegradable polymer nanoparticle. [0942] Clause 63. The pharmaceutical composition of any one of clauses 58-62, wherein the RNA polynucleotide is encapsulated in the transfer vehicle, optionally wherein the encapsulation efficiency is at least about 80%. [0943] Clause 64. The pharmaceutical composition of any one of clauses 58-63, wherein the RNA polynucleotide comprises an expression sequence. [0944] Clause 65. The pharmaceutical composition of clause 64, wherein the first expression sequence encodes a therapeutic protein. [0945] Clause 66. The pharmaceutical composition of clause 65, wherein the first expression sequence encodes a cytokine or a functional fragment thereof, a transcription factor, an immune checkpoint inhibitor, or a chimeric antigen receptor (CAR). [0946] Clause 67. The pharmaceutical composition of any one of clauses 58-66, wherein the RNA polynucleotide comprises, in the following order: a. a 5’ enhanced exon element, b. a core functional element, and c. a 3’ enhanced exon element. [0947] Clause 68. The pharmaceutical composition of clause 67, wherein the core functional element comprises a translation initiation element (TIE). [0948] Clause 69. The pharmaceutical composition of clause 68, wherein the TIE comprises an untranslated region (UTR) or fragment thereof. [0949] Clause 70. The pharmaceutical composition of clause 69, wherein the UTR or fragment thereof comprises a IRES or eukaryotic IRES. [0950] Clause 71. The pharmaceutical composition of any one of clauses 68-70, wherein the TIE comprises an aptamer complex, optionally wherein the aptamer complex comprises at least two aptamers. [0951] Clause 72. The pharmaceutical composition of any one of clauses 67-71, wherein the core functional element comprises a coding region. [0952] Clause 73. The pharmaceutical composition of clause 72, wherein the coding region encodes for a therapeutic protein. [0953] Clause 74. The pharmaceutical composition of clause 73, wherein the therapeutic protein is a chimeric antigen receptor (CAR). [0954] Clause 75. The pharmaceutical composition of any one of clause 67-74, wherein the core functional element comprises a noncoding region. [0955] Clause 76. The pharmaceutical composition of any one of clauses 67-75, wherein the 3’ enhanced exon element comprises a 5’ exon fragment, and optionally a 3’ internal spacer and/or a 3’ internal duplex element, wherein the 3’ internal spacer and/or 3’ internal duplex element are each independently located upstream to the 5’ exon fragment, optionally wherein the 3’ internal spacer is a polyA or polyA-C sequence of about 10 to about 60 nucleotides in length. [0956] Clause 77. The pharmaceutical composition of any one of clauses 67-76, wherein the RNA polynucleotide is made via circularization of a RNA polynucleotide comprising, in the following order: a. a 5’ enhanced intron element, b. a 5’ enhanced exon element, c. a core functional element, d. a 3’ enhanced exon element, and e. a 3’ enhanced intron element. [0957] Clause 78. The pharmaceutical composition of clause 77, wherein the 5’ enhanced intron element comprises: a 3’ intron fragment, comprising a first or a first and a second nucleotides of a 3’ group I intron splice site dinucleotide; and optionally a 5’ affinity tag located upstream to the 3’ intron fragment, a 5’ external spacer located upstream to the 3’ intron fragment, and/or a leading untranslated sequence located at the 5’ end of the said 5’ enhanced intron element. [0958] Clause 79. The pharmaceutical composition of any one of clauses 58-78, wherein the RNA polynucleotide is from about 100nt to about 10,000nt in length, such as about 100nt to about 15,000nt in length. [0959] Clause 80. The pharmaceutical composition of any one of clauses 58-79, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the composition has a duration of therapeutic effect in a human cell or in vivo in humans greater than or equal to that of a reference composition, wherein the reference comprises (1) instead of the circular RNA polynucleotide, a reference linear RNA polynucleotide having the same expression sequence as the circular RNA polynucleotide; and/or (2) an ionizable lipid that is not an ionizable of any one of clauses 1-56. [0960] Clause 81. The pharmaceutical composition of clause 80, wherein the pharmaceutical composition has a duration of therapeutic effect in vivo in humans of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 hours. [0961] Clause 82. The pharmaceutical composition of any one of clauses 58-81, wherein the RNA polynucleotide is a circular RNA polynucleotide, and wherein the composition has a functional half- life in a human cell or in vivo in human greater than or equal to that of a pre-determined threshold value. [0962] Clause 83. The pharmaceutical composition of clause 82, wherein the composition has a functional half-life of at least about 20 hours. [0963] Clause 84. The pharmaceutic composition of any one of clauses 57-83, wherein the transfer vehicle further comprises a structural lipid and a PEG-modified lipid. [0964] Clause 85. The pharmaceutical composition of any one of clause 84, wherein the structural lipid binds to C1q and/or promotes the binding of the transfer vehicle comprising said lipid to C1q compared to a control transfer vehicle lacking the structural lipid and/or increases uptake of C1q-bound transfer vehicle into an immune cell compared to a control transfer vehicle lacking the structural lipid. [0965] Clause 86. The pharmaceutical composition of clause 85, wherein the immune cell is a T cell, an NK cell, an NKT cell, a macrophage, or a neutrophil. [0966] Clause 87. The pharmaceutical composition of any one of clauses 84-86, wherein the structural lipid is cholesterol. [0967] Clause 88. The pharmaceutical composition of clause 87, wherein the structural lipid is beta- sitosterol. [0968] Clause 89. The pharmaceutical composition of clause 87, wherein the structural lipid is not beta-sitosterol. [0969] Clause 90. The pharmaceutical composition of any one of clauses 84-89, wherein the PEG- modified lipid is DSPE-PEG, DMG-PEG, PEG-DAG, PEG-S-DAG, PEG-PE, PEG-S-DMG, PEG-cer, PEG-dialkoxypropylcarbamate, PEG-OR, PEG-OH, PEG-c-DOMG, or PEG-1. [0970] Clause 91. The pharmaceutical composition of clause 90, wherein the PEG-modified lipid is DSPE-PEG(2000). [0971] Clause 92. The pharmaceutical composition of any one of clauses 57-91, wherein the transfer vehicle further comprises a helper lipid. [0972] Clause 93. The pharmaceutical composition of clause 92, wherein the helper lipid is DSPC or DOPE. [0973] Clause 94. The pharmaceutical composition of any one of clauses 57-93, wherein the transfer vehicle comprises DSPC, cholesterol, and DMG-PEG(2000). [0974] Clause 95. The pharmaceutical composition of any one of clauses 84-94, wherein the transfer vehicle comprises about 0.5% to about 4% PEG-modified lipids by molar ratio. [0975] Clause 96. The pharmaceutical composition of any one of clauses 84-95, wherein the transfer vehicle comprises about 1% to about 2% PEG-modified lipids by molar ratio. [0976] Clause 97. The pharmaceutical composition of any one of clauses 57-96, wherein the transfer vehicle comprises: a. an ionizable lipid selected from: OH N OH O O , , and , or a mixture thereof, b. a helper lipid selected from DOPE or DSPC, c. cholesterol, and d. a PEG-lipid selected from DSPE-PEG(2000) or DMG-PEG(2000). [0977] Clause 98. The pharmaceutical composition of any one of clauses 92-97, wherein the molar ratio of ionizable lipid:helper lipid:cholesterol:PEG-lipid is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. [0978] Clause 99. The pharmaceutical composition of any one of clauses 92-98, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. [0979] Clause 100. The pharmaceutical composition of clause 99, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is about 62:4:33:1. [0980] Clause 101. The pharmaceutical composition of clause 99, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DSPE-PEG(2000) is about 53:5:41:1. [0981] Clause 102. The pharmaceutical composition of any one of clauses 92-97, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. [0982] Clause 103. The pharmaceutical composition of clause 102, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 50:10:38.5:1.5. [0983] Clause 104. The pharmaceutical composition of clause 102, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 41:12:45:2. [0984] Clause 105. The pharmaceutical composition of clause 102, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DMG-PEG(2000), and wherein the molar ratio of ionizable lipid:DSPC:cholesterol:DMG-PEG(2000) is about 45:9:44:2. [0985] Clause 106. The pharmaceutical composition of any one of clauses 92-97, wherein the transfer vehicle comprises the helper lipid of DSPC and the PEG-lipid of DSPE-PEG(2000), and wherein the molar ratio of ionizable lipid: DSPC:cholesterol:DSPE-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. [0986] Clause 107. The pharmaceutical composition of any one of clauses 92-97, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid is C14-PEG(2000), and wherein the molar ratio of ionizable lipid:DOPE:cholesterol:C14-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. [0987] Clause 108. The pharmaceutical composition of any one of clauses 92-97, wherein the transfer vehicle comprises the helper lipid of DOPE and the PEG-lipid of DMG-PEG(2000), wherein the molar ratio of ionizable lipid:DOPE:cholesterol:DMG-PEG(2000) is about 45:9:44:2, about 50:10:38.5:1.5, about 41:12:45:2, about 62:4:33:1, or about 53:5:41:1. [0988] Clause 109. The pharmaceutical composition of any one of clauses 58-108, having a lipid to phosphate (IL:P) molar ratio of about 3 to about 9, such as about 3, about 4, about 4.5, about 5, about 5.5, about 5.7, about 6, about 6.2, about 6.5, or about 7. [0989] Clause 110. The pharmaceutical composition of any one of clauses 58-109, wherein the transfer vehicle is formulated for endosomal release of the RNA polynucleotide. [0990] Clause 111. The pharmaceutical composition of any one of clauses 57-110, wherein the transfer vehicle is capable of binding to apolipoprotein E (APOE) or is substantially free of APOE binding sites. [0991] Clause 112. The pharmaceutical composition of any one of clauses 57-111, wherein the transfer vehicle is capable of low density lipoprotein receptor (LDLR) dependent uptake or LDLR independent uptake into a cell. [0992] Clause 113. The pharmaceutical composition of any one of clauses 57-112, wherein the transfer vehicle has a diameter of less than about 120 nm and/or does not form aggregates with a diameter of more than 300 nm. [0993] Clause 114. The pharmaceutical composition of any one of clauses 60-113, wherein the pharmaceutical composition is substantially free of linear RNA. [0994] Clause 115. The pharmaceutical composition of any one of clauses 57-114, further comprising a targeting moiety operably connected to the transfer vehicle. [0995] Clause 116. The pharmaceutical composition of clause 115, wherein the targeting moiety specifically or indirectly binds an immune cell antigen, wherein the immune cell antigen is a T cell antigen selected from CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2 integrin, and C1qR. [0996] Clause 117. The pharmaceutical composition of clause 116, further comprising an adapter molecule comprising a transfer vehicle binding moiety and a cell binding moiety, wherein the targeting moiety specifically binds the transfer vehicle binding moiety, and the cell binding moiety specifically binds a target cell antigen, optionally wherein the target cell antigen is an immune cell antigen selected from a T cell antigen, an NK cell antigen, an NKT cell antigen, a macrophage antigen, or a neutrophil antigen. [0997] Clause 118. The pharmaceutical composition of any one of clauses 115-117, wherein the targeting moiety is a small molecule (e.g., mannose, a lectin, acivicin, biotin, or digoxigenin), and/or the targeting moiety is a single chain Fv (scFv) fragment, nanobody, peptide, peptide-based macrocycle, minibody, small molecule ligand such as folate, arginylglycylaspartic acid (RGD), or phenol-soluble modulin alpha 1 peptide (PSMA1), heavy chain variable region, light chain variable region or fragment thereof. [0998] Clause 119. The pharmaceutical composition of any one of clauses 58-118, wherein less than 1%, by weight, of the polynucleotides in the composition are double stranded RNA, DNA splints, or triphosphorylated RNA. [0999] Clause 120. The pharmaceutical composition of any one of clauses 58-119, wherein less than 1%, by weight, of the polynucleotides and proteins in the pharmaceutical composition are double stranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins, protein ligases, and capping enzymes. [1000] Clause 121. A method of treating or preventing a disease, disorder, or condition, comprising administering an effective amount of a pharmaceutical composition of any one of clauses 57-120. [1001] Clause 122. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 57-120. EXAMPLES [1002] Wesselhoeft et al., (2019) RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In vivo. Molecular Cell.74(3), 508-520 and Wesselhoeft et al., (2018) Engineering circular RNA for Potent and Stable Translation in Eukaryotic Cells. Nature Communications.9, 2629 are incorporated by reference in their entirety. [1003] The disclosure is further described in detail by reference to the following examples but are not intended to be limited to the following examples. These examples encompass any and all variations of the illustrations with the intention of providing those of ordinary skill in the art with complete disclosure and description of how to make and use the subject disclosure and are not intended to limit the scope of what is regarded as the disclosure. EXAMPLE 1 [1004] Example 1A: External duplex regions allow for circularization of long precursor RNA using the permuted intron exon (PIE) circularization strategy. [1005] A 1.1kb sequence containing a full-length encephalomyocarditis virus (EMCV) IRES, a Gaussia luciferase (GLuc) expression sequence, and two short exon fragments of the permuted intron- exon (PIE) construct were inserted between the 3’ and 5’ introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage. Precursor RNA was synthesized by run-off transcription. Circularization was attempted by heating the precursor RNA in the presence of magnesium ions and GTP, but splicing products were not obtained. [1006] Perfectly complementary 9 nucleotide and 19 nucleotide long duplex regions were designed and added at the 5’ and 3’ ends of the precursor RNA. Addition of these homology arms increased splicing efficiency from 0 to 16% for 9 nucleotide duplex regions and to 48% for 19 nucleotide duplex regions as assessed by disappearance of the precursor RNA band. [1007] The splicing product was treated with RNase R. Sequencing across the putative splice junction of RNase R-treated splicing reactions revealed ligated exons, and digestion of the RNase R-treated splicing reaction with oligonucleotide-targeted RNase H produced a single band in contrast to two bands yielded by RNase H-digested linear precursor. This shows that circular RNA is a major product of the splicing reactions of precursor RNA containing the 9 or 19 nucleotide long external duplex regions [1008] Example 1B: Spacers that conserve secondary structures of IRES and PIE splice sites increase circularization efficiency. [1009] A series of spacers was designed and inserted between the 3’ PIE splice site and the IRES. These spacers were designed to either conserve or disrupt secondary structures within intron sequences in the IRES, 3’ PIE splice site, and/or 5’ splice site. The addition of spacer sequences designed to conserve secondary structures resulted in 87% splicing efficiency, while the addition of a disruptive spacer sequences resulted in no detectable splicing. EXAMPLE 2 [1010] Example 2A: Internal duplex regions in addition to external duplex regions create a splicing bubble and allows for translation of several expression sequences. [1011] Spacers were designed to be unstructured, non-homologous to the intron and IRES sequences, and to contain spacer-spacer duplex regions. These were inserted between the 5’ exon and IRES and between the 3’ exon and expression sequence in constructs containing external duplex regions, EMCV IRES, and expression sequences for Gaussia luciferase (total length: 1289 nt), Firefly luciferase (2384 nt), eGFP (1451 nt), human erythropoietin (1313 nt), and Cas9 endonuclease (4934 nt). Circularization of all 5 constructs was achieved. Circularization of constructs utilizing T4 phage and Anabaena introns were roughly equal. Circularization efficiency was higher for shorter sequences. To measure translation, each construct was transfected into HEK293 cells. Gaussia and Firefly luciferase transfected cells produced a robust response as measured by luminescence, human erythropoietin was detectable in the media of cells transfected with erythropoietin circRNA, and EGFP fluorescence was observed from cells transfected with EGFP circRNA. Co-transfection of Cas9 circRNA with sgRNA directed against GFP into cells constitutively expressing GFP resulted in ablated fluorescence in up to 97% of cells in comparison to an sgRNA-only control. [1012] Example 2B: Use of CVB3 IRES increases protein production. [1013] Constructs with internal and external duplex regions and differing IRES containing either Gaussia luciferase or Firefly luciferase expression sequences were made. Protein production was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. The Coxsackievirus B3 (CVB3) IRES construct produced the most protein in both cases. [1014] Example 2C: Use of polyA or polyAC spacers increases protein production. [1015] Thirty nucleotide long polyA or polyAC spacers were added between the IRES and splice junction in a construct with each IRES that produced protein in example 2B. Gaussia luciferase activity was measured by luminescence in the supernatant of HEK293 cells 24 hours after transfection. Both spacers improved expression in every construct over control constructs without spacers. EXAMPLE 3 [1016] HEK293 or HeLa cells transfected with circular RNA produce more protein than those transfected with comparable unmodified or modified linear RNA. [1017] HPLC-purified Gaussia luciferase-coding circRNA (CVB3-GLuc-pAC) was compared with a canonical unmodified 5’ methylguanosine-capped and 3’ polyA-tailed linear GLuc mRNA, and a commercially available nucleoside-modified (pseudouridine, 5-methylcytosine) linear GLuc mRNA (from Trilink). Luminescence was measured 24 h post-transfection, revealing that circRNA produced 811.2% more protein than the unmodified linear mRNA in HEK293 cells and 54.5% more protein than the modified mRNA. Similar results were obtained in HeLa cells and a comparison of optimized circRNA coding for human erythropoietin with linear mRNA modified with 5-methoxyuridine. [1018] Luminescence data was collected over 6 days. In HEK293 cells, circRNA transfection resulted in a protein production half-life of 80 hours, in comparison with the 43 hours of unmodified linear mRNA and 45 hours of modified linear mRNA. In HeLa cells, circRNA transfection resulted in a protein production half-life of 116 hours, in comparison with the 44 hours of unmodified linear mRNA and 49 hours of modified linear mRNA. CircRNA produced substantially more protein than both the unmodified and modified linear mRNAs over its lifetime in both cell types. EXAMPLE 4 [1019] Example 4A: Purification of circRNA by RNase digestion, HPLC purification, and phosphatase treatment decreases immunogenicity. Completely purified circular RNA is significantly less immunogenic than unpurified or partially purified circular RNA. Protein expression stability and cell viability are dependent on cell type and circular RNA purity. [1020] Human embryonic kidney 293 (HEK293) and human lung carcinoma A549 cells were transfected with: a. products of an unpurified GLuc circular RNA splicing reaction, b. products of RNase R digestion of the splicing reaction, c. products of RNase R digestion and HPLC purification of the splicing reaction, or d. products of RNase digestion, HPLC purification, and phosphatase treatment of the splicing reaction. [1021] RNase R digestion of splicing reactions was insufficient to prevent cytokine release in A549 cells in comparison to untransfected controls. [1022] The addition of HPLC purification was also insufficient to prevent cytokine release, although there was a significant reduction in interleukin-6 (IL-6) and a significant increase in interferon-α1 (IFNα1) compared to the unpurified splicing reaction. [1023] The addition of a phosphatase treatment after HPLC purification and before RNase R digestion dramatically reduced the expression of all upregulated cytokines assessed in A549 cells. Secreted monocyte chemoattractant protein 1 (MCP1), IL-6, IFNα1, tumor necrosis factor α (TNFα), and IFNγ inducible protein-10 (IP-10) fell to undetectable or un-transfected baseline levels. [1024] There was no substantial cytokine release in HEK293 cells. A549 cells had increased GLuc expression stability and cell viability when transfected with higher purity circular RNA. Completely purified circular RNA had a stability phenotype similar to that of transfected 293 cells. [1025] Example 4B: Circular RNA does not cause significant immunogenicity and is not a RIG-I ligand. [1026] A549 cells were transfected with: a. unpurified circular RNA, b. high molecular weight (linear and circular concatenations) RNA, c. circular (nicked) RNA, d. an early fraction of purified circular RNA (more overlap with nicked RNA peak), e. a late fraction of purified circular RNA (less overlap with nicked RNA peak), f. introns excised during circularization, or g. vehicle (i.e. untransfected control). [1027] Precursor RNA was separately synthesized and purified in the form of the splice site deletion mutant (DS) due to difficulties in obtaining suitably pure linear precursor RNA from the splicing reaction. Cytokine release and cell viability was measured in each case. [1028] Robust IL-6, RANTES, and IP-10 release was observed in response to most of the species present within the splicing reaction, as well as precursor RNA. Early circRNA fractions elicited cytokine responses comparable to other non-circRNA fractions, indicating that even relatively small quantities of linear RNA contaminants are able to induce a substantial cellular immune response in A549 cells. Late circRNA fractions elicited no cytokine response in excess of that from untransfected controls. A549 cell viability 36 hours post-transfection was significantly greater for late circRNA fractions compared with all of the other fractions. [1029] RIG-I and IFN-β1 transcript induction upon transfection of A549 cells with late circRNA HPLC fractions, precursor RNA or unpurified splicing reactions were analyzed. Induction of both RIG- I and IFN-β1 transcripts were weaker for late circRNA fractions than precursor RNA and unpurified splicing reactions. RNase R treatment of splicing reactions alone was not sufficient to ablate this effect. Addition of very small quantities of the RIG-I ligand 3p-hpRNA to circular RNA induced substantial RIG-I transcription. In HeLa cells, transfection of RNase R-digested splicing reactions induced RIG-I and IFN-β1, but purified circRNA did not. Overall, HeLa cells were less sensitive to contaminating RNA species than A549 cells. [1030] A time course experiment monitoring RIG-I, IFN-β1, IL-6, and RANTES transcript induction within the first 8 hours after transfection of A549 cells with splicing reactions or fully purified circRNA did not reveal a transient response to circRNA. Purified circRNA similarly failed to induce pro- inflammatory transcripts in RAW264.7 murine macrophages. [1031] A549 cells were transfected with purified circRNA containing an EMCV IRES and EGFP expression sequence. This failed to produce substantial induction of pro-inflammatory transcripts. These data demonstrate that non-circular components of the splicing reaction are responsible for the immunogenicity observed in previous studies and that circRNA is not a natural ligand for RIG-I. EXAMPLE 5 [1032] Circular RNA avoids detection by TLRs. [1033] TLR 3, 7, and 8 reporter cell lines were transfected with multiple linear or circular RNA constructs and secreted embryonic alkaline phosphatase (SEAP) was measured. [1034] Linearized RNA was constructed by deleting the intron and homology arm sequences. The linear RNA constructs were then treated with phosphatase (in the case of capped RNAs, after capping) and purified by HPLC. [1035] None of the attempted transfections produced a response in TLR7 reporter cells. TLR3 and TLR8 reporter cells were activated by capped linearized RNA, polyadenylated linearized RNA, the nicked circRNA HPLC fraction, and the early circRNA fraction. The late circRNA fraction and m1ψ- mRNA did not provoke TLR-mediated response in any cell line. [1036] In a second experiment, circRNA was linearized using two methods: treatment of circRNA with heat in the presence of magnesium ions and DNA oligonucleotide-guided RNase H digestion. Both methods yielded a majority of full-length linear RNA with small amounts of intact circRNA. TLR3, 7, and 8 reporter cells were transfected with circular RNA, circular RNA degraded by heat, or circular RNA degraded by RNase H, and SEAP secretion was measured 36 hours after transfection. TLR8 reporter cells secreted SEAP in response to both forms of degraded circular RNA, but did not produce a greater response to circular RNA transfection than mock transfection. No activation was observed in TLR3 and TLR7 reporter cells for degraded or intact conditions, despite the activation of TLR3 by in vitro transcribed linearized RNA. EXAMPLE 6 [1037] Unmodified circular RNA produces increased sustained in vivo protein expression than linear RNA. [1038] Mice were injected and HEK293 cells were transfected with unmodified and m1ψ-modified human erythropoietin (hEpo) linear mRNAs and circRNAs. Equimolar transfection of m1ψ-mRNA and unmodified circRNA resulted in robust protein expression in HEK293 cells. hEpo linear mRNA and circRNA displayed similar relative protein expression patterns and cell viabilities in comparison to GLuc linear mRNA and circRNA upon equal weight transfection of HEK293 and A549 cells. [1039] In mice, hEpo was detected in serum after the injection of hEpo circRNA or linear mRNA into visceral adipose. hEpo detected after the injection of unmodified circRNA decayed more slowly than that from unmodified or m1ψ-mRNA and was still present 42 hours post-injection. Serum hEpo rapidly declined upon the injection of unpurified circRNA splicing reactions or unmodified linear mRNA. Injection of unpurified splicing reactions produced a cytokine response detectable in serum that was not observed for the other RNAs, including purified circRNA. EXAMPLE 7 [1040] Circular RNA can be effectively delivered in vivo or in vitro via lipid nanoparticles. [1041] Purified circular RNA was formulated into lipid nanoparticles (LNPs) with the ionizable lipidoid cKK-E12 (Dong et al., 2014; Kauffman et al., 2015). The particles formed uniform multilamellar structures with an average size, polydispersity index, and encapsulation efficiency similar to that of particles containing commercially available control linear mRNA modified with 5moU. [1042] Purified hEpo circRNA displayed greater expression than 5moU-mRNA when encapsulated in LNPs and added to HEK293 cells. Expression stability from LNP-RNA in HEK293 cells was similar to that of RNA delivered by transfection reagent, with the exception of a slight delay in decay for both 5moU-mRNA and circRNA. Both unmodified circRNA and 5moU-mRNA failed to activate RIG- I/IFN-β1 in vitro. [1043] In mice, LNP-RNA was delivered by local injection into visceral adipose tissue or intravenous delivery to the liver. Serum hEpo expression from circRNA was lower but comparable with that from 5moU-mRNA 6 hours after delivery in both cases. Serum hEpo detected after adipose injection of unmodified LNP-circRNA decayed more slowly than that from LNP-5moU-mRNA, with a delay in expression decay present in serum that was similar to that noted in vitro, but serum hEpo after intravenous injection of LNP-circRNA or LNP-5moU-mRNA decayed at approximately the same rate. There was no increase in serum cytokines or local RIG-I, TNFα, or IL-6 transcript induction in any of these cases. EXAMPLE 8 [1044] Expression and functional stability by IRES in HEK293, HepG2, and 1C1C7 cells. [1045] Constructs including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and varying IRES were circularized. 100 ng of each circularization reaction was separately transfected into 20,000 HEK293 cells, HepG2 cells, and 1C1C7 cells using Lipofectamine MessengerMax. Luminescence in each supernatant was assessed after 24 hours as a measure of protein expression. In HEK293 cells, constructs including Crohivirus B, Salivirus FHB, Aichi Virus, Salivirus HG-J1, and Enterovirus J IRES produced the most luminescence at 24 hours (FIG.1A). In HepG2 cells, constructs including Aichi Virus, Salivirus FHB, EMCV-Cf, and CVA3 IRES produced high luminescence at 24 hours (FIG. 1B). In 1C1C7 cells, constructs including Salivirus FHB, Aichi Virus, Salivirus NG-J1, and Salivirus A SZ-1 IRES produced high luminescence at 24 hours (FIG.1C). [1046] A trend of larger IRES producing greater luminescence at 24 hours was observed. Shorter total sequence length tends to increase circularization efficiency, so selecting a high expression and relatively short IRES may result in an improved construct. In HEK293 cells, a construct using the Crohivirus B IRES produced the highest luminescence, especially in comparison to other IRES of similar length (FIG.2A). Expression from IRES constructs in HepG2 and 1C1C7 cells plotted against IRES size are in FIGs.2B and 2C. [1047] Functional stability of select IRES constructs in HepG2 and 1C1C7 cells were measured over 3 days. Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after transfection of 20,000 cells with 100 ng of each circularization reaction, followed by complete media replacement. Salivirus A GUT and Salivirus FHB exhibited the highest functional stability in HepG2 cells, and Salivirus N-J1 and Salivirus FHB produced the most stable expression in 1C1C7 cells (FIGs.3A and 3B). EXAMPLE 9 [1048] Expression and functional stability by IRES in Jurkat cells. [1049] 2 sets of constructs including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized. 60,000 Jurkat cells were electroporated with 1 µg of each circularization reaction. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation. A CVB3 IRES construct was included in both sets for comparison between sets and to previously defined IRES efficacy. CVB1 and Salivirus A SZ1 IRES constructs produced the most expression at 24h. Data can be found in FIGs.4A and 4B. [1050] Functional stability of the IRES constructs in each round of electroporated Jurkat cells was measured over 3 days. Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after electroporation of 60,000 cells with 1 µg of each circularization reaction, followed by complete media replacement (FIGs.5A and 5B). [1051] Salivirus A SZ1 and Salivirus A BN2 IRES constructs had high functional stability compared to other constructs. EXAMPLE 10 [1052] Expression, functional stability, and cytokine release of circular and linear RNA in Jurkat cells. [1053] A construct including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a Salivirus FHB IRES was circularized. mRNA including a Gaussia luciferase expression sequence and a ~150nt polyA tail, and modified to replace 100% of uridine with 5-methoxy uridine (5moU) is commercially available and was purchased from Trilink. 5moU nucleotide modifications have been shown to improve mRNA stability and expression (Bioconjug Chem. 2016 Mar 16;27(3):849-53). Expression of modified mRNA, circularization reactions (unpure), and circRNA purified by size exclusion HPLC (pure) in Jurkat cells were measured and compared (FIG. 6A). Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 60,000 cells with 1 µg of each RNA species. [1054] Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after electroporation of 60,000 cells with 1ug of each RNA species, followed by complete media replacement. A comparison of functional stability data of modified mRNA and circRNA in Jurkat cells over 3 days is in FIG.6B. [1055] IFNγ (FIG.7A), IL-6 (FIG.7B), IL-2 (FIG.7C), RIG-I (FIG.7D), IFN-β1 (FIG.7E), and TNFα (FIG.7F) transcript induction was measured 18 hours after electroporation of 60,000 Jurkat cells with 1 µg of each RNA species described above and 3p-hpRNA (5’ triphosphate hairpin RNA, which is a known RIG-I agonist). EXAMPLE 11 [1056] Expression of circular and linear RNA in monocytes and macrophages. [1057] A construct including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a Salivirus FHB IRES was circularized. mRNA including a Gaussia luciferase expression sequence and a ~150 nt polyA tail, and modified to replace 100% of uridine with 5-methoxy uridine (5moU) was purchased from Trilink. Expression of circular and modified mRNA was measured in human primary monocytes (FIG. 8A) and human primary macrophages (FIG. 8B). Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 60,000 cells with 1 µg of each RNA species. Luminescence was also measured 4 days after electroporation of human primary macrophages with media changes every 24 hours (FIG.8C). The difference in luminescence was statistically significant in each case (p < 0.05). EXAMPLE 12 [1058] Expression and functional stability by IRES in primary T cells. [1059] Constructs including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 primary human CD3+ T cells were electroporated with 1 µg of each circRNA. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation (FIG.9A). Aichi Virus and CVB3 IRES constructs had the most expression at 24 hours. [1060] Luminescence was also measured every 24 hours after electroporation for 3 days in order to compare functional stability of each construct (FIG.9B). The construct with a Salivirus A SZ1 IRES was the most stable. EXAMPLE 13 [1061] Expression and functional stability of circular and linear RNA in primary T cells and PBMCs. [1062] Constructs including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a Salivirus A SZ1 IRES or Salivirus FHB IRES were circularized. mRNA including a Gaussia luciferase expression sequence and a ~150 nt polyA tail, and modified to replace 100% of uridine with 5-methoxy uridine (5moU) and was purchased from Trilink. Expression of Salivirus A SZ1 IRES HPLC purified circular and modified mRNA was measured in human primary CD3+ T cells. Expression of Salivirus FHB HPLC purified circular, unpurified circular and modified mRNA was measured in human PBMCs. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 150,000 cells with 1 µg of each RNA species. Data for primary human T cells is in FIGs.10A and 10B, and data for PBMCs is in FIG.10C. The difference in expression between the purified circular RNA and unpurified circular RNA or linear RNA was significant in each case (p < 0.05). [1063] Luminescence from secreted Gaussia luciferase in primary T cell supernatant was measured every 24 hours after electroporation over 3 days in order to compare construct functional stability. Data is shown in FIG.10B. The difference in relative luminescence from the day 1 measurement between purified circular RNA and linear RNA was significant at both day 2 and day 3 for primary T cells. EXAMPLE 14 [1064] Circularization efficiency by permutation site in Anabaena intron. [1065] RNA constructs including a CVB3 IRES, a Gaussia luciferase expression sequence, anabaena intron / exon regions, spacers, internal duplex regions, and homology arms were produced. Circularization efficiency of constructs using the traditional anabaena intron permutation site and 5 consecutive permutations sites in P9 was measured by HPLC. HPLC chromatograms for the 5 consecutive permutation sites in P9 are shown in FIG.11A. [1066] Circularization efficiency was measured at a variety of permutation sites. Circularization efficiency is defined as the area under the HPLC chromatogram curve for each of: circRNA / (circRNA + precursor RNA). Ranked quantification of circularization efficiency at each permutation site is in FIG.11B. 3 permutation sites (indicated in FIG.11B) were selected for further investigation. [1067] Circular RNA in this example was circularized by in vitro transcription (IVT) then purified via spin column. Circularization efficiency for all constructs would likely be higher if the additional step of incubation with Mg 2+ and guanosine nucleotide were included; however, removing this step allowed for comparison between, and optimization of, circular RNA constructs. This level of optimization is especially useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors. EXAMPLE 15 [1068] Circularization efficiency of alternative introns. [1069] Precursor RNA containing a permuted group 1 intron of variable species origin or permutation site and several constant elements including: a CVB3 IRES, a Gaussia luciferase expression sequence, spacers, internal duplex regions, and homology arms were created. Circularization data can be found in FIG. 12. FIG. 12A shows chromatograms resolving precursor, CircRNA and introns. Fig. 12B provides ranked quantification of circularization efficiency, based on the chromatograms shown in Fig. 12A, as a function of intron construct. [1070] Circular RNA in this example was circularized by in vitro transcription (IVT) then spin column purification. Circularization efficiency for all constructs would likely be higher if the additional step of incubation with Mg 2+ and guanosine nucleotide were included; however, removing this step allows for comparison between, and optimization of, circular RNA constructs. This level of optimization is especially useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors. EXAMPLE 16 [1071] Circularization efficiency by homology arm presence or length. [1072] RNA constructs including a CVB3 IRES, a Gaussia luciferase expression sequence, anabaena intron / exon regions, spacers, and internal duplex regions were produced. Constructs representing 3 anabaena intron permutation sites were tested with 30nt, 25% GC homology arms or without homology arms (“NA”). These constructs were allowed to circularize without the step of incubation with Mg 2+ . Circularization efficiency was measured and compared. Data can be found in FIG.13. Circularization efficiency was higher for each construct lacking homology arms. FIG. 13A provides ranked quantification of circularization efficiency; FIG. 13B provides chromatograms resolving precursor, circRNA and introns. [1073] For each of the 3 permutation sites, constructs were created with 10 nt, 20 nt, and 30 nt arm length and 25%, 50%, and 75% GC. Splicing efficiency of these constructs was measured and compared to constructs without homology arms (FIG. 14). Splicing efficiency is defined as the proportion of free introns relative to the total RNA in the splicing reaction. [1074] FIG.15 A (left) contains HPLC chromatograms showing the contribution of strong homology arms to improved splicing efficiency. Top left: 75% GC content, 10 nt homology arms. Center left: 75% GC content, 20 nt homology arms. Bottom left: 75% GC content, 30 nt homology arms. [1075] FIG.15 A (right) shows HPLC chromatograms indicating increased splicing efficiency paired with increased nicking, appearing as a shoulder on the circRNA peak. Top right: 75% GC content, 10 nt homology arms. Center right: 75% GC content, 20 nt homology arms. Bottom right: 75% GC content, 30 nt homology arms. [1076] FIG. 15 B (left) shows select combinations of permutation sites and homology arms hypothesized to demonstrate improved circularization efficiency. [1077] FIG. 15 B (right) shows select combinations of permutation sites and homology arms hypothesized to demonstrate improved circularization efficiency, treated with E. coli polyA polymerase. [1078] Circular RNA in this example was circularized by in vitro transcription (IVT) then spin-column purified. Circularization efficiency for all constructs would likely be higher if an additional Mg 2+ incubation step with guanosine nucleotide were included; however, removing this step allowed for comparison between, and optimization of, circular RNA constructs. This level of optimization is especially useful for maintaining high circularization efficiency with large RNA constructs, such as those encoding chimeric antigen receptors. EXAMPLE 17 [1079] Circular RNA encoding chimeric antigen receptors. [1080] Constructs including anabaena intron / exon regions, a Kymriah chimeric antigen receptor (CAR) expression sequence, and a CVB3 IRES were circularized. 100,000 human primary CD3+ T cells were electroporated with 500ng of circRNA and co-cultured for 24 hours with Raji cells stably expressing GFP and firefly luciferase. Effector to target ratio (E:T ratio) 0.75:1. 100,000 human primary CD3+ T cells were mock electroporated and co-cultured as a control (FIG.16). [1081] Sets of 100,000 human primary CD3+ T cells were mock electroporated or electroporated with 1 µg of circRNA then co-cultured for 48 hours with Raji cells stably expressing GFP and firefly luciferase. E:T ratio 10:1 (FIG.17). [1082] Quantification of specific lysis of Raji target cells was determined by detection of firefly luminescence (FIG. 18). 100,000 human primary CD3+ T cells either mock electroporated or electroporated with circRNA encoding different CAR sequences were co-cultured for 48 hours with Raji cells stably expressing GFP and firefly luciferase. % Specific lysis defined as 1-[CAR condition luminescence]/[mock condition luminescence]. E:T ratio 10:1. EXAMPLE 18 [1083] Expression and functional stability of circular and linear RNA in Jurkat cells and resting human T cells. [1084] Constructs including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 Jurkat cells were electroporated with 1 µg of circular RNA or 5moU-mRNA. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation (FIG. 19A left). 150,000 resting primary human CD3+ T cells (10 days post- stimulation) were electroporated with 1 µg of circular RNA or 5moU-mRNA. Luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation (FIG. 19A right). [1085] Luminescence from secreted Gaussia luciferase in supernatant was measured every 24 hours after electroporation, followed by complete media replacement. Functional stability data is shown in FIG. 19B. Circular RNA had more functional stability than linear RNA in each case, with a more pronounced difference in Jurkat cells. EXAMPLE 19 [1086] IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and TNFα transcript induction of cells electroporated with linear RNA or varying circular RNA constructs. [1087] Constructs including anabaena intron / exon regions, a Gaussia luciferase expression sequence, and a subset of previously tested IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 CD3+ human T cells were electroporated with 1 µg of circular RNA, 5moU- mRNA, or immunostimulatory positive control poly inosine:cytosine. IFN-β1 (FIG.20A), RIG-I (FIG. 20B), IL-2 (FIG.20C), IL-6 (FIG.20D), IFN-γ (FIG.20E), and TNF-α (FIG.20F) transcript induction was measured 18 hours after electroporation. EXAMPLE 20 [1088] Specific lysis of target cells and IFNγ transcript induction by CAR expressing cells electroporated with different amounts of circular or linear RNA; specific lysis of target and non-target cells by CAR expressing cells at different E:T ratios. [1089] Constructs including anabaena intron / exon regions, an anti-CD19 CAR expression sequence, and a CVB3 IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 human primary CD3+ T cells either mock electroporated or electroporated with different quantities of circRNA encoding an anti-CD19 CAR sequence were co-cultured for 12 hours with Raji cells stably expressing GFP and firefly luciferase at an E:T ratio of 2:1. Specific lysis of Raji target cells was determined by detection of firefly luminescence (FIG.21A). %Specific lysis was defined as 1-[CAR condition luminescence]/[mock condition luminescence]. IFNγ transcript induction was measured 24 hours after electroporation (FIG.21B). [1090] 150,000 human primary CD3+ T cells were either mock electroporated or electroporated with 500ng circRNA or m1ψ-mRNA encoding an anti-CD19 CAR sequence, then co-cultured for 24 hours with Raji cells stably expressing firefly luciferase at different E:T ratios. Specific lysis of Raji target cells was determined by detection of firefly luminescence (FIG.22A). Specific lysis was defined as 1- [CAR condition luminescence]/[mock condition luminescence]. [1091] CAR expressing T cells were also co-cultured for 24 hours with Raji or K562 cells stably expressing firefly luciferase at different E:T ratios. Specific lysis of Raji target cells or K562 non-target cells was determined by detection of firefly luminescence (FIG.22B). % Specific lysis is defined as 1- [CAR condition luminescence]/[mock condition luminescence]. EXAMPLE 21 [1092] Specific lysis of target cells by T cells electroporated with circular RNA or linear RNA encoding a CAR. [1093] Constructs including anabaena intron / exon regions, an anti-CD19 CAR expression sequence, and a CVB3 IRES were circularized and reaction products were purified by size exclusion HPLC. Human primary CD3+ T cells were electroporated with 500 ng of circular RNA or an equimolar quantity of m1ψ-mRNA, each encoding a CD19-targeted CAR. Raji cells were added to CAR-T cell cultures over 7 days at an E:T ratio of 10:1. % Specific lysis was measured for both constructs at 1, 3, 5, and 7 days (FIG.23). EXAMPLE 22 [1094] Specific lysis of Raji cells by T cells expressing an anti-CD19 CAR or an anti-BCMA CAR. [1095] Constructs including anabaena intron / exon regions, anti-CD19 or anti-BCMA CAR expression sequence, and a CVB3 IRES were circularized and reaction products were purified by size exclusion HPLC. 150,000 primary human CD3+ T cells were electroporated with 500ng of circRNA, then were co-cultured with Raji cells at an E:T ratio of 2:1. % Specific lysis was measured 12 hours after electroporation (FIG.24). EXAMPLE 23 [1096] Expression, functional stability, and cytokine transcript induction of circular and linear RNA expressing antigens. [1097] Constructs including one or more antigen expression sequences are circularized and reaction products are purified by size exclusion HPLC. Antigen presenting cells are electroporated with circular RNA or mRNA. [1098] In vitro antigen production is measured via ELISA. Optionally, antigen production is measured every 24 hours after electroporation. Cytokine transcript induction or release is measured 18 hours after electroporation of antigen presenting cells with circular or linear RNA encoding antigens. The tested cytokines may include IFN-β1, RIG-I, IL-2, IL-6, IFNγ, RANTES, and TNFα. [1099] In vitro antigen production and cytokine induction are measured using purified circRNA, purified circRNA plus antisense circRNA, and unpurified circRNA in order to find the ratio that best preserves expression and immune stimulation. EXAMPLE 24 [1100] In vivo antigen and antibody expression in animal models. [1101] To assess the ability of antigen encoding circRNAs to facilitate antigen expression and antibody production in vivo, escalating doses of RNA encoding one or more antigens is introduced into mice via intramuscular injection. [1102] Mice are injected once, blood collected after 28 days, then injected again, with blood collected 14 days thereafter. Neutralizing antibodies against antigen of interest is measured via ELISA. EXAMPLE 25 [1103] Protection against infection. [1104] To assess the ability of antigen encoding circRNAs to protect against or cure an infection, RNA encoding one or more antigens of a virus (such as influenza) is introduced into mice via intramuscular injection. [1105] Mice receive an initial injection and boost injections of circRNA encoding one or more antigens. Protection from a virus such as influenza is determined by weight loss and mortality over 2 weeks. EXAMPLE 26 [1106] Example 26A: Synthesis of compounds [1107] Synthesis of representative ionizable lipids of the invention are described in PCT applications PCT/US2016/052352, PCT/US2016/068300, PCT/US2010/061058, PCT/US2018/058555, PCT/US2018/053569, PCT/US2017/028981, PCT/US2019/025246, PCT/US2018/035419, PCT/US2019/015913, PCT/US2020/063494, and US applications with publication numbers 20190314524, 20190321489, and 20190314284, the contents of each of which are incorporated herein by reference in their entireties. [1108] Example 26B: Synthesis of compounds [1109] Synthesis of representative ionizable lipids of the invention are described in US patent publication number US20170210697A1, the contents of which is incorporated herein by reference in its entirety. EXAMPLE 27 [1110] Protein expression by organ [1111] Circular or linear RNA encoding FLuc was generated and loaded into transfer vehicles with the following formulation: 50% ionizable lipid represented by , 10% DSPC, 1.5% PEG-DMG, 38.5% cholesterol. CD-1 mice were dosed at 0.2 mg/kg and luminescence was measured at 6 hours (live IVIS) and 24 hours (live IVIS and ex vivo IVIS). Total Flux (photons/second over a region of interest) of the liver, spleen, kidney, lung, and heart was measured. EXAMPLE 28 [1112] Distribution of expression in the spleen [1113] Circular or linear RNA encoding GFP is generated and loaded into transfer vehicles with the following formulation: 50% ionizable Lipid represented by , 10% DSPC, 1.5% PEG-DMG, 38.5% cholesterol. The formulation is administered to CD-1 mice. Flow cytometry is run on spleen cells to determine the distribution of expression across cell types. EXAMPLE 29 [1114] EXAMPLE 29A: Production of nanoparticle compositions [1115] In order to investigate safe and efficacious nanoparticle compositions for use in the delivery of circular RNA to cells, a range of formulations are prepared and tested. Specifically, the particular elements and ratios thereof in the lipid component of nanoparticle compositions are optimized. [1116] Nanoparticles can be made in a 1 fluid stream or with mixing processes such as microfluidics and T-junction mixing of two fluid streams, one of which contains the circular RNA and the other has the lipid components. [1117] Lipid compositions are prepared by combining an ionizable lipid, optionally a helper lipid (such as DOPE, DSPC, or oleic acid obtainable from Avanti Polar Lipids, Alabaster, AL), a PEG lipid (such as 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, AL), and a structural lipid such as cholesterol at concentrations of about, e.g., 40 or 50 mM in a solvent, e.g., ethanol. Solutions should be refrigerated for storage at, for example, -20 °C. Lipids are combined to yield desired molar ratios (see, for example, Tables 17a and 17b below) and diluted with water and ethanol to a final lipid concentration of e.g., between about 5.5 mM and about 25 mM. Table 17a Formulation Description number 1 Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, Chol and DMG- PEG2K (40:30:25:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. 2 Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE, cholesterol and DMG- PEG2K (18:56:20:6) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.35 mg/mL EPO circRNA (encapsulated). Zave=75.9 nm (Dv(50)=57.3 nm; Dv(90)=92.1 nm). 3 Aliquots of 50 mg/mL ethanolic solutions of HGT4003, DOPE, cholesterol and DMG-PEG2K (50:25:20:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. 4 Aliquots of 50 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K (70:25:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. 5 Aliquots of 50 mg/mL ethanolic solutions of HGT5000, DOPE, cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.82 mg/mL EPO mRNA (encapsulated). Zave=105.6 nm (Dv(50)=53.7 nm; Dv(90)=157 nm). 6 Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. 7 Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (35:16:46.5:2.5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. 8 Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (40:10:40:10) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous circRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. [1118] In some embodiments, transfer vehicle has a formulation as described in Table 17a. Table 17b
[1119] In some embodiments, transfer vehicle has a formulation as described in Table 17b. [1120] For nanoparticle compositions including circRNA, solutions of the circRNA at concentrations of 0.1 mg/ml in deionized water are diluted in a buffer, e.g., 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution. Alternatively, solutions of the circRNA at concentrations of 0.15 mg/ml in deionized water are diluted in a buffer, e.g., 6.25 mM sodium acetate buffer at a pH between 3 and 4.5 to form a stock solution. [1121] Nanoparticle compositions including a circular RNA and a lipid component are prepared by combining the lipid solution with a solution including the circular RNA at lipid component to circRNA wt:wt ratios between about 5:1 and about 50:1. The lipid solution is rapidly injected using, e.g., a NanoAssemblr microfluidic based system at flow rates between about 10 ml/min and about 18 ml/min or between about 5 ml/min and about 18 ml/min into the circRNA solution, to produce a suspension with a water to ethanol ratio between about 1:1 and about 4:1. [1122] Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange. Formulations are dialyzed twice against phosphate buffered saline (PBS), pH 7.4, at volumes 200 times that of the primary product using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL) with a molecular weight cutoff of 10 kDa or 20 kDa. The formulations are then dialyzed overnight at 4° C. The resulting nanoparticle suspension is filtered through 0.2 μm sterile filters (Sarstedt, Nümbrecht, Germany) into glass vials and sealed with crimp closures. Nanoparticle composition solutions of 0.01 mg/ml to 0.15 mg/ml are generally obtained. [1123] The method described above induces nano-precipitation and particle formation. [1124] Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nano-precipitation. B. Characterization of nanoparticle compositions [1125] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the nanoparticle compositions in 1×PBS in determining particle size and 15 mM PBS in determining zeta potential. [1126] Ultraviolet-visible spectroscopy can be used to determine the concentration of circRNA in nanoparticle compositions. 100 μL of the diluted formulation in 1×PBS is added to 900 μL of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of circRNA in the nanoparticle composition can be calculated based on the extinction coefficient of the circRNA used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm. [1127] A QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of circRNA by the nanoparticle composition. The samples are diluted to a concentration of approximately 5 μg/mL or 1 μg/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted samples are transferred to a polystyrene 96 well plate and either 50 μL of TE buffer or 50 μL of a 2-4% Triton X-100 solution is added to the wells. The plate is incubated at a temperature of 37° C for 15 minutes. The RIBOGREEN® reagent is diluted 1:100 or 1:200 in TE buffer, and 100 μL of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free circRNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100). [1128] EXAMPLE 29B: In vivo formulation studies [1129] In order to monitor how effectively various nanoparticle compositions deliver circRNA to targeted cells, different nanoparticle compositions including circRNA are prepared and administered to rodent populations. Mice are intravenously, intramuscularly, intraarterially, or intratumorally administered a single dose including a nanoparticle composition with a lipid nanoparticle formulation. In some instances, mice may be made to inhale doses. Dose sizes may range from 0.001 mg/kg to 10 mg/kg, where 10 mg/kg describes a dose including 10 mg of a circRNA in a nanoparticle composition for each 1 kg of body mass of the mouse. A control composition including PBS may also be employed. [1130] Upon administration of nanoparticle compositions to mice, dose delivery profiles, dose responses, and toxicity of particular formulations and doses thereof can be measured by enzyme- linked immunosorbent assays (ELISA), bioluminescent imaging, or other methods. Time courses of protein expression can also be evaluated. Samples collected from the rodents for evaluation may include blood and tissue (for example, muscle tissue from the site of an intramuscular injection and internal tissue); sample collection may involve sacrifice of the animals. [1131] Higher levels of protein expression induced by administration of a composition including a circRNA will be indicative of higher circRNA translation and/or nanoparticle composition circRNA delivery efficiencies. As the non-RNA components are not thought to affect translational machineries themselves, a higher level of protein expression is likely indicative of a higher efficiency of delivery of the circRNA by a given nanoparticle composition relative to other nanoparticle compositions or the absence thereof. EXAMPLE 30 [1132] Characterization of nanoparticle compositions [1133] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the transfer vehicle compositions in 1×PBS in determining particle size and 15 mM PBS in determining zeta potential. [1134] Ultraviolet-visible spectroscopy can be used to determine the concentration of a therapeutic and/or prophylactic (e.g., RNA) in transfer vehicle compositions. 100 μL of the diluted formulation in 1×PBS is added to 900 μL of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of therapeutic and/or prophylactic in the transfer vehicle composition can be calculated based on the extinction coefficient of the therapeutic and/or prophylactic used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm. [1135] For transfer vehicle compositions including RNA, a QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of RNA by the transfer vehicle composition. The samples are diluted to a concentration of approximately 5 μg/mL or 1 μg/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted samples are transferred to a polystyrene 96 well plate and either 50 μL of TE buffer or 50 μL of a 2-4% Triton X- 100 solution is added to the wells. The plate is incubated at a temperature of 37° C for 15 minutes. The RIBOGREEN® reagent is diluted 1:100 or 1:200 in TE buffer, and 100 μL of this solution is added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, MA) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are subtracted from that of each of the samples and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X- 100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100). EXAMPLE 31 [1136] T cell targeting [1137] To target transfer vehicles to T-cells, T cell antigen binders, e.g., anti-CD8 antibodies, are coupled to the surface of the transfer vehicle. Anti-T cell antigen antibodies are mildly reduced with an excess of DTT in the presence of EDTA in PBS to expose free hinge region thiols. To remove DTT, antibodies are passed through a desalting column. The heterobifunctional cross-linker SM(PEG)24 is used to anchor antibodies to the surface of circRNA-loaded transfer vehicles (Amine groups are present in the head groups of PEG lipids, free thiol groups on antibodies were created by DTT, SM(PEG)24 cross-links between amines and thiol groups). Transfer vehicles are first incubated with an excess of SM(PEG)24 and centrifuged to remove unreacted cross-linker. Activated transfer vehicles are then incubated with an excess of reduced anti-T cell antigen antibody. Unbound antibody is removed using a centrifugal filtration device. EXAMPLE 32 [1138] RNA containing transfer vehicle using RV88. [1139] In this example RNA containing transfer vehicles are synthesized using the 2-D vortex microfluidic chip with the cationic lipid RV88 for delivery of circRNA. Table 18a [1140] RV88, DSPC, and cholesterol all being prepared in ethanol at a concentration of 10 mg/ml in borosilica vials. The lipid 14:0-PEG2K PE is prepared at a concentration of 4 mg/ml also in a borosilica glass vial. Dissolution of lipids at stock concentrations is attained by sonication of the lipids in ethanol for 2 min. The solutions are then heated on an orbital tilting shaker set at 170 rpm at 37 °C for 10 min. Vials are then equilibrated at 26 °C for a minimum of 45 min. The lipids are then mixed by adding volumes of stock lipid as shown in Table 18b. The solution is then adjusted with ethanol such that the final lipid concentration was 7.92 mg/ml. Table 18b [1141] RNA is prepared as a stock solution with 75 mM Citrate buffer at pH 6.0 and a concentration of RNA at 1.250 mg/ml. The concentration of the RNA is then adjusted to 0.1037 mg/ml with 75 mM citrate buffer at pH 6.0, equilibrated to 26 °C. The solution is then incubated at 26 °C for a minimum of 25 min. [1142] The microfluidic chamber is cleaned with ethanol and neMYSIS syringe pumps are prepared by loading a syringe with the RNA solution and another syringe with the ethanolic lipid. Both syringes are loaded and under the control of neMESYS software. The solutions are then applied to the mixing chip at an aqueous to organic phase ratio of 2 and a total flow rate of 22 ml/min (14.67 ml/min for RNA and 7.33 ml/min for the lipid solution. Both pumps are started synchronously. The mixer solution that flowed from the microfluidic chip is collected in 4x1 ml fractions with the first fraction being discarded as waste. The remaining solution containing the RNA-liposomes is exchanged by using G-25 mini desalting columns to 10 mM Tris-HCI, 1 mM EDTA, at pH 7.5. Following buffer exchange, the materials are characterized for size, and RNA entrapment through DLS analysis and Ribogreen assays, respectively. EXAMPLE 33 [1143] RNA containing transfer vehicle using RV94.
[1144] In this example, RNA containing liposome are synthesized using the 2-D vortex microfluidic chip with the cationic lipid RV94 for delivery of circRNA. Table 19 [1145] The lipids were prepared as in Example 29 using the material amounts named in Table 20 to a final lipid concentration of 7.92 mg/ml.
Table 20 [1146] The aqueous solution of circRNA is prepared as a stock solution with 75 mM Citrate buffer at pH 6.0 the circRNA at 1.250 mg/ml. The concentration of the RNA is then adjusted to 0.1037 mg/ml with 75 mM citrate buffer at pH 6.0, equilibrated to 26 °C. The solution is then incubated at 26 °C for a minimum of 25 min. [1147] The microfluidic chamber is cleaned with ethanol and neMYSIS syringe pumps are prepared by loading a syringe with the RNA solution and another syringe with the ethanolic lipid. Both syringes are loaded and under the control of neMESYS software. The solutions are then applied to the mixing chip at an aqueous to organic phase ratio of 2 and a total flow rate of 22 ml/min (14.67 ml/min for RNA and 7.33 ml/min forthe lipid solution. Both pumps are started synchronously. The mixer solution that flowed from the microfluidic chip is collected in 4x1 ml fractions with the first fraction being discarded as waste. The remaining solution containing the circRNA-transfer vehicles is exchanged by using G-25 mini desalting columns to 10 mM Tris-HCI, 1 mM EDTA, at pH 7.5, as described above. Following buffer exchange, the materials are characterized for size, and RNA entrapment through DLS analysis and Ribogreen assays, respectively. The biophysical analysis of the liposomes is shown in Table 21. Table 21 EXAMPLE 34 [1148] General protocol for in line mixing. [1149] Individual and separate stock solutions are prepared - one containing lipid and the other circRNA. Lipid stock containing a desired lipid or lipid mixture, DSPC, cholesterol and PEG lipid is prepared by solubilized in 90% ethanol. The remaining 10% is low pH citrate buffer. The concentration of the lipid stock is 4 mg/mL. The pH of this citrate buffer can range between pH 3 and pH 5, depending on the type of lipid employed. The circRNA is also solubilized in citrate buffer at a concentration of 4 mg/mL.5 mL of each stock solution is prepared. [1150] Stock solutions are completely clear and lipids are ensured to be completely solubilized before combining with circRNA. Stock solutions may be heated to completely solubilize the lipids. The circRNAs used in the process may be unmodified or modified oligonucleotides and may be conjugated with lipophilic moieties such as cholesterol. [1151] The individual stocks are combined by pumping each solution to a T-junction. A dual-head Watson-Marlow pump was used to simultaneously control the start and stop of the two streams. A 1.6mm polypropylene tubing is further downsized to 0.8mm tubing in order to increase the linear flow rate. The polypropylene line (ID = 0.8mm) are attached to either side of a T-junction. The polypropylene T has a linear edge of 1.6mm for a resultant volume of 4.1 mm 3 . Each of the large ends (1.6mm) of polypropylene line is placed into test tubes containing either solubilized lipid stock or solubilized circRNA. After the T-junction, a single tubing is placed where the combined stream exited. The tubing is then extended into a container with 2x volume of PBS, which is rapidly stirred. The flow rate for the pump is at a setting of 300 rpm or 110 mL/min. Ethanol is removed and exchanged for PBS by dialysis. The lipid formulations are then concentrated using centrifugation or diafiltration to an appropriate working concentration. [1152] C57BL/6 mice (Charles River Labs, MA) receive either saline or formulated circRNA via tail vein injection. At various time points after administration, serum samples are collected by retroorbital bleed. Serum levels of Factor VII protein are determined in samples using a chromogenic assay (Biophen FVTI, Aniara Corporation, OH). To determine liver RNA levels of Factor VII, animals are sacrificed and livers are harvested and snap frozen in liquid nitrogen. Tissue lysates are prepared from the frozen tissues and liver RNA levels of Factor VII are quantified using a branched DNA assay (QuantiGene Assay, Panomics, CA). [1153] FVII activity is evaluated in FVTI siRNA-treated animals at 48 hours after intravenous (bolus) injection in C57BL/6 mice. FVII is measured using a commercially available kit for determining protein levels in serum or tissue, following the manufacturer’s instructions at a microplate scale. FVII reduction is determined against untreated control mice, and the results are expressed as % Residual FVII. Two dose levels (0.05 and 0.005 mg/kg FVII siRNA) are used in the screen of each novel liposome composition. EXAMPLE 36 [1154] circRNA formulation using preformed vesicles. [1155] Cationic lipid containing transfer vehicles are made using the preformed vesicle method. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol at a molar ratio of 40/10/40/10, respectively. The lipid mixture is added to an aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol and lipid concentration of 30% (vol/vol) and 6.1 mg/mL respectively and allowed to equilibrate at room temperature for 2 min before extrusion. The hydrated lipids are extruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22°C using a Lipex Extruder (Northern Lipids, Vancouver, BC) until a vesicle diameter of 70-90 nm, as determined by Nicomp analysis, is obtained. For cationic lipid mixtures which do not form small vesicles, hydrating the lipid mixture with a lower pH buffer (50mM citrate, pH 3) to protonate the phosphate group on the DSPC headgroup helps form stable 70-90 nm vesicles. [1156] The FVII circRNA (solubilised in a 50mM citrate, pH 4 aqueous solution containing 30% ethanol) is added to the vesicles, pre-equilibrated to 35°C, at a rate of ~5mL/min with mixing. After a final target circRNA/lipid ratio of 0.06 (wt wt) is achieved, the mixture is incubated for a further 30 min at 35°C to allow vesicle re-organization and encapsulation of the FVII RNA. The ethanol is then removed and the external buffer replaced with PBS (155mM NaCl, 3mM Na2HP04, ImM KH2P04, pH 7.5) by either dialysis or tangential flow diafiltration. The final encapsulated circRNA-to-lipid ratio is determined after removal of unencapsulated RNA using size-exclusion spin columns or ion exchange spin columns. EXAMPLE 37 [1157] Example 37A: Expression of trispecific antigen binding proteins from engineered circular RNA [1158] Circular RNAs are designed to include: (1) a 3′ post splicing group I intron fragment; (2) an Internal Ribosome Entry Site (IRES); (3) a trispecific antigen-binding protein coding region; and (4) a 3′ duplex region. The trispecific antigen-binding protein regions are constructed to produce an exemplary trispecific antigen-binding protein that will bind to a target antigen, e.g., GPC3. [1159] Example 37B: Generation of a scFv CD3 binding domain [1160] The human CD3epsilon chain canonical sequence is Uniprot Accession No. P07766. The human CD3gamma chain canonical sequence is Uniprot Accession No. P09693. The human CD3delta chain canonical sequence is Uniprot Accession No. P043234. Antibodies against CD3epsilon, CD3gamma or CD3delta are generated via known technologies such as affinity maturation. Where murine anti-CD3 antibodies are used as a starting material, humanization of murine anti-CD3 antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti-mouse antigen (HAMA) response in subjects who receive treatment of a trispecific antigen- binding protein described herein. Humanization is accomplished by grafting CDR regions from murine anti-CD3 antibody onto appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions. [1161] Human or humanized anti-CD3 antibodies are therefore used to generate scFv sequences for CD3 binding domains of a trispecific antigen-binding protein. DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. The order in which the VL and VH domains appear in the scFv is varied (i.e. VL-VH, or VH-VL orientation), and three copies of the "G4S" or "G 4 S" subunit (G 4 S) 3 connect the variable domains to create the scFv domain. Anti-CD3 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD3-expressing cells. [1162] Example 37C: Generation of a scFv Glypican-3 (GPC3) binding domain [1163] Glypican-3 (GPC3) is one of the cell surface proteins present on Hepatocellular Carcinoma but not on healthy normal liver tissue. It is frequently observed to be elevated in hepatocellular carcinoma and is associated with poor prognosis for HCC patients. It is known to activate Wnt signalling. GPC3 antibodies have been generated including MDX-1414, HN3, GC33, and YP7. [1164] A scFv binding to GPC-3 or another target antigen is generated similarly to the above method for generation of a scFv binding domain to CD3. [1165] Example 37D: Expression of trispecific antigen-binding proteins in vitro [1166] A CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968; 60(4):1275- 81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies. [1167] For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO. [1168] Recombinant CHO cell lines stably expressing secreted trispecific antigen-binding proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0.1×10 6 viable cells/mL. Cell pools stably expressing trispecific antigen-binding proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium. [1169] Trispecific antigen-binding proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use. [1170] Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS- PAGE. [1171] Example 37E: Purification of trispecific antigen-binding proteins [1172] Trispecific antigen-binding proteins are purified from CHO cell culture supernatants in a two- step procedure. The constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step. Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL Purity and homogeneity (typically >90%) of final samples are assessed by SDS PAGE under reducing and non- reducing conditions, followed by immunoblotting using an anti-(half-life extension domain) or anti idiotype antibody as well as by analytical SEC, respectively. Purified proteins are stored at aliquots at -80 °C until use. EXAMPLE 38 [1173] Expression of engineered circular RNA with a half-life extension domain has improved pharmacokinetic parameters than without a half-life extension domain [1174] The trispecific antigen-binding protein encoded on a circRNA molecule of Example 37 is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection intramuscularly. Another cynomolgus monkey group receives a comparable protein encoded on a circRNA molecule in size with binding domains to CD3 and GPC-3 but lacking a half-life extension domain. A third and fourth group receive a protein encoded on a circRNA molecule with CD3 and half-life extension domain binding domains and a protein with GPC-3 and half-life extension domains, respectively. Both proteins encoded by circRNA are comparable in size to the trispecific antigen-binding protein. Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD3 and/or GPC-3. [1175] Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi -exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae ~at +Be ~pt , where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and a and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The a-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(a-k21)/(a-p), B=D/V(p-k21)/(a-p), and a and β (for α>β) are roots of the quadratic equation: r 2 +(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2 and k21=transfer rate from compartment 2 to compartment 1, and D=the administered dose. [1176] Data analysis: Graphs of concentration versus time profiles are made using KaleidaGraph (KaleidaGraph™ V.3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, EST: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D C. [1177] It is expected that the trispecific antigen-binding protein encoded on a circRNA molecule of Example 37 has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a half-life extension domain. EXAMPLE 39 [1178] Cytotoxicity of the Trispecific Antigen-Binding Protein [1179] The trispecific antigen-binding protein encoded on a circRNA molecule of Example 37 is evaluated in vitro on its mediation of T cell dependent cytotoxicity to GPC-3+ target cells. [1180] Fluorescence labeled GPC3 target cells are incubated with isolated PBMC of random donors or T-cells as effector cells in the presence of the trispecific antigen-binding protein of Example 37. After incubation for 4 h at 37 °C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorimeter. Target cells incubated without the trispecific antigen-binding protein of Example 37 and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively. [1181] Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1-(number of living targets(sample)/number of living targets(spontaneous))] x 100%. Sigmoidal dose response curves and EC50 values are calculated by non- linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software. EXAMPLE 40 [1182] Lipid nanoparticle formulation with circular RNA [1183] Lipid Nanoparticles (LNPs) were formed using a Precision Nanosystems Ignite instrument with a ‘NextGen’ mixing chamber. Ethanol phase contained ionizable Lipid 10c-7, DSPC, Cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio was combined with an aqueous phase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNP then were dialyzed in 1L of water and exchanged 2 times over 18 hours. Dialyzed LNPs were filtered using 0.2 µm filter. Prior to in vivo dosing, LNPs were diluted in PBS. LNP sizes were determined by dynamic light scattering. A cuvette with 1 mL of 20 µg/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded. [1184] 40.1 Formulation of Lipids 10c-7 and 10c-8 [1185] Lipid Nanoparticles (LNPs) were formed using a Precision Nanosystems Ignite instrument with a ‘NextGen’ mixing chamber. Ethanol phase contained ionizable Lipid 10c-7 or Lipid 10c-28, DOPE, Cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio was combined with an aqueous phase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNPs were then dialyzed in 1L of water and exchanged 2 times over 18 hours. Dialyzed LNPs were filtered using 0.2 µm filter. Prior to in vivo dosing, LNPs were diluted in PBS. LNP sizes were determined by dynamic light scattering. A cuvette with 1 mL of 20 µg/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded. EXAMPLE 41 [1186] In Vitro Delivery of Green Fluorescent Protein (GFP) or Chimeric Antigen Receptor (CAR) [1187] Human PBMCs (Stemcell Technologies) were transfected with LNP encapsulating GFP and examined by flow cytometry. PBMCs from five different donors (PBMC A-E) were incubated in vitro with one LNP composition, containing circular RNA encoding either GFP or CD19-CAR (200 ng), at 37°C in RPMI, 2% human serum, IL-2 (10 ng/mL), and 50 uM BME. PBMCs incubated without LNP were used as a negative control. After 24, 48, or 72 hours post-LNP incubation, cells were analyzed for CD3, CD19, CD56, CD14, CD11b, CD45, fixable live dead, and payload (GFP or CD19-CAR). [1188] Representative data are presented in FIGs.27A and 27B, showing that the tested LNP is capable of delivering circular RNA into primary human immune cells resulting in protein expression. EXAMPLE 42 [1189] Multiple IRES variants can mediate expression of murine CD19 CAR in vitro [1190] Multiple circular RNA constructs, encoding anti-murine CD19 CAR, contains unique IRES sequences and were lipotransfected into 1C1C7 cell lines. Prior to lipotransfection, 1C1C7 cells are expanded for several days in complete RPMI Once the cells expanded to appropriate numbers, 1C1C7 cells were lipotransfected (Invitrogen RNAiMAX) with four different circular RNA constructs. After 24 hours, 1C1C7 cells were incubated with His-tagged recombinant murine CD19 (Sino Biological) protein, then stained with a secondary anti-His antibody. Afterwards, the cells were analyzed via flow cytometry. [1191] Representative data are presented in FIGs. 26, showing that IRES sourced from the indicated virus (apodemus agrarius picornavirus, caprine kobuvirus, parabovirus, and salivirus) are capable of driving expression of an anti-mouse CD19 CAR in murine T cells. EXAMPLE 43 [1192] Murine CD19 CAR mediates tumor cell killing in vitro [1193] Circular RNA encoding anti-mouse CD19 CAR were electroporated into murine T cells to evaluate CAR-mediated cytotoxicity. For electroporation, T cells were electroporated with circular RNA encoding anti-mouse CD19 CAR using ThermoFisher’s Neon Transfection System then rested overnight. For the cytotoxicity assay, electroporated T cells were co-cultured with Fluc+ target and non- target cells at 1:1 ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37°C. Cytotoxicity was measured using a luciferase assay system 24 hours post- co-culture (Promega Brightglo Luciferase System) to detect lysis of Fluc+ target and non-target cells. Values shown are calculated relative to the untransfected mock signal. [1194] Representative data are presented in FIG.27, showing that an anti-mouse CD19 CAR expressed from circular RNA is functional in murine T cells in vitro. EXAMPLE 44 [1195] CD19 CAR expressed from circular RNA has higher yield and greater cytotoxic effect compared to that expressed from mRNA [1196] Circular RNA encoding encoding anti-CD19 chimeric antigen antigen receptor, which includes, from N-terminus to C-terminus, a FMC63-derived scFv, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular domain, were electroporated into human peripheral T cells to evaluate surface expression and CAR-mediated cytotoxicity. For comparison, circular RNA- electroporated T cells were compared to mRNA-electroporated T cells in this experiment. For electroporation, CD3+ T cells were isolated from human PBMCs using commercially available T cell isolation kits (Miltenyi Biotec) from donor human PBMCs. After isolation, T cells were stimulated with anti-CD3/anti-CD28 (Stemcell Technologies) and expanded over 5 days at 37 ° C in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME. Five days post stimulation, T cells were electroporated with circular RNA encoding anti-human CD19 CAR using ThermoFisher’s Neon Transfection System and then rested overnight. For the cytotoxicity assay, electroporated T cells were co-cultured with Fluc+ target and non-target cells at 1:1 ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37 ° C. Cytotoxicity was measured using a luciferase assay system 24 hours post-co-culture (Promega Brightglo Luciferase System) to detect lysis of Fluc+ target and non-target cells. Furthermore, an aliquot of electroporated T cells were taken and stained for live dead fixable staining, CD3, CD45, and chimeric antigen receptors (FMC63) at the day of analysis. [1197] Representative data are presented in FIGs. 28 and 29. FIGs. 28A and 28B show that an anti- human CD19 CAR expressed from circular RNA is expressed at higher levels and longer than an anti- human CD19 CAR expressed from linear mRNA. FIGs.29A and 29B show that an anti-human CD19 CAR expressed from circular RNA is exerts a greater cytotoxic effect relative to anti-human CD19 CAR expressed from linear mRNA. EXAMPLE 45 [1198] Functional Expression of Two CARs from a Single Circular RNA [1199] Circular RNA encoding chimeric antigen receptors were electroporated into human peripheral T cells to evaluate surface expression and CAR-mediated cytotoxicity. The purpose of this study is to evaluate if circular RNA encoding for two CARs can be stochastically expressed with a 2A (P2A) or an IRES sequence. For electroporation, CD3+ T cells were commercially purchased (Cellero) and stimulated with anti-CD3/anti-CD28 (Stemcell Technologies) and expanded over 5 days at 37 ° C in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME. Four days post stimulation, T cells were electroporated with circular RNA encoding anti-human CD19 CAR, anti-human CD19 CAR-2A-anti-human BCMA CAR, and anti-human CD19 CAR-IRES-anti-human BCMA CAR using ThermoFisher’s Neon Transfection System then rested overnight. For the cytotoxicity assay, electroporated T cells were co-cultured with Fluc+ K562 cells expressing human CD19 or BCMA antigens at 1:1 ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37 ° C. Cytotoxicity was measured using a luciferase assay system 24 hours post- co-culture (Promega BrightGlo Luciferase System) to detect lysis of Fluc+ target cells. [1200] Representative data are presented in FIG. 30, showing that two CARs can be functionally expressed from the same circular RNA construct and exert cytotoxic effector function. EXAMPLE 46 [1201] Example 46A: Built-in polyA sequences and affinity-purification to produce immue-silent circular RNA [1202] PolyA sequences (20-30nt) were inserted into the 5’ and 3’ ends of the RNA construct (precursor RNA with built-in polyA sequences in the introns). Precursor RNA and introns can alternatively be polyadenylated post-transcriptionally using, e.g., E coli. polyA polymerase or yeast polyA polymerase, which requires the use of an additional enzyme. [1203] Circular RNA in this example was circularized by in vitro transcription (IVT) and affinity- purified by washing over a commercially available oligo-dT resin to selectively remove polyA-tagged sequences (including free introns and precursor RNA) from the splicing reaction. The IVT was performed with a commercial IVT kit (New England Biolabs) or a customerized IVT mix (Orna Therapeutics), containing guanosine monophosphate (GMP) and guanosine triphosphate (GTP) at different ratios (GMP:GTP = 8, 12.5, or 13.75 ). In some embodiments, GMP at a high GMP:GTP ratio may be preferentially included as the first nucleotide, yielding a majority of monophosphate-capped precursor RNAs. As a comparison, the circular RNA product was alternatively purified by the treatment with Xrn1, Rnase R, and Dnase I (enzyme purification). [1204] Immunogenicity of the circular RNAs prepared using the affinity purification or enzyme purification process were then assessed. Briefly, the prepared circular RNAs were transfected into A549 cells. After 24 hours, the cells were lysed and interferon beta-1 induction relative to mock samples was measured by qPCR. 3p-hpRNA, a triphosphorylated RNA, was used as a positive control. [1205] FIGs.31B and 31C show that the negative selection affinity purification removes non-circular products from splicing reactions when polyA sequences are included on elements that are removed during splicing and present in unspliced precursor molecules. FIG.31D shows circular RNAs prepared with tested IVT conditions and purification methods are all immunoquiescent. These results suggest the negative selection affinity purification is equivalent or superior to enzyme purification for circular RNA purification and that customized circular RNA synthesis conditions (IVT conditions) may reduce the reliance on GMP excess to achieve maximal immunoquiescence. [1206] Example 46B: Dedicated binding site and affinity-purification for circular RNA production [1207] Instead of polyA tags, one can include specifically design sequences (DBS, dedicated binding site). [1208] Instead of a polyA tag, a dedicated binding site (DBS), such as a specifically designed complementary oligonucleotide that can bind to a resin, may be used to selectively deplete precursor RNA and free introns. In this example, DBS sequences (30nt) were inserted into the 5’ and 3’ ends of the precursor RNA. RNA was transcribed and the transcribed product was washed over a custom complementary oligonucleotide linked to a resin. [1209] FIGs. 32B and 32C demonstrates that including the designed DBS sequence in elements that are removed during splicing enables the removal of unspliced precursor RNA and free intron components in a splicing reaction, via negative affinity purification. [1210] Example 46C: Production of a circular RNA encoding dystrophin [1211] A 12kb12,000nt circular RNA encoding dystrophin was produced by in vitro transcription of RNA precursors followed by enzyme purification using a mixture of Xrn1, DNase 1, and RNase R to degrade remaining linear components. FIG.33 shows that the circular RNA encoding dystrophin was successfully produced. EXAMPLE 47 [1212] 5’ spacer between 3’ intron fragment and the IRES improves circular RNA expression [1213] Expression level of purified circRNAs with different 5’ spacers between the 3’ intron fragment and the IRES in Jurkat cells were compared. Briefly, luminescence from secreted Gaussia luciferase in supernatant was measured 24 hours after electroporation of 60,000 cells with 250ng of each RNA. [1214] Additionally, stability of purified circRNAs with different 5’ spacers between the 3’ intron fragment and the IRES in Jurkat cells were compared. Briefly, luminescence from secreted Gaussia luciferase in supernatant was measured over 2 days after electroporation of 60,000 cells with 250ng of each RNA and normalized to day 1 expression. [1215] The results are shown in FIGs.34A and 34B, indicating that adding a spacer can enhance IRES function and the importance of sequence identity and length of the added spacer. A potential explanation is that the spacer is added right before the IRES and likely functions by allowing the IRES to fold in isolation from other structured elements such as the intron fragments. EXAMPLE 48 [1216] This example describes deletion scanning from 5’ or 3’ end of the caprine kobuvirus IRES. IRES borders are generally poorly characterized and require empirical analysis, and this example can be used for locating the core functional sequences required for driving translation. Briefly, circular RNA constructs were generated with truncated IRES elements operably linked to a gaussia luciferase coding sequence. The truncated IRES elements had nucleotide sequences of the indicated lengths removed from the 5’ or 3’ end. Luminescence from secreted gaussia luciferase in supernatant was measured 24 and 48 hours after electroporation of primary human T cells with RNA. Stability of expression was calculated as the ratio of the expression level at the 48-hour time point relative to that at the 24-hour time point. [1217] As shown in FIG.35, deletion of more than 40 nucleotides from the 5’ end of the IRES reduced expression and disrupted IRES function. Stability of expression was relatively unaffected by the truncation of the IRES element but expression level was substantially reduced by deletion of 141 nucleotides from the 3’ end of the IRES, whereas deletion of 57 or 122 nucleotides from the 3’ end had a positive impact on the expression level. [1218] It was also observed that deletion of the 6-nucleotide pre-start sequence reduced the expression level of the luciferase reporter. Replacement of the 6-nucleotide sequence with a classical kozak sequence (GCCACC) did not have a significant impact but at least maintained expression. EXAMPLE 49 [1219] This example describes modifications (e.g., truncations) of selected selected IRES sequences, including Caprine Kobuvirus (CKV) IRES, Parabovirus IRES, Apodemus Picornavirus (AP) IRES, Kobuvirus SZAL6 IRES, Crohivirus B (CrVB) IRES, CVB3 IRES, and SAFV IRES. The sequences of the IRES elements are provided in SEQ ID NOs: 348-389. Briefly, circular RNA constructs were generated with truncated IRES elements operably linked to a gaussia luciferase coding sequence. HepG2 cells were transfected with the circular RNAs. Luminescence in the supernatant was assessed 24 and 48 hours after transfection. Stability of expression was calculated as the ratio of the expression level at the 48-hour time point relative to that at the 24-hour time point. [1220] As shown in FIG. 36, truncations had variable effects depending on the identity of the IRES, which may depend on the initiation mechanism and protein factors used for translation, which often differs between IRESs. 5’ and 3’ deletions can be effectively combined, for example, in the context of CKV IRES. Addition of a canonical Kozak sequence in some cases significantly improved expression (as in SAFV, Full vs Full+K) or diminished expression (as in CKV, 5d40/3d122 vs 5d40/3d122+K). EXAMPLE 50 [1221] This example describes modifications of CK-739, AP-748, and PV-743 IRES sequences, including mutations at the translation initiation elements. Briefly, circular RNA constructs were generated with modified IRES elements operably linked to a gaussia luciferase coding sequence. Luminescence from secreted gaussia luciferase in supernatant was measured 24 and 48 hours after transfection of 1C1C7 cells with RNA. [1222] CUG was the most commonly found alternative start site but many others were also characterized. These triplets can be present in the IRES scanning tract prior to the start codon and can affect translation of correct polypeptides. Four alternative start site mutations were created, with the IRES sequnces provided in SEQ ID NOs: 378-380. As shown in FIG. 37, mutations of alternative translation initiation sites in the CK-739 IRES affected translation of correct polypeptides, positively in some instances and negatively in other instances. Mutation of all the alternative translation initiation sites reduced the level of translation. [1223] Alternative Kozak sequences, 6 nucleotides before start codon, can also affect expression levels. The 6-nucleotide sequence upstream of the start codon were gTcacG, aaagtc, gTcacG, gtcatg, gcaaac, and acaacc, respectively, in CK-739 IRES and Sample Nos.1-5 in the “6nt Pre-Start” group. As shown in FIG. 37, substitution of certain 6-nucleotide sequences prior to the start codon affected translation. [1224] It was also observed that 5’ and 3’ terminal deletions in AP-748 and PV-743 IRES sequences reduced expression. However, in the CK-739 IRES, which had a long scanning tract, translation was relatively unaffected by deletions in the scanning tract. EXAMPLE 51 [1225] This example describes modifications of selected IRES sequences by inserting 5’ and/or 3’ untranslated regions (UTRs) and creating IRES hybrids. Briefly, circular RNA constructs were generated with modified IRES elements operably linked to a gaussia luciferase coding sequence. Luminescence from secreted gaussia luciferase in supernatant was measured 24 and 48 hours after transfection of HepG2 cells with RNA. [1226] IRES sequences with UTRs inserted are provided in SEQ ID NOs: 390-401. As shown in FIG. 53, insertion of 5’ UTR right after the 3’ end of the IRES and before the start codon slightly increased the translation from Caprine Kobuvirus (CK) IRES but in some instances abrogated translation from Salivirus SZ1 IRES. Insertion of 3’ UTR right after the stop cassette had no impact on both IRES sequences. [1227] Hybrid CK IRES sequences are provided in SEQ ID NOs: 390-401. CK IRES was used as a base, and specific regions of the CK IRES were replaced with similar-looking structures from other IRES sequences, for example, SZ1 and AV (Aichivirus). As shown in FIG.38, certain hybrid synthetic IRES sequences were functional, indicating that hybrid IRES can be constructed using parts from distinct IRES sequences that show similar predicted structures while deleting these structures completely abrogates IRES function. EXAMPLE 52 [1228] This example describes modifications of circular RNAs by introducing stop codon or cassette variants. Briefly, circular RNA constructs were generated with IRES elements operably linked to a gaussia luciferase coding sequence followed by variable stop codon cassettes, which included a stop codon in each frame and two stop codons in the reading frame of the gaussia luciferase coding sequence. 1C1C7 cells were transfected with the circular RNAs. Luminescence in supernatant was assessed 24 and 48 hours after transfection. [1229] The sequences of the stop codon cassettes are set forth in SEQ ID NOs: 406-412. As shown in FIG. 39, certain stop codon cassettes improved expression levels, although they had little impact on expression stability. In particular, a stop cassette with two frame 1 (the reading frame of the gaussia luciferase coding sequence) stop codons, the first being TAA, followed by a frame 2 stop codon and a frame 3 stop codon, is effective for promoting functional translation. EXAMPLE 53 [1230] This example describes modifications of circular RNAs by inserting 5’ UTR variants. Briefly, circular RNA constructs were generated with IRES elements with 5’ UTR variants inserted between the 3’ end of the IRES and the start codon, the IRES being operably linked to a gaussia luciferase coding sequence. 1C1C7 cells were transfected with the circular RNAs. Luminescence in supernatant was assessed 24 and 48 hours after transfection. [1231] The sequences of the 5’ UTR variants are set forth in SEQ ID NOs: 402-405. As shown in FIG. 40, a CK IRES with a canonical Kozak sequence (UTR4) was more effective when a 36-nucleotide unstructured/low GC spacer sequence was added (UTR2), suggesting that the GC-rich Kozak sequences may interfere with core IRES folding. Using a higher-GC/structured spacer with a kozak sequence did not show the same benefit (UTR3), possibly due to interference with IRES folding by the spacer itself. Mutating the kozak sequence to gTcacG (UTR1) enhanced translation to the same level as the Kozak+spacer alternative without the need for a spacer. EXAMPLE 54 [1232] This example describes the impact of miRNA target sites in circular RNAs on expression levels. Briefly, circular RNA constructs were generated with IRES elements operably linked to a human erythropoietin (hEPO) coding sequence, where 2 tandem miR-122 target sites were inserted into the construct. miR-122-expressing Huh7 cells were transfected with the circular RNAs. hEPO expression in supernatant was assessed 24 and 48 hours after transfection by sandwich ELISA. [1233] As shown in FIG.41, the hEPO expression level was obrogated where the miR-122 target sites were inserted into the circular RNA. This result demonstrates that expression from circular RNA can be regulated by miRNA. As such, cell type- or tissue-specific expression can be achieved by incorporating target sites of the miRNAs expressed in the cell types in which expression of the recombinant protein is undesirable. EXAMPLE 55 [1234] LNP and circular RNA construct containing anti-CD19 CAR reduces B cells in the blood and spleen in vivo. [1235] Circular RNA constructs encoding an anti-CD19 CAR expression were encapsulated within lipid nanoparticles as described above. For comparison, circular RNAs encoding luciferase expression were encapsulated within separate lipid nanoparticle. [1236] C57BL/6 mice at 6 to 8 weeks old were injected with either LNP solution every other day for a total of 4 LNP injections within each mouse. 24 hours after the last LNP injection, the mice’s spleen and blood were harvested, stained, and analyzed via flow cytometry. As shown in FIG.42A and FIG. 42B, mice containing LNP-circular RNA constructs encoding an anti-CD19 CAR led to a statistically significant reduction in CD19+ B cells in the peripheral blood and spleen compared to mice treated with LNP-circular RNA encoding a luciferase. EXAMPLE 56 [1237] IRES sequences contained within circular RNA encoding CARs improves CAR expressions and cytotoxicity of T-Cells. [1238] Activated murine T-cells were electroporated with 200ng of circular RNA constructs containing a unique IRES and a murine anti-CD19 1D3ζ CAR expression sequence. The IRES contained in these constructs were derived either in whole or in part from a Caprine Kobuvirus, Apodemus Picornavirus, Parabovirus, or Salivirus. A Caprine Kobuvirus derived IRES was additionally codon optimized. As a control, a circular RNA containing a wild-type zeta mouse CAR with no IRES was used for comparison. The T-cells were stained for the CD-19 CAR 24 hours post electroporation to evaluate for surface expression and then co-cultured with A20 Fluc target cells. The assay was then evaluated for cytotoxic killing of the Fluc+ A20 cells 24 hours after co-culture of the T- cells with the target cells. [1239] As seen in FIGs.43A, 43B, 43C, and 44, the unique IRES were able to increase the frequency that the T-cells expressed the CAR protein and level of CAR expression on the surface of the cells. The increase frequency of expression of the CAR protein and level of CAR expression on the surface of cells lead to an improved anti-tumor response. EXAMPLE 57 [1240] Cytosolic and surface proteins expressed from circular RNA construct in primary human T- cells. [1241] Circular RNA construct contained either a sequence encoding for a fluorescent cytosolic reporter or a surface antigen reporter. Fluorescent reporters included green fluorescent protein, mCitrine, mWasabi, Tsapphire. Surface reporters included CD52 and Thy1.1 bio . Primary human T- cells were activated with an anti-CD3/anti-CD28 antibody and electroporated 6 days post activation of the circular RNA containing a reporter sequence. T-cells were harvested and analyzed via flow cytometry 24 hours post electroporation. Surface antigens were stained with commercially available antibodies (e.g., Biolegend, Miltenyi, and BD). [1242] As seen in FIG. 45A and FIG. 45B, cytosolic and surface proteins can be expressed from circular RNA encoding the proteins in primary human T-cells. EXAMPLE 58 [1243] Circular RNAs containing unique IRES sequences have improved translation expression over linear mRNA. [1244] Circular RNA constructs contained a unique IRES along with an expression sequence for Firefly luciferase (FLuc). [1245] Human T-cells from 2 donors were enriched and stimulated with anti-CD3/anti-CD28 antibodies. After several days of proliferation, activated T cells were harvested and electroporated with equal molar of either mRNA or circular RNA expressing FLuc payloads. Various IRES sequences, including those derived from Caprine Kobuvirus, Apodemus Picornavirus, and Parabovirus, were studied to evaluate expression level and durability of the payload expression across 7 days. Across the 7 days, the T-cells were lysed with Promega Brightglo to evaluate for bioluminsences. [1246] As shown in FIGs.46C, 46D, 46E, 46F, and 46G, the presence of an IRES within a circular RNA can increase translation and expression of a cytosolic protein by orders of magnitude and can improve expression compared to linear mRNA. This was found consistent across multiple human T- cell donors. EXAMPLE 59 [1247] Example 59A: LNP-circular RNA encoding anti-CD19 mediates human T-cell killing of K562 cells. [1248] Circular RNA constructs contained a sequence encoding for anti-CD19 antibodies. Circular RNA constructs were then encapsulated within a lipid nanoparticle (LNP). [1249] Human T-cells were stimulated with anti-CD3/anti-CD28 and left to proliferate up to 6 says. At day 6, LNP-circular RNA and ApoE3 (1µg/mL) were co-cultured with the T-cells to mediate transfection. 24 hours later, Fluc+ K562 cells were electroporated with 200ng of circular RNA encoding anti-CD19 antibodies and were later co-cultured at day 7. 48 hours post co-culture, the assay was assessed for CAR expression and cytotoxic killing of K562 cells through Fluc expression. [1250] As shown in FIG.47A and FIG.47B, there is T-cell expression of anti-CD19 CAR from the LNP-mediated delivery of a CAR in vitro to T-cells and its capability to lyse tumor cells in a specific, antigen dependent manner in engineered K562 cells. [1251] Example 59B: LNP-circular RNA encoding anti-BCMA antibody mediates human T-cell killing of K562 cells. [1252] Circular RNA constructs contained a sequence encoding for anti-BCMA antibodies. Circular RNA constructs were then encapsulated within a lipid nanoparticle (LNP). [1253] Human T-cells were stimulated with anti-CD3/anti-CD28 and left to proliferate up to 6 says. At day 6, LNP-circular RNA and ApoE3 (1µg/mL) were co-cultured with the T-cells to mediate transfection. 24 hours later, Fluc+ K562 cells were electroporated with 200ng of circular RNA encoding anti-BCMA antibodies and were later co-cultured at day 7. 48 hours post co-culture, the assay was assessed for CAR expression and cytotoxic killing of K562 cells through Fluc expression. [1254] As shown in FIG. 47B, there is T-cell expression of BCMA CAR from the LNP-mediated delivery of a CAR in vitro to T-cells and its capability to lyse tumor cells in a specific, antigen dependent manner in engineered K562 cells. EXAMPLE 60 [1255] Anti-CD19 CAR T-cells exhibit anti-tumor activity in vitro. [1256] Human T-cells were activated with anti-CD3/anti-CD28 and electroporated once with 200ng of anti-CD19 CAR-expressing circular RNA. Electroporated T-cells were co-cultured with FLuc+ Nalm6 target cells and non-target Fluc+K562 cells to evaluate CAR-mediated killing. After 24 hours post co-culture, the T-cells were lysed and examined for remanent FLuc expression by target and non- target cells to evaluate expression and stability of expression across 8 days total. [1257] As shown in FIGs. 48A and 48B, T-cells express circular RNA CAR constructs in specific, antigen-dependent manner. Results also shows improved cytotoxicity of circular RNAs encoding CARs compared to linear mRNA encoding CARs and delivery of a functional surface receptor. EXAMPLE 61 [1258] Effective LNP transfection of circular RNA mediated with ApoE3 [1259] Human T-cells were stimulated with anti-CD3/anti-CD28 and left to proliferate up to 6 days. At day 6, lipid nanoparticle (LNP) was and circular RNA expressing green fluorescence protein solution with or without ApoE3 (1µg/mL) were co-cultured with the T-cells. 24 hours later, the T-cells were stained for live/dead T-cells and the live T-cells were analyzed for GFP expression on a flow cytometer. [1260] As shown by FIGs.49A, 49B, 49C, 49D, and 49E, efficient LNP transfection can be mediated by ApoE3 on activated T-cells, followed by significant payload expression. These results were exhibited across multiple donors. EXAMPLE 62 [1261] This example illustrates expression of SARS-CoV2 spike protein expression in vitro. Circular RNA encoding SARS-CoV2 stabilized spike protein was transfected into 293 cells using MessengerMax Transfection Reagent. 24 hours after transfection, the 293 cells were stained with a CR3022 anti-spike primary antibody and APC-labeled secondary antibody. [1262] FIG. 50A shows circularization efficiency of roughly 4.5kb SARS-Cov2 stabilized spike protein-encoding RNA resulting from an in vitro transcription reaction. FIG.50B and FIG.73C show SARS-CoV2 stabilized spike protein expression on 293 cells after the circular RNA transfection with MessengerMax Transfection Reagent relative to mock transfected cells. [1263] FIG.54A and FIG.54B show SARS-CoV2 stabilized spike protein expression by percentage of cells and gMFI on 293 cells after transfection of a panel of circular RNAs, containing variable IRES sequences, codon optimized coding regions, and stabilized SARS-CoV2 spike proteins, using MessengerMax Transfection Reagent. FIG.54C shows the relationship between MFI and percentage. EXAMPLE 63 [1264] This example shows in vivo cytokine response after IV injection of 0.2mg/kg circRNA preparations encapsulated in a lipid nanoparticle formulation. circRNA splicing reactions synthesized with GTP as a precursor RNA initiator and splicing nucleotide incited greater cytokine responses than purified circRNA and linear m1ψ-mRNA due to the presence of triphosphorylated 5’ termini in the splicing reaction. Levels of IL-1β, IL-6, IL-10, IL-12p70, RANTES, TNFα were measured from blood drawn 6 hours following intravenous injection of the LNP-formulated circRNA preparation. Mice injected with PBS were used as a control. [1265] As seen in FIG. 51, circRNA splicing reactions synthesized with GTP as a precursor RNA initiator and splicing nucleotide incite greater cytokine responses than purified circRNA and linear m1ψ-mRNA due to the presence of triphosphorylated 5’ termini in the splicing reaction. EXAMPLE 64 [1266] This example illustrates intramuscular delivery of varying doses of lipid nanoparticle containing circular RNAs. Mice were dosed with either 0.1µg, 1 µg, or 10 µg of circRNA formulated in lipid nanoparticles. Whole body IVIS imagine was conducted at 6 hours following an injection of luciferin (FIG.52A and FIG.52B). Ex vivo IVIS imaging was conducted at 24-hour. Flux values for liver, quad, and calf are shown in FIG.52C. FIG.53B and FIG.53C show that the expression of the circular RNA is present in the muscle tissue of the mice. EXAMPLE 65 [1267] This example illustrates expression of multiple circular RNAs in LNP formulations. Circular RNA constructs encoding either hEPO or fLuc were dosed in a single and mixed set of LNPs. hEPO concentration in the serum (FIG. 53A) and total flux by IVIS imaging (FIG. 53B) was determined. The results show that the circular RNA hEPO or fLuc constructs individually formulated or co- formulated expressed protein efficiently. EXAMPLE 66 [1268] Example 66A: Hepatocyte plating and culture [1269] Primary human hepatocytes (PHH), primary mouse hepatocytes (PMH), primary cynomolgus monkey hepatocytes (PCH) were thawed and resuspended in hepatocyte thawing medium (Xenotech, cat# K8600/K8650) followed by centrifugation. The supernatant was discarded, and the pelleted cells were resuspended in hepatocyte plating medium (Xenotech, cat# K8200). Cells were counted via hemocytometer and plated on Bio-coat collagen-I coated 96-well plates at a density of 25,000 cells/well for PHH, 25,000 cells/well for PMH, and 50,000 cells/well for PCH in 100uL of plating media. Plated cells were allowed to settle and adhere for 6 hours in a tissue culture incubator at 37°C and 5% CO2 atmosphere. After incubation cells were checked for monolayer formation after which the plating media was aspirated and replaced with 100ul of culture media (Xenotech, cat# K8300). Media was replaced every 24 hours for the duration of the experiment. [1270] Example 66B: In vitro screening of LNP formulated circular RNA encoding firefly luciferase in primary human, mouse, and cynomolgus monkey hepatocytes [1271] A circular RNA construct comprising a TIE and a coding element encoding for firefly luciferase was produced and transfected into LNPs. Various concentrations of LNPs formulated with the circularized RNA (oRNA) were diluted in hepatocyte media supplemented with 3% fetal bovine serum (FBS) (ThermoFisher, cat# A3160401). Media was aspirated from the cells prior to addition of 100uL of LNP/FBS/media mixture to the cells. [1272] Luciferase activity was detected in primary human (FIG. 67A), mouse (FIG. 67B), and cynomolgus monkey (FIG.67C) hepatocyte. 24 hours post-transfection plates were removed from the incubator and allowed to equilibrate to room temperature for 15mins. A volume of 100µL of Firefly Luciferase one-step glow assay working solution (Pierce, cat# 16196) was added to each well. The plate was placed on a microplate shaker (ThermoFisher, cat# S72050) and mixed at 300rpm for 3min. Post-mixing, the plate was allowed to incubate at room temperature for 10min. Luminescence was read using a Varioskan or Bio-Tek Cytation5 instrument. [1273] As seen in FIG.67A, FIG.67B, and FIG.67C, TIE containing circular RNAs are capable of driving firefly luciferase protein expression in primary hepatocytes from multiple species in a dose- dependent manner when transfected in vitro with an LNP. [1274] Example 66C: In vitro screening of LNP formulated circular RNA encoding firefly luciferase in multiple primary human hepatocyte donors [1275] A circular RNA construct comprising a TIE and a coding element encoding for firefly luciferase was produced and transfected into LNPs. Various concentrations of LNPs formulated with the circularized RNA (oRNA) were diluted in hepatocyte media supplemented with 3% fetal bovine serum (FBS) (ThermoFisher, cat# A3160401). Media was aspirated from the cells prior to addition of 100ul of LNP/FBS/media mixture to the cells. [1276] Luciferase activity was detected in primary human (FIG. 68A), mouse (FIG. 68B), and cynomolgus monkey (FIG.68C) hepatocyte. 24 hours post-transfection plates were removed from the incubator and allowed to equilibrate to room temperature for 15mins. A volume of 100µL of Firefly Luciferase one-step glow assay working solution (Pierce, cat# 16196) was added to each well. The plate was placed on a microplate shaker (ThermoFisher, cat# S72050) and mixed at 300rpm for 3min. Post-mixing, the plate was allowed to incubate at room temperature for 10min. Luminescence was read using a Varioskan or Bio-Tek Cytation5 instrument. [1277] As seen in FIG.68A, FIG.68B, and FIG.68C, TIE containing circular RNAs are capable of driving firefly luciferase protein expression in primary hepatocytes from multiple human donors in a dose-dependent manner when transfected in vitro with an LNP. EXAMPLE 67 [1278] In vitro expression of LNP formulated with circular RNA encoding for GFP in multiple human cell models. [1279] A circular RNA construct was produced comprising a TIE and coding element encoding for a GFP protein. LNP were formulated with the circular RNA construct. Then various concentrations of the LNP containing the circular RNA construct were diluted in hepatocyte media supplemented with 3% fetal bovine (FBS) (ThermoFisher, cat# A3160401). Media was aspirated from the cells prior to addition of 100ul of LNP/FBS/media mixture to the cells. [1280] HeLa (human cervical adenocarcinoma; ATCC, cat# CCL-2), HEK293 (human embryonic kidney; ATCC, cat# CRL-1573), and HUH7 (human liver hepatocellular carcinoma; JCRB, cat# JCRB0403) were transfected as previously described with LNP formulated oRNAs. Twenty-four hours post-transfection, the media was removed and the cells were trypsinized. The trypsinized cells were neutralized with PBS supplemented with 10% FBS, harvested, and transferred to a tube. The tube was centrifuged to pellet the cells and the supernatant was aspirated. The pellet was stored at -80°C prior to lysis. For lysis the cells were thawed on ice and were lysed with 100μL/well RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshly added 1 mM DTT, and 250 U/mL Benzonase (EMD Millipore, cat# 71206-3), and protease inhibitor mixture consisting of complete protease inhibitor cocktail (Sigma, cat# 11697498001). Cells were kept on ice for 30 minutes at which time NaCl (1M final concentration) was added. Cell lysates were thoroughly mixed and retained on ice for 30min. The whole cell extracts (WCE) were centrifuged to pellet debris. A Bradford assay (Bio-Rad, cat# 500-0001) was used to assess protein content of the lysates. The Bradford assay procedure was completed according to the manufacturer’s protocol. Extracts were stored at −20°C prior to use. Western blots were performed to assess GFP protein levels. Whole cell extract lysates were mixed with Laemmli buffer and denatured at 95°C for 10min. Western blots were run using the NuPage system on 4-12% Bis-Tris gels (ThermoFisher, cat# NP0335BOX) according to the manufacturer’s protocol followed by wet transfer onto 0.45μm nitrocellulose membrane (ThermoFisher, cat# LC2001). After transfer membranes were rinsed thoroughly with water and stained with Ponceau S solution (Boston Bio Products, cat# ST-180) to confirm complete and even transfer. Blots were blocked using 5% Dry Milk in TBS for 30 minutes on a lab rocker at room temperature. Blots were rinsed with 1X TBST (Boston BioProducts, cat# IBB- 180) and probed with mouse dylight 680-tagged anti-GFP monoclonal antibody (ThermoFisher, cat# MA515256D680) at 1:1,000 in 1X TBST. Anti-β-actin or GAPDH was used as a loading control (ThermoFisher, cat# AM4302/AM4300) at 1:4,000 in 1X TBST and incubated simultaneously with the GFP primary antibody. Blots were sealed in a bag and kept overnight at 4°C on a lab rocker. After incubation, blots were rinsed 3 times for 5 minutes each in 1X TBST and probed with mouse secondary antibodies (ThermoFisher, cat# PI35519) at 1:25,000 each in 1X TBST for 30 minutes at room temperature. After incubation, blots were rinsed 3 times for 5 minutes each in 1X TBST. Blots were visualized and analyzed using a Licor Odyssey system. [1281] As shown in FIG. 69, TIE-containing circular RNA is capable of expressing GFP protein in diverse human cell lines (e.g., HeLa, HEK293, and HUH7 cells) in a dose dependent manner when transfected in vitro with an LNP. EXAMPLE 68 [1282] In vitro expression of LNP formulated with circular RNA encoding for GFP in primary human hepatocytes. [1283] A circular RNA construct was produced comprising a TIE and coding element encoding for a GFP protein. Various concentrations of LNP containing circularized RNA (oRNA) were diluted in hepatocyte media supplemented with 3% fetal bovine serum (FBS) (ThermoFisher, cat# A3160401). Media was aspirated from the cells prior to addition of 100µL of LNP/FBS/media mixture to the cells. [1284] Primary human hepatocytes (PHH) were thawed and resuspended in hepatocyte thawing medium (Xenotech, cat# K8600/K8650) followed by centrifugation. The supernatant was discarded, and the pelleted cells were resuspended in hepatocyte plating medium (Xenotech, cat# K8200). Cells were counted via hemocytometer and plated on Bio-coat collagen-I coated 96-well plates at a density of 25,000 cells/well for PHH, 25,000 cells/well for PMH, and 50,000 cells/well for PCH in 100ul of plating media. Plated cells were allowed to settle and adhere for 6 houra in a tissue culture incubator at 37°C and 5% CO 2 atmosphere. After incubation cells were checked for monolayer formation after which the plating media was aspirated and replaced with 100ul of culture media (Xenotech, cat# K8300). Media was replaced every 24 hours for the duration of the experiment. [1285] Primary human hepatocytes were transfected as previously described with LNP formulated oRNAs. Twenty-four hours post-transfection, the media was removed and the cells were trypsinized. The trypsinized cells were neutralized with PBS supplemented with 10% FBS, harvested, and transferred to a tube. The tube was centrifuged to pellet the cells and the supernatant was aspirated. The pellet was stored at -80°C prior to lysis. For lysis the cells were thawed on ice and were lysed with 100μL/well RIPA buffer (Boston Bio Products, Cat. BP-115) plus freshly added 1 mM DTT, and 250 U/ml Benzonase (EMD Millipore, cat# 71206-3), and protease inhibitor mixture consisting of complete protease inhibitor cocktail (Sigma, cat# 11697498001). Cells were kept on ice for 30 minutes at which time NaCl (1M final concentration) was added. Cell lysates were thoroughly mixed and retained on ice for 30min. The whole cell extracts (WCE) were centrifuged to pellet debris. A Bradford assay (Bio- Rad, cat# 500-0001) was used to assess protein content of the lysates. The Bradford assay procedure was completed according to the manufacturer’s protocol. Extracts were stored at −20°C prior to use. Western blots were performed to assess GFP protein levels. Whole cell extract lysates were mixed with Laemmli buffer and denatured at 95°C for 10min. Western blots were run using the NuPage system on 4-12% Bis-Tris gels (ThermoFisher, cat# NP0335BOX) according to the manufacturer’s protocol followed by wet transfer onto 0.45μm nitrocellulose membrane (ThermoFisher, cat# LC2001). After transfer membranes were rinsed thoroughly with water and stained with Ponceau S solution (Boston Bio Products, cat# ST-180) to confirm complete and even transfer. Blots were blocked using 5% Dry Milk in TBS for 30 minutes on a lab rocker at room temperature. Blots were rinsed with 1X TBST (Boston BioProducts, cat# IBB-180) and probed with mouse dylight 680-tagged anti-GFP monoclonal antibody (ThermoFisher, cat# MA515256D680) at 1:1,000 in 1X TBST. Anti-β-actin or GAPDH was used as a loading control (ThermoFisher, cat# AM4302/AM4300) at 1:4,000 in 1X TBST and incubated simultaneously with the GFP primary antibody. Blots were sealed in a bag and kept overnight at 4°C on a lab rocker. After incubation, blots were rinsed 3 times for 5 minutes each in 1X TBST and probed with mouse secondary antibodies (ThermoFisher, cat# PI35519) at 1:25,000 each in 1X TBST for 30 minutes at room temperature. After incubation, blots were rinsed 3 times for 5 minutes each in 1X TBST. Blots were visualized and analyzed using a Licor Odyssey system. [1286] As shown in the western blot in FIG. 70, circular RNAs containing a TIE is capable of successfully encoding a GFP protein in primary human hepatocytes when transfected in vivo with an LNP. EXAMPLE 69 [1287] In vitro expression of firefly luciferase in circular RNA encoding firefly luciferase in mouse myoblast and primary human skeletal muscle myoblast cells using lipofectamine. [1288] A circular RNA construct comprising a TIE and coding element encoding firefly luciferase protein. [1289] Primary human skeletal muscle (HSkM) cells (Lonza, cat# 20TL356514) were thawed in a 37°C water bath and plated at recommended seeding density (3,000 to 5,000 per cm 2 ) in SkGM-2 BulletKit growth media (Lonza, cat# CC-3245) and allowed to grow overnight. Cells were detached using ReagentPack subculture reagents (Lonza, cat# CC-5034) and plated on tissue culture grade 96- well plates at recommended seeding density and allowed to grow overnight in a tissue culture incubator at 37°C and 5% CO2 atmosphere or to 70-80% confluency with growth media changed every 2 days. [1290] For one 96-well plate reaction, 0.3µL of Lipofectamine-3000 transfection reagent (Lipo3K) (ThermoFisher, cat# L3000015) was mixed with 5 µL Opti-MEM reduced serum media (ThermoFisher, cat# 51985091). In a separate tube, per reaction, firefly luciferase (f.luc) oRNA (at 10-200ng) was combined with 5 µL Opti-MEM and 0.2 µL P3000TM enhancer reagent (ThermoFisher, cat# L3000015). Equal volumes of Lipo3K/Opti-MEM mix was combined with oRNA/Opti-MEM mix and incubated at room temperature for 15min. The Lipo3K/oRNA mixture was added to each well to be transfected and placed in a tissue culture incubator at 37°C and 5% CO 2 atmosphere for 24 hours. [1291] After 24 hours, the transfection plates were removed from the incubator and allowed to equilibrate to room temperature for 15 minutes. A volume of 100 µL of firefly luciferase one-step glow assay working solution (Pierce, cat# 16196) was added to each well. The plate was placed on a microplate shaker (ThermoFisher, cat# S72050) and mixed at 300rpm for 3 minutes. Post-mixing, the plate was allowed to incubate at room temperature for 10 minutes. Luminescence was read using a Varioskan or Bio-Tek Cytation5 instrument. [1292] As shown in FIG.71A and FIG.71B, circular RNAs comprising a TIE is capable of driving firefly luciferase protein expression in myoblasts from different species in a dose-dependent manner when transfected in vitro with lipofectamine. EXAMPLE 70 [1293] In vitro expression of firefly luciferase in circular RNA encoding firefly luciferase in differentiated primary human skeletal muscles myotubes [1294] A circular RNA construct comprising a TIE and coding element encoding firefly luciferase protein. [1295] Primary human skeletal muscle (HSkM) cells (Lonza, cat# 20TL356514) were thawed in a 37°C water bath and plated at recommended seeding density (3,000 to 5,000 per cm 2 ) in SkGM-2 BulletKit growth media (Lonza, cat# CC-3245) and allowed to grow overnight. Cells were detached using ReagentPack subculture reagents (Lonza, cat# CC-5034) and plated on tissue culture grade 96- well plates at recommended seeding density and allowed to grow overnight in a tissue culture incubator at 37°C and 5% CO2 atmosphere or to 70-80% confluency with growth media changed every 2 days. Once cells reached 70-80% confluency, growth media was removed, cells were washed twice in 1X PBS (Gibco, cat# 10010023) and changed to differentiation media consisting of F-10 (1X) (Gibco, cat# 11550-043) supplemented with 2% Horse Serum (Gibco, cat# 26050088) and 1% Pen-Strep (Gibco, cat# 15140-122). Media was changed daily for 5 to 6 days until nearly all myoblasts had fused to form myotubes. [1296] For one 96-well plate reaction, 0.3 µL of Lipofectamine-3000 transfection reagent (Lipo3K) (ThermoFisher, cat# L3000015) was mixed with 5ul Opti-MEM reduced serum media (ThermoFisher, cat# 51985091). In a separate tube, per reaction, firefly luciferase (f.luc) oRNA (at 10-200ng) was combined with 5µL Opti-MEM and 0.2 µL P3000TM enhancer reagent (ThermoFisher, cat# L3000015). Equal volumes of Lipo3K/Opti-MEM mix was combined with oRNA/Opti-MEM mix and incubated at room temperature for 15min. The Lipo3K/oRNA mixture was added to each well to be transfected and placed in a tissue culture incubator at 37°C and 5% CO 2 atmosphere for 24 hours. [1297] After 24 hours, transfection plates were removed from the incubator and allowed to equilibrate to room temperature for 15 minutes. A volume of 100 µL of Firefly Luc one-step glow assay working solution (Pierce, cat# 16196) was added to each well. The plate was placed on a microplate shaker (ThermoFisher, cat# S72050) and mixed at 300rpm for 3minutes. Post-mixing, the plate was allowed to incubate at room temperature for 10 minutes. Luminescence was read using a Varioskan or Bio-Tek Cytation5 instrument. [1298] As shown in FIG.72A and FIG 72B, a circular RNA comprising a TIE is capable of driving firefly luciferase protein expression in primary muscles cells through differentiated states (e.g., in myoblast and differentiated myotubes) in multiple human donors in a dose-dependent manner when transfected in vitro with lipofectamine. EXAMPLE 71 [1299] Cell-free in vitro translation of circular RNAs containing TIEs [1300] A cell-free rabbit reticulocyte in vitro translation assay (Promega, cat# L4540) was completed to characterize protein products from various RNA templates. Both linear mRNA and circular oRNA templates were used in the assay and reaction components were assembled according to the manufacturer’s protocol. Prior to assay, RNA templates were denatured at 65°C for 3 minutes and immediately cooled on ice. All reaction components were assembled on ice. Flexi Rabbit Reticulocyte kit components, complete amino acid mixture (Promega, cat# L5061), RNAsin TIEuclease inhibitor (Promega, cat# N2111), and transcend tRNA (Promega, cat# L5061) were added to denatured RNA templates. The reaction was vortexed to mix and incubated at 30°C for 60 minutes and then placed on ice. [1301] The reaction mixture was added to 1x sample buffer (ThermoFisher, cat# NP0007) and heated at 70°C for 15 minutes. The denatured protein sample was loaded onto 4-12% Bis-Tris gels (ThermoFisher, cat# NPO335BOX). Gel electrophoresis was completed, and the gel was wet transferred onto 0.45 μm nitrocellulose membrane (ThermoFisher, cat# LC2001). Post-transfer, the membrane was blocked for 1 hour with rocking in freshly made TBS with 0.5% Tween-20 (Boston Bioproducts Inc Cat # IBB-180). The membrane was incubated for 45min with rocking with streptavidin-AP (Promega, cat# V5591) at a 1:2,500 dilution. The membrane was rinsed with four cycles of: twice with TBST and twice with deionized water for 1min per rinse. The membrane was incubated with Western Blue substrate (Promega, cat# S3841) for 45 minutes and the membrane was rinsed in water and scanned on a LICOR Odyssey CLx imaging system. [1302] As shown in FIG.73A and FIG.73B, a circular RNA comprising a TIE is capable of driving protein expression in a cell-free lysate, independent of any cell type. FIG.73A illustrates expression of firefly luciferase from a linear or circular RNA input. FIG.73B illustrates expression of human and mouse ATP7B proteins with different codon optimization (co) approaches compared to wild-type native sequence (WT). The codon optimized-circular RNAs expressing ARP7B protein and the circular RNA expressing firefly luciferase shoed protein full-length protein expression. EXAMPLE 72 [1303] TIE selection methodology [1304] Putative TIEs were identified for activity assessment from sequences in GenBank. Briefly, Riboviria and Unclassified Virus sequences greater than 1kb in length were identified.5’ and intergenic UTRs were extracted based on putative CDS start and end sites with a minimum length cutoff of 250nt. Reverse sequences were also collected for negative sense CDS annotations. For genuses not expected to contain TIE sequences, a few noncoding regions per genus were selected at random. Duplicates, >10nt repeat-containing sequences, sequences with both XbaI and BamHI sites, and low-quality sequences (non acgt) were culled, then sequences were clustered through CDHit with an 80% sequence similarity cutoff for clustering; representative sequences from each cluster were selected for further study. For unclassified sequences or sequences expected to contain a TIE, all 5’ and intergenic UTRs were selected; duplicates, >10nt repeat-containing sequences, sequences with both XbaI and BamHI sites, and low-quality sequences (non acgt) were culled, then sequences were clustered through CDHit with a 95% (low risk, known IRESs) sequence similarity cutoff for clustering; representative sequences from each cluster were selected for further study. For both strategies, sequences shorter than 300nt or unable to be synthesized due to sequence complexity were eliminated. EXAMPLE 73 [1305] Example 73A: TIE activity in primary human T cells [1306] Nucleic acid sequences containing putative TIEs were inserted into a circular RNA (oRNA) construct prior to the start codon of a gaussia luciferase reporter sequence. oRNA containing the TIE was synthesized and purified. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNA was transfected into T cells in vitro. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine- containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours indicates higher oRNA stability due to TIE function. [1307] Example 73B: TIE activity in primary human hepatocytes [1308] Nucleic acid sequences containing putative TIEs were inserted into a circular RNA (oRNA) construct prior to the start codon of a gaussia luciferase reporter sequence. oRNA containing the TIE was synthesized and purified. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNA was transfected into hepatocytes in vitro. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine- containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours indicates higher oRNA stability due to TIE function. [1309] Example 73C: TIE activity in primary human myotubes [1310] Nucleic acid sequences containing putative TIEs were inserted into a circular RNA (oRNA) construct prior to the start codon of a gaussia luciferase reporter sequence. oRNA containing the TIE was synthesized and purified. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNA was transfected into human myotubes in vitro. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine- containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours indicates higher oRNA stability due to TIE function. EXAMPLE 74 [1311] TIE tissue tropism [1312] Select TIE-containing oRNAs were formulated into LNPs. LNP-oRNAs were transfected into T cells, hepatocytes, and myotubes. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine- containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours may indicate higher oRNA stability due to TIE function. TIE activity was compared between cell types and differences resulting from TIE tissue preference were noted. Differences may be a result of the TIE engaging proteins that show tissue- specific expression and promoting enhanced translation initiation, degradation, or stability. EXAMPLE 75 [1313] Example 75A: TIE deletion scanning [1314] Select TIE sequences with progressive deletions from either the 5’ end or 3’ end of the TIE were inserted into a circular RNA (oRNA) construct prior to the start codon of a gaussia luciferase reporter sequence. oRNA containing the TIE variant was synthesized and purified. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNA was transfected into human primary T cells. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours may indicate higher oRNA stability due to TIE function. Expression or stability impairment due to progressive deletions identifies the core functional unit of the TIE. [1315] Example 75B: TIE variant generation and identification [1316] Select TIE-containing oRNA synthesis plasmids were subjected to error-prone PCR to introduce random mutations into the PCR product. PCR product was used as a template for oRNA synthesis. Purified oRNA was formulated into LNPs and transfected into primary human T cells. Polysome fractions were harvested from T cells at 6, 24, 48, and 72 hours post-transfection by HPLC. RNA associated with each polysome fraction was extracted from polysome fractions and sequenced by NGS. TIE mutation enrichment in each polysome fraction at each time point was analyzed to identify mutations that 1) maintain or improve translation activity from the TIE and/or 2) improve stability of the oRNA. [1317] Example 75C: TIE single and multi-variant validation [1318] Nucleic acid sequences containing putative beneficial TIE mutations from example 6 alone or in combination were inserted into a circular RNA (oRNA) construct prior to the start codon of a gaussia luciferase reporter sequence. oRNA containing the TIE variant was synthesized and purified. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNA was transfected into human primary T cells. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours indicates higher oRNA stability due to TIE function. EXAMPLE 76 [1319] Example 76A: Selection of eukaryotic TIEs [1320] Selection of eukaryotic TIEs. Putative eukaryotic TIEs were identified using several databases. TIEs selected include sequences 40-1578 nucleotides in length and may or may not contain identified modification (m6A) sites. [1321] Example 76B: TIEs containing modified nucleotides (m6A) [1322] Nucleic acid sequences containing putative TIEs were inserted into a circular RNA (oRNA) construct preceding the coding region of a gaussia luciferase reporter sequence. oRNAs were synthesized with a titration of modified nucleotide. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNAs were transfected into T cells, hepatocytes, and myotubes. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence of modified nucleotide containing TIEs at 24 hours indicates necessity for modification for enhanced function. Higher luminescence at 48 hours relative to 24 hours indicates modified nucleotide containing TIEs enhance stability of oRNA. EXAMPLE 77 [1323] Expression of TIEs in cells undergoing oxidative and/or hypoxic stress [1324] Nucleic acid sequences containing putative TIEs were inserted into a circular RNA (oRNA) construct preceding the coding region of a gaussia luciferase reporter sequence. Purified oRNA was formulated into lipid nanoparticles. Hepatocytes were treated with hydrogen peroxide to induce oxidative stress or CoCl2 to induce hypoxic stress. LNP-oRNA was transfected into hepatocytes (under hypoxic stress, oxidative stress, or untreated) in vitro. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours may indicate higher oRNA stability due to TIE function. EXAMPLE 78 [1325] Aptamer as a TIE [1326] Nucleic acid sequences containing aptamers against translation initiation factors (ie eIF4E, eIF4G, eIF4a) were inserted into a circular RNA (oRNA) construct preceding the coding region of a gaussia luciferase reporter sequence. Purified oRNA was formulated into lipid nanoparticles. LNP- oRNAs were transfected into hepatocytes. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine- containing detection reagent and luminometer. Higher luminescence at 24 hours indicates higher TIE function. Higher luminescence at 48 hours relative to 24 hours may indicate higher oRNA stability due to TIE function. EXAMPLE 79 [1327] Tandem TIEs [1328] Select combinations of viral, eukaryotic, and/or aptamer TIEs were inserted into a circular RNA (oRNA) construct preceding the coding region of a gaussia luciferase reporter sequence. oRNAs were synthesized with a titration of modified nucleotide. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNAs were transfected into T cells, hepatocytes, and myotubes. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence of constructs containing multiple TIEs at 24 hours indicates a synergy of TIEs. Higher luminescence at 48 hours relative to 24 hours may indicate having multiple TIEs in one construct enhance stability of oRNA. EXAMPLE 80 [1329] Example 80A: Coding aptamers to enhance cap-independent translation [1330] Certain aptamers that bind to eIF4E, eIF4a, and other translation initiators are known to inhibit translation by forcing the proteins to adopt a non-functional conformation. Nucleic acid sequences containing aptamers against translation initiation factors (ie eIF4E, eIF4a) were inserted into a circular RNA (oRNA) construct preceding a functional TIE and the coding region of a gaussia luciferase reporter sequence. Purified oRNA was formulated into lipid nanoparticles. LNP-oRNAs were transfected into hepatocytes. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence at 24 hours indicates a preference for cap- independent translation. Higher luminescence at 48 hours relative to 24 hours indicates higher oRNA stability due to inhibition of cap-dependent translation. [1331] Example 80B: Coding aptamers to enhance cap-independent translation [1332] Cotransfection of oRNA and aptamers. Certain aptamers that bind to eIF4E, eIF4a, and other translation initiators are known to inhibit translation by forcing the proteins to adopt a non-functional conformation. Nucleic acid sequences containing aptamers against translation initiation factors (ie eIF4E, eIF4a) were co-transfected with a circular RNA (oRNA) containing a TIE and the coding region of a gaussia luciferase reporter. Purified aptamer and oRNA were formulated into lipid nanoparticles together. LNP-oRNAs were transfected into hepatocytes. Supernatant was harvested and replaced 24 and 48 hours after transfection, and gaussia luciferase expression from oRNA was determined using a coelenterazine-containing detection reagent and luminometer. Higher luminescence at 24 hours indicates a preference for cap-independent translation. Higher luminescence at 48 hours relative to 24 hours may indicate higher oRNA stability due to inhibition of cap-dependent translation. EXAMPLE 81 [1333] Example 81.1 Synthesis of heptadecan-9-yl 8-((3-hydroxypropyl)(2- hydroxytetradecyl)amino)octanoate (Lipid 10e-1)
[1334] Example 81.1.1 Synthesis of heptadecan-9-yl 8-bromooctanoate (3) [1335] To a mixture of 8-bromooctanoic acid 2 (10 g, 44.82 mmol) and heptadecan-9-ol 1 (9.6 g, 37.35 mmol) in CH2Cl2 (300 mL) was added DMAP (900 mg, 7.48 mmol), DIPEA (26 mL, 149.7 mmol) and EDC (10.7 g, 56.03 mmol). The reaction was stirred at room temperature overnight. After concentration of the reaction mixture, the crude residue was dissolved in ethyl acetate (300 mL), washed with 1N HCl, sat. NaHCO 3 , water and Brine. The organic layer was dried over anhydrous Na 2 SO 4 . The solvent was evaporated, and the crude residue was purified by flash chromatography (SiO 2 : Hexane = 100% to 30% of EtOAc in Hexane) and colorless oil product 3 was obtained (5 g, 29%). [1336] 1 H NMR (300 MHz, CDCl 3 ): δ ppm 4.86 (m, 1H), 3.39 (t, J = 7.0 Hz, 2H), 2.27 (t, J = 7.6 Hz, 2H), 1.84 (m, 2H), 1.62 (m, 2H), 1.5-1.4 (m, 8H), 1.35-1.2 (m, 26H), 0.87 (t, J = 6.7 Hz, 6H). [1337] Example 81.1.2 Synthesis of heptadecan-9-yl 8-((3-hydroxypropyl)amino)octanoate (5) [1338] A solution of 1-octylnonyl 8-bromooctanoate 3 (7.4 g, 16.03 mmol) in EtOH (200 mL) was added 3-amino-1-propanol 4 (24.4 mL, 320 mmol) and the reaction solution was heated at 70 °C overnight. MS showed the expected product: [APCI]: [MH] + 456.4. After concentration of the reaction mixture, the crude residue was dissolved in methyl tert-butyl ether (500 mL), washed with sat. NaHCO3, water and Brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue was purified by flash chromatography (SiO2: CH2Cl2 = 100% to 10% of MeOH in CH2Cl2 with 1% NH4OH) and colorless oil product 5 was obtained (6.6 g, 88%). [1339] 1 H NMR (300 MHz, CDCl3): δ ppm 4.84 (m, 1H), 3.80 (t, J = 5.5 Hz, 2H), 2.87 (t, J = 5.76 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.27 (t, J = 7.6 Hz, 2H), 1.68 (m, 2H), 1.62 (m, 2H), 1.5-1.4 (m, 5H), 1.35-1.2 (m, 32H), 0.87 (t, J = 6.7 Hz, 6H). MS (APCI+): 456.4 (M+1). [1340] Example 81.1.3 Synthesis of heptadecan-9-yl 8-((3-hydroxypropyl)(2- hydroxytetradecyl)amino)octanoate (7) [1341] A mixture of compound 5 (6.6 g, 14.5 mmol) and 1,2-epoxytetradecane (3.68 g, 17.4 mmol) in isopropanol (150 mL) was heated to reflux for overnight. MS showed the expected product: [APCI]: [MH] + 668.6. The reaction mixture was concentrated, and crude product was purified flash chromatography (SiO2: CH2Cl2 = 100% to 10% of MeOH in CH2Cl2 with 1% NH4OH) to obtained Lipid 10e-1 as colorless oil (6.34 g, 65%). [1342] 1 H NMR (300 MHz, CDCl3): δ ppm 4.85 (m, 1H), 3.76 (t, J = 5.49 Hz, 2H), 3.68 (m, 1H), 2.75 (m, 1H), 2.59 (m, 2H), 2.38 (m, 3H), 2.27 (m, 2H), 1.58-1.68 (m, 2H), 1.48 (m, 6H),1.24 (m, 56H), 0.87 (m, 9H). MS (APCI+): 668.6 (M+1). [1343] Example 81.2 Synthesis of Di(undecan-3-yl) 8,8’-((3-hydroxypropyl)azanediyl)bis(7- hydroxyoctanoate) (10e-7) [1344] Example 81.2.1 Synthesis of undecan-3-yl oct-7-enoate (3) [1345] To a mixture of oct-7-enoic acid 2 (10 g, 70.3 mmol) and undecan-3-ol 1 (10 g, 58.6 mmol) in CH2Cl2 (300 mL) was added DMAP (1.4 g, 11.6 mmol), DIPEA (40 mL, 232 mmol) and EDC (16.9 g, 87.9 mmol). The reaction was stirred at room temperature overnight. After concentration of the reaction mixture, the crude residue was dissolved in tert-butylmethyl ether (500 mL), washed with 1N HCl, sat. NaHCO 3 , water and Brine. The organic layer was dried over anhydrous Na 2 SO 4 . The solvent was evaporated and the crude residue was purified by flash chromatography (SiO2: Hexane = 100% to 20% of EtOAc in Hexane) and colorless oil product 3 was obtained (17.2 g, 98%). [1346] 1 H NMR (300 MHz, CDCl 3 ): δ ppm 5.88-5.72 (m, 1H), 5.02-4.91 (m, 1H), 4.80 (m, 1H), 2.28 (t, J = 7.4 Hz, 2H), 2.05-2.03 (m, 2H), 1.62-1.49 (m, 6H), 1.37-1.25 (m, 16H), 0.87 (t, J = 7.4 Hz, 6H). [1347] Example 81.2.2 Synthesis of undecan-3-yl 6-(oxiran-2-yl)hexanoate (4) [1348] To a mixture of undecan-3-yl oct-7-enoate 3 (17.2 g, 58.1 mmol) in CH 2 Cl 2 (300 mL) was added meta-chloroperoxybenzoic acid (mCPBA, <77%) (19.5 g, 87 mmol) in one portion at 0 ºC ice- water bath. The reaction was stirred at room temperature overnight. The white precipitate (meta-benzoic acid) was filtered and the filtrate was diluted with CH2Cl2 (200 mL), washed with 10% Na2S2O3, sat. NaHCO 3 , water and Brine. The organic layer was dried over anhydrous Na 2 SO 4 . The solvent was evaporated and the crude residue was purified by flash chromatography (SiO 2 : Hexane = 100% to 30% of EtOAc in Hexane) and colorless oil product 3 was obtained (17.1 g, 97%). [1349] 1 H NMR (300 MHz, CDCl3): δ ppm 4.80 (m, 1H), 2.89-2.86 (m, 1H), 3.39 (t, J = 7.0 Hz, 2H), 2.74 (t, J = 4.7 Hz, 1H), 2.47 (dd, J = 4.9, 2.2 Hz, 1H), 2.28 (t, J = 7.4 Hz, 1H), 1.74-1.46 (m, 10H), 1.35-1.2 (m, 13H) 0.87 (m, 6H). [1350] Example 81.2.3 Synthesis of Di(undecan-3-yl) 8,8’-((3-hydroxypropyl)azanediyl)bis(7- hydroxyoctanoate) (10e-7) [1351] A solution of undecan-3-yl 6-(oxiran-2-yl)hexanoate 4 (8 g, 25.6 mmol) in isopropanol (50 mL) was added 3-amino-1-propanol (769.1 mg, 10.2 mmol) and the reaction solution was heated at 90 °C overnight. MS showed the expected product: [APCI]: [MH] + 700.6. After concentration of the reaction mixture, the crude residue was purified by flash chromatography (SiO2: CH2Cl2 = 100% to 10% of MeOH in CH 2 Cl 2 ) and colorless oil product was obtained (5.1 g, 71%). [1352] 1 H NMR (300 MHz, CDCl3): δ ppm 4.81 (m, 2H), 3.80 (m, 2H), 3.73 (m, 2H), 2.78 (m, 2H), 2.52-2.43 (m, 4H), 2.28 (t, J = 7.3 Hz, 2H), 1.68-1.48 (m, 15H), 1.35-1.17 (m, 37H), 0.88-0.83 (m, 12H). MS (APCI + ): 700.6 (M+1). [1353] Example 81.3 Synthesis of Nonyl 8-((8-((7-ethyl-2-methylundecan-4-yl)oxy)-8- oxooctyl)(3-hydroxypropyl) amino)-7-hydroxyoctanoate (10e-177)
O EDCI, DMAP D iPEA, DCM O m-CPBA OH + HO O DCM 1 2 3 H 2 N OH O O OH O 5 H N OH O O 4 IPA, reflux 6 O EDCI, DMAP O DiPEA, DCM + OH Br Br HO O 7 8 9 O OH H O N OH IPA, reflux O + Br O 6 9 O OH O N OH O O Lipid 10e-177 [1354] Example 81.3.1: Synthesis of nonyl oct-7-enoate 3 [1355] To a mixture of 7-octenoic acid 2 (9.2 g, 64.7 mmol) and 1-nonanol 1 (9.3 g, 64.7 mmol) in CH 2 Cl 2 (500 mL) was added DMAP (1.58 g, 13 mmol), DIPEA (22.5 mL, 129.4 mmol), and EDC (18.6 g, 97 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was washed with Brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated. The crude residue was purified by flash chromatography (SiO2: Hexane to 30% of EtOAc in Hexane) and a colorless oil product, nonyl oct-7-enoate 3 was obtained (15.3 g, 88%). [1356] 1 H NMR (300 MHz, CDCl3): δ ppm 5.9-5.7 (m, 1H), 5.05-4.9 (m, 2H), 4.07 (t, J = 6.3 Hz. 2H), 2.38-2.24 (m, 2), 2.14-2.02 (m, 2H), 1.7-1.2 (m, 20H), 0.87 (t, J = 7.4 Hz, 3H). [1357] Example 81.3.2: Synthesis of nonyl 6-(oxiran-2-yl) hexanoate 4 [1358] To a solution of nonyl oct-7-enoate 3 (15.3 g, 57 mmol) in CH 2 Cl 2 (300 mL) was added meta-chloroperoxybenzoic acid (mCPBA, <77%) (16.6 g, 74 mmol) in one portion at 0 ºC (ice-water bath). The reaction was stirred at room temperature overnight. Na2S2O3 (1.2M, 600 mL), sat. NaHCO3 (600 mL), and CH2Cl2 (600 mL) were added to the reaction mixture. The organic phase was separated and was washed with Brine (300 mL). The organic layer was dried over anhydrous Na 2 SO 4 . The solvent was evaporated, and the crude residue was purified by flash chromatography (SiO 2 : Hexane to 30% of EtOAc in Hexane) and a colorless oil product, nonyl 6-(oxiran-2-yl) hexanoate 4 was obtained (15.1 g, 93%). [1359] 1 H NMR (300 MHz, CDCl3): δ ppm 4.07 (t, J = 6.6 Hz.2H), 2.96-2.86 (m, 1H), 2.74 (t, J = 4.1 Hz, 1H), 2.46 (dd, J = 5.0, 2.3 Hz, 1H), 2.36-2.24 (m, 2H), 1.75-1.1 (m, 20 H), 0.87 (t, J = 7.4 Hz, 3H). [1360] Example 81.3.3: Synthesis of nonyl 7-hydroxy-8-((3-hydroxypropyl)amino)octanoate 6 [1361] A solution of 3-aminopropanol 5 (5.4 mL, 71 mmol) and nonyl 6-(oxiran-2-yl) hexanoate 4 (20 g, 70 mmol) in isopropanol (250 mL) was heated to reflux overnight. The reaction mixture was concentrated, the crude product was purified flash chromatography (SiO2: CH2Cl2 to 10% of MeOH in CH 2 Cl 2 with 1% NH 4 OH), and colorless oil product, nonyl 7-hydroxy-8-((3- hydroxypropyl)amino)octanoate 6 (8.7 g, 34%) was obtained. [1362] 1 H NMR (400 MHz, CDCl3): δ ppm 4,03 (t, J = 6.8 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.86 (t, J = 5.6 Hz, 2H), 2.57 (t, J = 7.2, 2H), 2.26 (t, J = 7.2, 2H), 1.8-1.1 (m, 26H), 0.88 (t, J = 6.4 Hz, 3H). MS (APCI+): 360.3 (M+1). [1363] Example 81.3.4: Synthesis of 7-ethyl-2-methylundecan-4-yl 8-bromooctanoate 9 [1364] To a mixture of 8-bromooctanoic acid 8 (5.8 g, 26 mmol) and 7-ethyl-2-methylundecan-4- ol 7 (5.4 g, 25 mmol) in CH 2 Cl 2 (250 mL) was added DMAP (0.6 g, 5 mmol), DIPEA (8.6 mL, 50 mmol) and EDC (7.5 g, 39 mmol). The reaction was stirred at room temperature for two days. The reaction mixture was diluted with CH2Cl2 (250 mL) and washed with brine. The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, and the crude residue was purified by flash chromatography (SiO 2 : Hexane = 100% to 30% of EtOAc in Hexane) and colorless oil product, 7-ethyl- 2-methylundecan-4-yl 8-bromooctanoate 9 was obtained (6.34 g, 60%, mixture of Br and Cl analogs). [1365] 1 H NMR (400 MHz, CDCl 3 ): δ ppm 4.95-4.90 (m, 1H), 3.51 (t, J = 6.4 Hz, 1.1H), 3.39 (t, J = 6.4 Hz, 0.9H), 2.27 (t, J = 7.6 Hz, 2H), 1.9-1.1 (m, 26H), 1.0-0.7 (m, 12H). [1366] Example 81.3.5: Synthesis of Nonyl 8-((8-((7-ethyl-2-methylundecan-4-yl)oxy)-8- oxooctyl)(3-hydroxypropyl) amino)-7-hydroxyoctanoate (10e-177) [1367] A mixture of nonyl 7-hydroxy-8-((3-hydroxypropyl)amino)octanoate 6 (3.0 g, 8.34 mmol), 7-ethyl-2-methylundecan-4-yl 8-bromooctanoate 9 (6.34 g, 13.4 mmol), and KI (0.9 g, 5.4 mmol) in isopropanol (100 mL) was heated to reflux for three days. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (SiO2: CH2Cl2 to 10% of MeOH in CH2Cl2 with 1% NH 4 OH). Lipid 10e-177 (2.44 g, 42%) was obtained as a colorless oil. [1368] 1 H NMR (400 MHz, CDCl3): δ ppm 4.95-4.9 (m, 1H), 4.03 (t, J = 6.8 Hz, 2H), 3.75 (t, J = 5.6 Hz, 2H), 3.7-3.55 (m,1H), 2.8-2.65 (m, 1H), 2.6-2.45 (m, 2H), 2.4-2.2 (m, 7H), 1.9-1.1 (m, 50H), 0.95-0.7 (m, 15H). MS (APCI+): 698.6 (M+1). [1369] Example 81.4: Synthesis of heptadecan-9-yl 8-((3-(decyloxy)-3-oxopropyl)(3- hydroxypropyl)amino)7-hydroxyoctanoate (10e-175)
[1370] Example 81.4.1: Synthesis of decyl acrylate 3 [1371] To an ice cooled solution of decan-1-ol 1 (5 g, 31.6 mmol) in THF (80 mL, + 1 crystal of p-methoxyphenol) was added triethylamine (8.8 mL, 63.2 mmol), followed by dropwise addition of acryloyl chloride 14 (3.8 mL, 47.4 mmol) in THF (20 mL). The resulting mixture was stirred at room temperature overnight. Water (200 mL) was added and the mixture was extracted with DCM (2 x 200 mL). The organic extracts were washed with brine (100 mL), were dried (Na2SO4), and the solvent was evaporated. The crude residue was purified by flash chromatography (SiO2: hexanes to 30% of ethyl acetate in hexanes) and colorless oil product, decyl acrylate 3 (4.5 g, 67%) was obtained. [1372] 1 H NMR (300 MHz, CDCl 3 ): δ ppm 6.37 (dd, J = 17.3, 1.6 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.80(dd, J = 10.4, 1.6 Hz, 1H), 4.14 (t, J = 6.6 Hz, 2H), 1.8-1.0 (m, 16H), 0.87 (t, J = 6.6 Hz, 3H). [1373] Example 81.4.2: Synthesis of heptadecane-9-yl oct-7-enoate 6 [1374] To a mixture of 7-octenoic acid 5 (25 g, 1175.8 mmol) and heptadecane-9-ol 4 (37.6 g, 146.5 mmol) in CH2Cl2 (500 mL) was added DMAP (3.6 g, 29.3 mmol), DIPEA (102 mL, 586.4 mmol) and EDC (42.2 g, 219.8 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was washed with brine and the organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, the crude residue was purified by flash chromatography (SiO 2 : Hexane to 30% of EtOAc in Hexane) and a colorless oil product, heptadecane-9-yl oct-7-enoate 6 was obtained (34 g, 87%). [1375] 1 H NMR (300 MHz, CDCl3): δ ppm 5.9-5.7 (m, 1H), 5.05-4.75 (m, 3H), 2.28 (t, J = 7.4 Hz, 2H), 2.15-2.0 (m, 2H), 1.7-1.2 (m, 34H), 0.87 (t, J = 7.4 Hz, 6H). [1376] Example 81.4.3: Synthesis of heptadecane-9-yl 6-(oxiran-2-yl) hexanoate 7 [1377] To a solution of heptadecane-9-yl oct-7-enoate 6 (22.8 g, 60 mmol) in CH2Cl2 (300 mL) was added meta-chloroperoxybenzoic acid (mCPBA, <77%) (17.5 g, 78 mmol) in one portion at 0 ºC (ice-water bath). The reaction was stirred at room temperature overnight. Na 2 S 2 O 3 (1.2M, 600 mL), sat. NaHCO 3 (600 mL), and CH 2 Cl 2 (600 mL) were added to the reaction mixture. The organic phase was separated and was washed with Brine (300 mL). The organic layer was dried over anhydrous Na2SO4. The solvent was evaporated, the crude residue was purified by flash chromatography (SiO2: Hexane to 30% of EtOAc in Hexane), and a colorless oil product, heptadecane-9-yl 6-(oxiran-2-yl) hexanoate 7 was obtained (22.1 g, 93%). [1378] 1 H NMR (300 MHz, CDCl3): δ ppm 4.95-4.75 (m, 1H), 2.96-2.86 (m, 1H), 2.74 (t, J = 4.1 Hz, 1H), 2.46 (dd, J = 5.0, 2.3 Hz, 1H), 2.28 (t, J = 7.4 Hz, 2H), 1.75-1.1 (m, 34H), 0.87 (t, J = 7.4 Hz, 6H). [1379] Example 81.4.4: Synthesis of heptadecane-9-yl 7-hydroxy-8-((3-hydroxypropyl)amino) octanoate 9 [1380] A solution of 3-aminopropanol 8 (15.7 g, 209 mmol) and heptadecane-9-yl 6-(oxiran-2-yl) hexanoate 7 (16.6 g, 41.9 mmol) in isopropanol (200 mL) was heated to reflux for four days. The reaction mixture was concentrated, the crude product was purified flash chromatography (SiO 2 : CH 2 Cl 2 to 10% of MeOH in CH 2 Cl 2 with 1% NH 4 OH), and colorless oil product, heptadecane-9-yl 7-hydroxy- 8-((3-hydroxypropyl)amino) octanoate 9 (14.3 g, 73%) was obtained. [1381] 1 H NMR (300 MHz, CDCl 3 ): δ ppm 4.95-4.75 (m, 1H), 3.8 (t, J = 5.2 Hz, 2H), 3.75-3.55 (m, 1H) 2.85 (t, J = 5.8 Hz, 2H), 2.69 (dd, J = 12.0, 3.0Hz, 1H), 2.48 (dd, J = 12.0, 8.8 Hz, 1H), 2.5- 2.8 (bb, 3H), 2.28 (t, J = 7.6 Hz, 2H), 1.8-1.1 (m, 38H), 0.87 (t, J = 6.6 Hz, 6H). MS (APCI+): 472.4 (M+1). [1382] Example 81.4.5: Synthesis of heptadecan-9-yl 8-((3-(decyloxy)-3-oxopropyl)(3- hydroxypropyl)amino)7-hydroxyoctanoate (10e-175) [1383] A mixture of heptadecane-9-yl 7-hydroxy-8-((3-hydroxypropyl)amino) octanoate 9 (2.83 g, 6 mmol), decyl acrylate 3 (1.4 g, 6.6 mmol), one crystal of p-methoxy phenol, and one crystal of boric acid was heated in an oil bath (100-110 °C) overnight. The reaction mixture was purified flash chromatography (SiO 2 : hexanes to 100% ether) and colorless oil Lipid 10e-175 (2.4 g, 59%) was obtained. [1384] 1 H NMR (300 MHz, CDCl 3 ): δ ppm 4.95-4.75 (m, 1H), 4.08 (t, J = 6.6 Hz, 2H), 3.8-3.6 (m, 3H), 3.0-2.85 (m, 1H), 2.8-2.2 (m, 9H), 1.8-1.1 (m, 56H), 0.8 (t, J = 6.6 Hz, 9H). MS (APCI+): 684.6 (M+1). EXAMPLE 82 [1385] Lipid Nanoparticle Formulation Procedure [1386] A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) was used to determine the particle size, the polydispersity index (PDI), and zeta potential of the nanoparticle compositions in 1×PBS in determining particle size and 15 mM PBS in determining zeta potential. A cuvette with 1 mL of 20 µg/mL LNPs in PBS (pH 7.4) was measured for Z-average using the Malvern Panalytical Zetasizer Pro. The Z-average and polydispersity index were recorded. LNP sizes were determined by dynamic light scattering. [1387] Ultraviolet-visible spectroscopy can be used to determine the concentration of circRNA in nanoparticle compositions. 100 μL of the diluted formulation in 1×PBS is added to 900 μL of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA). The concentration of circRNA in the nanoparticle composition can be calculated based on the extinction coefficient of the circRNA used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm. [1388] For the transfer vehicle’s pK a , a TNS assay was conducted. 5 µL of 60 µg/mL 2-(p- toluidino) naphthalene-6-sulfonic acid (TNS) and 5 µL of 30 µg of RNA/mL lipid nanoparticles were added in to wells with HEPES buffer ranging from pH 2 – 12. The mixture was then shaken at room temperature for 5 minutes, and read for fluorescence (excitation 322 nm, emission 431 nm) using a plate reader. The inflection point of the fluorescence signal was calculated to determine the particle’s pK a . [1389] For transfer vehicle compositions including RNA, a QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluate the encapsulation of RNA by the transfer vehicle composition. Nanoparticle solutions were diluted in tris-ethylenediaminetetraacetic acid (TE) buffer at a theoretical oRNA concentration of 2 µg/mL. Standard oRNA solutions diluted in TE buffer were made ranging from 2 µg/mL to 0.125 µg/mL. The particles and standards were plated in a black 96-well plate with both TE buffer and 4% Triton-X separately (Triton-X was used as a surfactant to lyse the nanoparticles). After an incubation (37 ℃ at 350 rpm for 15 minutes), Quant- iT™ RiboGreen™ RNA reagent was added to all wells and a second incubation was performed (37 ℃ at 350 rpm for 3 minutes). Fluorescence was measured using a SPECTRAmax® GEMINI XS microplate spectrofluorometer (Molecular Devices Corporation Sunnyvale, CA). The concentration of oRNA in each particle solution was calculated using the standard curve. The encapsulation efficiency was calculated from the ratio of oRNA detected between lysed and unlysed particles. EXAMPLE 83 [1390] Expression of mOX40L in splenic immune cells. [1391] Lipid nanoparticles comprising Lipid 1 of Table 10e and Lipid 15 of Table 10f were formulated with circular RNA encoding for mOX40L at an ionizable lipid to phosphate ratio (IL:P) of 5.7. The ionizable lipid: helper lipid: cholesterol: PEG-lipid molar ratio of these LNPs was 50:10:38.5:1.5. Dialysis of the LNPs were performed using PBS. C57BL/6 female mice (6-8 weeks, n=4) were dosed at either LNP comprising Lipid 1 of Table 10e or the Lipid 15 of Table 10f at 1 mg/kg intravenously. At 24 hours, the spleen from the mice were collected for flow cytometry analysis. mOX40L transfection of Lipid 1 of Table 10e and Lipid 15 of Table 10f were compared in T cells, myeloid cells, B cells, and NK cells. [1392] As shown in FIG.74, Lipid 1 of Table 10e resulted in comparable or higher levels of mOX40L transfection in splenic immune cells compared to those comprising Lipid 15 of Table 10f. Formulation Ionizable Helper PEG-Lipid Dialysis Z-Average PDI RNA Encapsulation Lipid Lipid (nm) Efficiency (%) 10e-1 (5.7A) Table 10e, DSPC DMG- 1X PBS 79 0.03 95 Lipid 1 PEG(2000) 10f-15 (5.7A) Table 10f, DSPC DMG- 1X PBS 69 0.09 98 Lipid 15 PEG(2000) EXAMPLE 84 [1393] Fluorescent expression of circular RNA encompassed within LNP formulations compared to linear RNA encompassed within LNP formulations. [1394] LNPs were made to either contain circular RNAs encoding for firefly luciferase or linear RNAs (mRNA) encoding for firefly luciferase. The LNPs containing linear RNAs were also modified with 5- methoxyuridine (5-moU). These LNPs were formulated to contain Lipid 1 or Lipid 7 of Table 10e, wherein the ionizable lipid: helper lipid: cholesterol: PEG-lipid molar ratio of these LNPs was 50:10:38.5:1.5. Dialysis of the LNP was performed using 1X PBS. Size of the LNP construct, polydispersion index (PDI), and RNA entrapment was determined. [1395] As seen in the table below, encapsulation efficiency of circular RNA in each of the LNP formulations was greater than that of linear RNA in the same formulations. Formulation Ionizable Helper PEG- RNA Z- PDI RNA Lipid Lipid Lipid Average Encapsulation (nm) Efficiency (%) 10e-1 (5.7A) Table 10e, DSPC DMG- FLuc oRNA 82 0.05 95 Lipid 1 PEG(2000) FLuc mRNA 5MoU 89 0.05 90 10e-7 (5.7A) Table 10e, DSPC DMG- oRNA 86 0.02 97 Lipid 7 PEG(2000) FLuc mRNA 5MoU 86 0.04 94 EXAMPLE 85 [1396] Formulated LNPs undergoing dialysis using either PBS or TSS. [1397] LNPs were formulated with circular RNA at a ionizable lipid to phosphate ratio (IL:P) of 5.7 and a ionizble lipid:helper lipid: cholesterol: PEG-lipid ratio of 50:10:38.5:1.5. These LNPs then underwent dialysis using either 1X PBS or 1X TSS. Both formulated LNPs had greater than 90% encapsulation efficiency ratio of the circular RNAs. Formulation Ionizable Helper PEG- Dialysis Z-Average PDI RNA Encapsulation Lipid Lipid Lipid (nm) Efficiency (%) 10e-1 (5.7A) Table 10e, DSPC DMG- 1X PBS 87 0.06 94 Lipid 1 PEG(2000) 1X TSS 68 0.04 92 EXAMPLE 86 [1398] Mouse splenic protein expression post-treatment of LNP-circular RNAs encoding for firefly luciferase with varying Β-hydroxyl groups in the ionizable lipid [1399] C57BL/6 mice (female, 6-8 weeks, n = 4 per group) were injected intravenously with 0.5 mg/kg circular RNA encoding for firefly luciferase encapsulated in LNPs or PBS control. The LNPs were formulated with different ionizable lipids (Table 10e, Lipid 85, 86, 89, 90, 155, 124, 130, 129, 132, 126, 151, 147, 135, 175, 172, 170, 171, 173, 169, 166, or 165 or Table 10f Lipid 22) and formulated as described in the table below. After 6 hr, mice were injected intraperitoneally with D-luciferin (200 μL at 15 mg/mL). After 15 minutes, mice were euthanized and their spleens were collected. Whole tissue luminescence was measured ex vivo using an IVIS Spectrum In Vivo Imaging system (PerkinElmer) and total flux was quantified using Living Image® software (PerkinElmer). [1400] FIG.75A illustrates splenic expression of firefly luciferase delivered via LNPs formulated with various ionizable lipids from Table 22 post intravenous administration. Splenic expression was measured based on total luciferase flux (p/s) from ex vivo IVIS analysis. As shown in FIG.75A, the LNP-circular RNA constructs were able to express firefly luciferase in the spleen. [1401] As shown in FIG. 75B, increasing luciferase expression in the spleen was correlated with increasing numbers of Β-hydroxyl groups present in the ionizable lipid component of the LNP. Table 22 Formulation Ionizable Helper PEG-Lipid Ionizable lipid Z- PDI RNA Lipid Lipid : Helper lipid: Average Encapsulation Cholesterol : (nm) Efficiency (%) PEG-lipid (mol %) 10e-85 (5.7A) Table DSPC DMG- 50 : 10 : 38.5 : 81 0.08 91 10e, Lipid PEG(2000) 1.5 85 10e-89 (5.7A) Table DSPC DMG- 50 : 10 : 38.5 : 82 0.04 92 10e, Lipid PEG(2000) 1.5 89 10f-22 (5.7A) Table 10f, DSPC DMG- 50 : 10 : 38.5 : 67 0.18 88 (H) Lipid 22 PEG(2000) 1.5 10e-86 (5.7A) Table DSPC DMG- 50 : 10 : 38.5 : 79 0.1 92 (T) 10e, Lipid PEG(2000) 1.5 86 10e-90 (5.7A) Table DSPC DMG- 50 : 10 : 38.5 : 86 0.04 92 10e, Lipid PEG(2000) 1.5 90 10e-155 Table DSPC DMG-PEG 50 : 10 : 38.5 : 72 0.06 93 (5.7A) 10e, Lipid (2000) 1.5 (E) 155 10e-124 Table DSPC DMG-PEG 50 : 10 : 38.5 : 67 0.08 92 (5.7A) 10e, Lipid (2000) 1.5 (J) 124 10e-130 Table DSPC DMG-PEG 50 : 10 : 38.5 : 72 0.04 92 (5.7A) 10e, Lipid (2000) 1.5 130 (K) 10e-129 Table DSPC DMG-PEG 50 : 10 : 38.5 : 74 0.07 92 (5.7A) 10e, Lipid (2000) 1.5 (M) 129 10e-132 Table DSPC DMG-PEG 50 : 10 : 38.5 : 70 0.06 92 (5.7A) 10e, Lipid (2000) 1.5 (N) 132 10e-126 Table DSPC DMG-PEG 50 : 10 : 38.5 : 71 0.07 92 (5.7A) 10e, Lipid (2000) 1.5 (P) 126 10e-151 Table DSPC DMG-PEG 50 : 10 : 38.5 : 72 0.1 86 (5.7A) 10e, Lipid (2000) 1.5 (Q) 151 10e-147 Table DSPC DMG-PEG 50 : 10 : 38.5 : 72 0.11 90 (5.7A) 10e, Lipid (2000) 1.5 (R) 147 10e-135 Table DSPC DMG-PEG 50 : 10 : 38.5 : 72 0.11 91 (5.7A) 10e, Lipid (2000) 1.5 (W) 135 10e-175 Table DSPC DMG-PEG 50 : 10 : 38.5 : 67 0.18 88 (5.7A) 10e, Lipid (2000) 1.5 (A) 175 10e-172 Table DSPC DMG-PEG 50 : 10 : 38.5 : 107.7 0.026 83 (5.7A) 10e, Lipid (2000) 1.5 (B) 172 10e-170 Table DSPC DMG-PEG 50 : 10 : 38.5 : 60 0.07 89 (5.7A) 10e, Lipid (2000) 1.5 (D) 170 10e-171 Table DSPC DMG-PEG 50 : 10 : 38.5 : 97.74 0.069 86 (5.7A) 10e, Lipid (2000) 1.5 (F) 171 10e-173 Table DSPC DMG-PEG 50 : 10 : 38.5 : 79 0.03 88 (5.7A) 10e, Lipid (2000) 1.5 (G) 173 10e-169 Table DSPC DMG-PEG 50 : 10 : 38.5 : 70 0.04 89 (5.7A) 10e, Lipid (2000) 1.5 (I) 169 10e-166 Table DSPC DMG-PEG 50 : 10 : 38.5 : 66.57 0.08 87 (5.7A) 10e, Lipid (2000) 1.5 (O) 166 10e-165 Table DSPC DMG-PEG 50 : 10 : 38.5 : 70.9 0.07 87 (5.7A) 10e, Lipid (2000) 1.5 (V) 165 10e-168 Table DSPC DMG-PEG 50 : 10 : 38.5 : 74.1 0.06 88 (5.7A) 10e, Lipid (2000) 1.5 (X) 168 10e-167 Table DSPC DMG-PEG 50 : 10 : 38.5 : 72.2 0.05 87 (5.7A) 10e, Lipid (2000) 1.5 (Y) 167 EXAMPLE 87 [1402] Mouse whole splenic protein expression post-treatment of LNP-circular RNAs encoding for firefly luciferase and comprising ionizable lipids from Table 10e [1403] C57BL/6 mice (female, 6-8 weeks, n = 4 per group) were injected intravenously with 0.5 mg/kg circular RNA encoding for firefly luciferase encapsulated in LNPs or PBS control. The LNPs were formulated with different ionizable lipids from Table 10e Lipid 1, Lipid 85, Lipid 38, Lipid 34, Lipid 45, Lipid 86, Lipid 88, Lipid 89, Lipid 90). LNPs were formulated with circular RNA at a ionizable lipid to phosphate ratio (IL:P) of 5.7 and a ionizble lipid:helper lipid: cholesterol: PEG-lipid ratio of 50:10:38.5:1.5. After 6 hr, mice were injected intraperitoneally with D-luciferin (200 μL at 15 mg/mL). After 15 minutes, mice were euthanized and their spleens were collected. Whole tissue luminescence was measured ex vivo using an IVIS Spectrum In Vivo Imaging system (PerkinElmer) and total flux was quantified using Living Image® software (PerkinElmer). [1404] As seen in FIG.76 the LNP-circular RNAs were able to express firefly luciferase in spleen of the mice post intravenous administration of the construct. EXAMPLE 88 [1405] Expression of mOX40L in splenic T cells. [1406] Lipid nanoparticles comprising ionizable lipids from Table 10e (Lipid 1, 16, 85, 34, 45, 86, 88, 89, 90) or PBS (negative control) formulated with circular RNA encoding for mOX40L at an ionizable lipid to phosphate ratio (IL:P) of 5.7. The ionizable lipid: helper lipid: cholesterol: PEG-lipid molar ratio of these LNPs was 50:10:38.5:1.5. C57BL/6 female mice (6-8 weeks, n=4) were dosed at at 1 mg/kg intravenously. At 24 hours, the spleen from the mice were collected for flow cytometry analysis, tested for weight loss and measured for serum alanine aminotransferase (ALT) after blood collection. mOX40L expression was measured in splenic T cells. [1407] As shown in FIG. 77, Lipid 1 of Table 10e resulted in expression of mOX40L in splenic T cells. No substantial adverse effects were measured pertaining to weight loss or ALT. Table 23 Formulation Ionizable Helper PEG-Lipid Ionizable Z- PDI RNA Lipid Lipid lipid : Average Encapsulation Helper lipid: (nm) Efficiency (%) Cholesterol : PEG-lipid (mol %) 10e-1 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 73 0.05 94 Lipid 1 PEG(2000) : 1.5 10e-16 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 76 0.10 95 Lipid 16 PEG(2000) : 1.5 10e-85 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 74 0.04 95 Lipid 85 PEG(2000) : 1.5 10e-34 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 72 0.03 97 Lipid 34 PEG(2000) : 1.5 10e-45 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 75 0.04 98 Lipid 45 PEG(2000) : 1.5 10e-86 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 79 0.05 94 Lipid 86 PEG(2000) : 1.5 10e-88 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 67 0.01 95 Lipid 88 PEG(2000) : 1.5 10e-89 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 73 0.01 95 Lipid 89 PEG(2000) : 1.5 10e-90 (5.7A) Table 10e, DSPC DMG- 50 : 10 : 38.5 87 0.02 94 Lipid 90 PEG(2000) : 1.5 EXAMPLE 89 [1408] Level of B Cell Depletion post treatment of LNP-circular RNAs encoding for aCD19-CAR [1409] C57BL/6 mice (female, 6-8 weeks, n = 5 per group) were injected intravenously with 1 mg/kg circular RNA encoding for an aCD19-CAR encapsulated in LNPs or control circular RNA encoding for mWasabi encapsulated in LNPs on Days 0, 2, 5, and 7. The LNPs were formed with different ionizable lipids (Table 10e, Lipid 1, 16, 85, 45, 86, 90, 124, 129, 147, 151, 130, or 135). LNPs were formulated with circular RNA at an ionizable lipid to phosphate ratio (IL:P) of 5.7 and an ionizable lipid:helper lipid: cholesterol: PEG-lipid ratio of 50:10:38.5:1.5. On day 8, Cardiac punctures were performed to collect blood, and blood was stained, fixed, and lysed with BD FACS Lysis Solution per the manufacturer’s protocol. To assess the frequency of B cells in the blood, single cell suspensions were stained for dead cells (LiveDead Near IR, Invitrogen) and stained with anti-mouse antibodies (CD45, 30-F11, or BUV563 [blood], BD; CD3, 17A2, APC, Biolegend; B220, RA3-6B2, PE, Biolegend; CD11b, M1/70, BV421, Biolegend) at 1:200. Flow cytometry was performed using a BD FACSSymphony flow cytometer. B Cell depletion was defined by the percentage of B220+ B cells of live, CD45+ immune cells. [1410] Resulting blood B cell aplasia is illustrated in FIG.78A and FIG.78B. As shown in FIG.78A and FIG.78B, the LNP-circular RNA constructs were able to express an aCD19-CAR. The dotted line on the figures indicates Wasabi control B cell aplasia. Percentage B cell was normalized to the Wasabi control. [1411] FIG. 78C illustrates mWasabi equivalent construct comprising the same ionizable lipid. oWasabi on FIG. 78C refers to the data associated with the circular RNA encoding mWasabi. omuCD191-CAR refs to the data associate with a circular RNA encoding an antiCD19-CAR. Table 24 Formulation Ionizable Helper PEG- Ionizable RNA Z- PDI RNA Lipid Lipid Lipid lipid: Average Encapsulation Helper (nm) Efficiency (%) lipid: Cholesterol: PEG-lipid (mol %) 10e-86 Table DSPC DMG- 50 : 10 : oWasabi 73 0.1 93 (5.7A) 10e, Lipid PEG 38.5 : 1.5 86 (2000) omuCD19- 75 0.09 92 CAR 10e-16 Table DSPC DMG- 50 : 10 : oWasabi 76 0.06 95 (5.7A) 10e, Lipid PEG 38.5 : 1.5 16 (2000) omuCD19- 68 0.12 95 CAR 10e-147 Table DSPC DMG- 50 : 10 : oWasabi 79 0.09 92 (5.7A) 10e, Lipid PEG 38.5 : 1.5 147 (2000) omuCD19- 67 0.11 91 CAR 10e-151 Table DSPC DMG- 50 : 10 : oWasabi 74 0.11 89 (5.7A) 10e, Lipid PEG 38.5 : 1.5 151 (2000) omuCD19- 66 0.13 85 CAR 10e-175 Table DSPC DMG- 50 : 10 : oWasabi 69 0.05 91 (5.7A) 10e, Lipid PEG 38.5 : 1.5 175 (2000) omuCD19- 66 0.07 91 CAR 10e-176 Table DSPC DMG- 50 : 10 : oWasabi 69 0.10 93 (5.7A) 10e, Lipid PEG 38.5 : 1.5 176 (2000) omuCD19- 63 0.05 93 CAR 10e-155 Table DSPC DMG- 50 : 10 : oWasabi 70 0.08 92 (5.7A) 10e, Lipid PEG 38.5 : 1.5 155 (2000) omuCD19- 85 0.21 95 CAR EXAMPLE 90 [1412] Tumor growth kinetics post administration of LNP-oRNA construct in a Nalm6 model [1413] NSG mice were engrafted with Nalm6-luciferase tumor cells and 3 days later were engrafted with human PBMCs. Starting the following day, the mice were treated 4 times every other day with vehicle (PBS) or anti-CD19 LNP-oCAR compounds at a dose of 2 mg/kg. LNPs were formulated with circular RNA at a ionizable lipid to phosphate ratio (IL:P) of 5.7 and a ionizble lipid:helper lipid: cholesterol: PEG-lipid ratio of 50:10:38.5:1.5. Animals were then whole-body imaged via IVIS to monitor luciferase expression from Nalm6 cells. Nalm6 tumor burden is plotted as total flux of luciferase expression at each imaging timepoint. [1414] As shown in FIG. 79, all three anti-CD19 oCAR LNPs each comprising of a different ionizable lipid were capable of slowing tumor growth in this Nalm6 model. EXAMPLE 91 [1415] Lipid Expression Across Various Organs Post in vitro Administration of circular RNA-LNP Constructs [1416] Circular RNAs were designed to encode firefly luciferase and diluted in 10mM sodium acetate buffer to reach a final mass of 800 µg. Lipid nanoparticles (LNPs) were formed from dissolving lipids 175, 177, and 178 from Table 10e in an ethanol solution with a molar ratio of 10% DSPC / 38.5% cholesterol / 50% ionizable lipid / 1.5% DMG-PEG-2000. The circular RNAs were formulated into the lipid nanoparticles at 800 µg/mL using a commercially available LNP mixer (e.g., NanoAssembler Ignite System).^ The solutions were loaded into a syringe and formulated on the NanoAssembler Ignite system at a 3:1 ethanol:aqueous phase ratio to achieve a final ionizable lipid : RNA (N : P) ratio of 5.4. The resulting nanoprecipitate was loaded into 3 mL 20kDa dialysis cassettes and dialyzed in 3L of 1X PBS overnight at 4 °C. Sizing was confirmed via DLS post-dialysis and RNA concentration was determined using a Ribogreen assay according to manufacturer instructions (Table 25). LNP-circular RNA constructs were diluted to 50 µg/mL in 1X PBS. LNP-circular RNA constructs were then dosed at 200 µL via intravenous tail vein injection into female, 6-8 week old C57BL6 mice. Six hours later, the mice were administered with 200 µL of 15mg/mL D-luciferin via intraperitoneal injection. Fifteen minutes later, mice were euthanized via CO2 asphyxiation then cervical dislocation. Organs were harvested and arranged on black paper and then imaged on auto-exposure with an In Vivo Imaging System. Total flux values for each organ were analyzed using Living Image software as shown in FIGs. 80 and 81. Table 25 Formulation^ Helper PEG- Ionizable RNA RNA EE% Z-Avg. PDI Lipid^^ Lipid^ lipid: (ug/mL) Helper lipid: Cholesterol: PEG-lipid (mol %)^ 10e-175 DSPC^ DMG- 50 : 10 : Firefly 391 88 67 0.18 PEG 38.5 : 1.5^ luciferase (2000)^ 10e-177 DSPC^ DMG- 50 : 10 : Firefly 230 91 75 0.09 PEG 38.5 : 1.5^ luciferase (2000)^ 10e-178 DSPC^ DMG- 50 : 10 : Firefly 254 92 85 0.07 PEG 38.5 : 1.5^ luciferase (2000)^ INCORPORATION BY REFERENCE [1417] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated by reference herein, including, for example, U.S. provisional patent application nos. 63/250,932; 63/277,055; 63/382,816; and 63/492,971 and International patent application nos. PCT/US2022/045408 and PCT/US2022/049313.