PEPTIDE NUCLEIC ACIDS COMPRISING 2- AMINOPYRIDINE

AND RELATED METHODS OF USE THEREOF

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63/241,802, filed September 8, 2021, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Peptide nucleic acid (PNA) oligomers are polymeric nucleic acid mimics that can bind to nucleic acids with high affinity and sequence specificity. These compounds have been used in a number of applications, including those that entail binding to specific nucleic acid sequences. In order to design PNA oligomers with customizable binding properties, it would be helpful to have an expanded set of PNA monomers to access for incorporation into the PNA oligomer sequence.

SUMMARY

[0003] The present disclosure features peptide nucleic acids (PNA) oligomers comprising at least one 2-aminopyridine nucleobase, as well as compositions and related methods and kits. PNA oligomers are polymeric nucleic acid mimics that can bind to a target sequence in the genome with high affinity to form a stable PNA/DNA/PNA triplex. The presence of PNA/DNA/PNA triplexes in the genome has been shown to recruit a cell’s endogenous DNA repair systems, resulting in target-specific binding when a template DNA is provided (Bahai et al. (2016) Nat. Commun., 7: 13304; Ricciardi et al., Nat. Commun. (2018) 9:248). The present disclosure additionally provides the surprising and unexpected result that incorporation of 2- aminopyridine into certain PNA oligomers (e.g., tail-clamp PNA oligomers, or gammasubstituted tail-clamp PNA oligomers) provides improved target sequence binding (e.g., DNA binding) relative to analogous PNA oligomers that do not contain a 2-aminopyridine nucleobase. [0004] In one aspect, the present disclosure features a PNA oligomer, e.g., a PNA clamp oligomer, e.g., a tail-clamp PNA oligomer (tcPNA), comprising a first region linked to a second region via a linker or a covalent bond, wherein:: a) the first region comprises a plurality of PNA subunits having Hoogsteen complementarity with a target sequence, wherein the first region comprises a monomer comprising an 2-aminopyridine nucleobase; and b) the second region comprises a subregion, wherein the subregion consists of a plurality of PNA subunits having complete Watson Crick complementarity with the target sequence; wherein the PNA oligomer comprises a gamma modified PNA subunit. In some embodiments, the PNA oligomer further comprises: c) a first positively charged region linked to the first region by a second linker or covalent bond, wherein the first positively charged region comprises a positively charged amino acid subunit, e.g., a lysine subunit; or d) a second positively charged region linked to the second region via a third linker or covalent bond, wherein the second positively charged region comprises a positively charged amino acid subunit, e.g., a lysine subunit. In some embodiments, the PNA oligomer is a PNA tail-clamp oligomer (tcPNA).

[0005] In some embodiments, the PNA oligomer comprises a PNA subunit of Formula (I-a): salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof, wherein P ¹ , P ⁵ , L, X, R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ^9a , R ^9b and subvariables thereof are described herein. In some embodiments, the PNA oligomer comprises a PNA subunit having the structure of any one of Formulas (I-b), (I-c), (I-d), (I-e), (I- f), (I-g), (I-h), or a salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof.

[0006] In another aspect, the present disclosure features preparations of PNA oligomers comprising at least one 2-aminopyridine nucleobase. In another aspect, the present disclosure features compositions of PNA oligomers comprising at least one 2-aminopyridine nucleobase, and optionally an excipient. In some embodiments, the PNA oligomer comprises a PNA subunit having the structure of Formula (I-a) (e.g., any one of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I- f), (I-g), (I-h), or a salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof). The preparations and compositions may be useful for treating and/or preventing a disease in a subject.

[0007] In another aspect, the present disclosure features a kit comprising a PNA oligomer, e.g., as described herein. In an embodiment, the kit further comprises a load component, e.g., a nucleic acid. In an embodiment, the PNA oligomer is disposed in a first container. In an embodiment, the load component (e.g., nucleic acid) is disposed in a second container. In an embodiment, the PNA oligomer and the load component (e.g., nucleic acid) are disposed in the same container. In some embodiments, the PNA oligomer comprises a PNA subunit having the structure of Formula (I-a) (e.g., any one of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I- h), or a salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof). [0008] In another aspect, the present disclosure features a method of altering the structure of a target nucleic acid, the method comprising administering to the subject a peptide nucleic acid (PNA) oligomer described herein. In some embodiments, the method comprises an in vitro method. In some embodiments, the method comprises an in vivo method. In some embodiments, the method comprises an ex vivo method.

[0009] In another aspect, the present disclosure features a method of treating a disease in a subject, the method comprising administering to the subject a peptide nucleic acid (PNA) oligomer described herein. In some embodiments, the method comprises an in vivo method. In some embodiments, the method comprises an ex vivo method. In some embodiments, the disease comprises a blood disorder. In some embodiments, the blood disorder is a red blood cell disorder (e.g., beta-thalassemia or a sickle cell disease, e.g., sickle cell anemia).

[0010] In another aspect, the present disclosure features a method of making a PNA oligomer (e.g., a PNA oligomer described herein), comprising forming a covalent bond between a first component of a PNA oligomer and a second component of the PNA oligomer to thereby form a PNA oligomer (e.g., a PNA oligomer described herein). In some embodiments, the PNA oligomer comprises a PNA subunit having the structure of Formula (I-a) (e.g., any one of Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), or a salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof).

[0011] Embodiments of the PNA oligomers disclosed herein can be used in various applications such as diagnostic assays, nucleic acid sequencing (e.g. nanopore sequencing) and antisense applications. The PNA oligomers and methods disclosed herein can exhibit improved ease of administration and relatively low off target effects.

[0012] Additional details of one or more embodiments of the disclosure are set forth herein. Other features, objects, and advantages of the disclosure will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is an illustration of a generic peptide nucleic acid (PNA) subunit where B represents a nucleobase, and a, P, and y represent optionally substituted positions on the PNA backbone.

[0014] FIGS. 2A-2B are images of an acrylamide gel showing the results of an assay to visualize DNA binding and strand invasion of exemplary PNA oligomers described herein.

DETAILED DESCRIPTION

[0015] Described herein are peptide nucleic acids (PNA) oligomers comprising a 2- aminopyridine nucleobase, as well as compositions and related methods of use thereof. Definitions

[0016] “Peptide nucleic acid,” “PNA,” or “PNA oligomer” as used herein, refer to a non-natural polymer composition comprising linked nucleobases capable of sequence specifically hybridizing to a target nucleic acid. A PNA oligomer is comprised of PNA subunits (e.g., a PNA monomer), each of which comprises a backbone moiety and, optionally, a nucleobase moiety that can form hydrogen bonds with the nucleobase of the target nucleic acid. Exemplary PNA oligomers are disclosed in or otherwise claimed in any of the following: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049,5,714,331, 5,736,336, 5,773,571 or 5,786,461; (each of the foregoing are herein incorporated herein by reference in its entirety). The term "peptide nucleic acid", "PNA", or “PNA oligomer” shall also apply to polymers comprising two or more PNA subunits of kind described in the following publications: Diderichsen et al., Tetrahedron Lett. 37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7:637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:687-690(1997); Krotz et al., Tetrahedron Lett. 36:6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) l :539-546;Lowe et al., J. Chem. Soc. Perkin Trans. 1, 1 :547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1 :555-560 (1997); Mitra R. and Ganesh, K.N. Chem Commun (Camb) 47:1198-1200 (2011); Petersen et al., Bioorg. Med. Chem. Lett. 6:793- 796 (1996);Diederichsen, U., Bioorg. Med. Chem. Lett., 8: 165-168 (1998); Cantin et al., Tetrahedron Lett., 38:4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997);

Lagriffoule et aC,Chem. Eur. J., 3:912-919 (1997); Mann A. et al, Methods Mol Biol. 1050: 1-12 (2014); Sugiyama T. and Kittaka, A. Molecules 18:287-310 (2012); and International Patent Publication No. W096/04000. Additional PNA oligomers include phosphono-PNA analogues (pPNAs)as described in van der Laan, A. C. et al., Tetrahedron Lett. 37:7857-7860 (1996); trans-4-hydroxy-L-proline nucleic acids (HypNAs) as described in Efimov et al., Nucleic Acids Ae .34(8):2247-2257 (2006); and (lS,2R/lR,2S)-cis-cyclopentyl PNAs (cpPNAs) as described in Govindaraju, T. et al., J. Org. Chem. 69(17):5725-34 (2004); each of the foregoing is herein incorporated herein by reference in its entirety.

[0017] A “PNA monomer,” as used herein, refers to a single discrete building block for PNA synthesis. A PNA monomer comprises a backbone moiety and a nucleobase moiety that can form hydrogen bonds with the nucleobase of a target nucleic acid. To form a PNA oligomer, a first PNA monomer may be activated, for example, by exposure to an activating group (e.g., a carboxyl activating group such as PyBOP or HATU). The PNA monomer may then be coupled to a particular reactive moiety (e.g., a free amine terminus (i.e., N-terminus)) on second deprotected PNA monomer or a PNA oligomer to form a growing PNA oligomer chain.

Exemplary PNA monomers include Fmoc/Bhoc PNA monomers, Fmoc/t-boc PNA monomers, boc/Z PNA monomers, boc/cbz PNA monomers, and others. Additional exemplary PNA monomers are included in WO2012/138955; WO2018/175927; Eriksson and Nielsen Quart Rev Biophys 29(4):369-394 (1996); and Sugiyama et al., Bioorg Med Chem Lett 27(15):3337-3341 (2017); which are incorporated herein by reference in their entirety.

[0018] A “PNA subunit,” as used herein, refers to a subunit within a PNA oligomer. A PNA subunit comprises a backbone moiety and, optionally, a nucleobase moiety that can form hydrogen bonds with the nucleobase of a target nucleic acid, e.g., as shown in FIG. 1. In an embodiment, a PNA subunit comprises an aminoethylglycine backbone with an amine terminus (i.e., N-terminus) and a carboxyl terminus (i.e., C-terminus), as well as a nucleobase moiety attached to the backbone through a linker (e.g., a methylene carbonyl linker). PNA subunits can include Watson-Crick (i.e., WC-binding) PNA subunits which mediate WC-binding to nucleobases in a target nucleic acid sequence, and can include Hoogsteen (i.e., HG-binding) PNA subunits that mediate HG-binding to nucleobases in a target nucleic acid sequence. The nucleobases within a PNA subunit may be naturally occurring or non-naturally occurring. Exemplary nucleobases include adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine,

2-aminoadenine (or 2,6-diaminopurine), 2-thiouracil,2-thiothymine, 2-thiocytosine, 5- chlorouracil, 5-bromouracil, 5-iodouracil, 5-chlorocytosine,5-bromocytosine, 5-iodocytosine, 5- propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 7- methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,

3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, 5-propynyl uracil and 2-thio-5-propynyl, pyridazin-3(2H)-one (E), pyrimidin-2(lH)-one (P) and 2- aminopyridine (M), as well as tautomeric forms thereof.

[0019] A “tail-clamp PNA,” “tail-clamp PNA oligomer,” or “tcPNA,” as used herein, refers to a PNA oligomer capable of forming a PNA/DNA/PNA triplex upon binding to a target sequence (e.g., a target nucleic acid sequence, e.g. a target DNA sequence, e.g. a double stranded target DNA sequence). A tcPNA comprises: i) a first region comprising a plurality of PNA subunits that participate in binding to the Hoogsteen face of a target nucleic acid sequence and ii) a second region comprising a subregion that consists of a plurality of PNA subunits that participate in binding to the Watson-Crick face of the target nucleic acid sequence. In an embodiment, the first region and second region of PNA subunits are linked by a linker (e.g., a polyethylene glycol linker). The second region can further comprise another subregion, wherein the another subregion consists of a plurality of PNA that participate in binding to the Watson- Crick face of a tail target sequence, wherein the tail target sequence is a sequence contiguous with the target sequence and is not bound by the first region. The tcPNA oligomer can further comprise a positively charged region comprising one or more positively charged moieties (e.g., positively charged amino acids such as lysine, ornithine or arginine) which may be present on a terminus of the tcPNA. In an embodiment, a tcPNA comprises a 2-aminopyridine nucleobase in the first region.

[0020] As used herein, “Watson Crick complementarity” refers to the relation between a pair of nucleobase-bearing oligomers (e.g., a pair of PNA oligomers, or a PNA oligomer and a nucleic acid) that can engage in stable hydrogen bonding with one another via the Watson Crick face of at least one nucleobase of the pair. For example, the set of nucleobases “ACTC” has antiparallel Watson Crick complementarity with the set of nucleobases “TGAG.”

[0021] As used herein, “Hoogsteen complementarity” refers to the relation between a pair of nucleobase-bearing oligomers (e.g., a PNA oligomer or a nucleic acid) that can engage in stable hydrogen bonding with one another via the Hoogsteen face of at least one nucleobase of the pair. In some embodiments, a first nucleobase-bearing oligomer has Hoogsteen complementarity with a second nucleobase-bearing oligomer if the first nucleobase-bearing oligomer preferentially binds the Hoogsteen face of the second nucleobase-bearing oligomer when the second nucleobase-bearing oligomer is bound to a third nucleobase-bearing oligomer in a Watson-Crick duplex.

[0022] Complementarity can be complete or partial between two nucleobase-bearing oligomers. For example, a nucleobase-bearing oligomer (e.g., a PNA oligomer or a nucleic acid) has “complete Watson-Crick complementarity” with a second nucleobase-bearing oligomer (e.g., a PNA oligomer or a nucleic acid) if each nucleobase of the first oligomer can engage in stable hydrogen bonding via the Watson-Crick face of a nucleobase in a corresponding position in the second oligomer. Analogously, a nucleobase-bearing oligomer (e.g., a PNA oligomer) has “complete Hoogsteen complementarity” with a second nucleobase-bearing oligomer (e.g., a nucleic acid) if each nucleobase of the first oligomer can engage in stable hydrogen bonding via the Hoogsteen face of a nucleobase in a corresponding position in the second oligomer. Complementarity also can be complete or partial between a subset of nucleobase-bearing subunits (e.g., a plurality of PNA subunits within a PNA oligomer) and a second subset of nucleobase-bearing subunits (e.g., a plurality of nucleotides within a nucleic acid).

[0023] In some embodiments, a first nucleobase-bearing oligomer (e.g., a PNA oligomer or a nucleic acid) has partial Watson-Crick complementarity with a second nucleobase-bearing oligomer (e.g., a PNA oligomer or a nucleic acid). In some embodiments, the first nucleobase- bearing oligomer has about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% about 90%, about

90% to about 95%, or about 95% to about 100% Watson-Crick complementarity with the second nucleobase-bearing oligomer (e.g., a PNA oligomer or nucleic acid).

[0024] In some embodiments, a nucleobase-bearing oligomer (e.g., a PNA oligomer or a nucleic acid) has partial Hoogsteen complementarity with a second nucleobase-bearing oligomer (e.g., a PNA oligomer or a nucleic acid). In some embodiments, the first nucleobase-bearing oligomer has about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% about 90%, about 90% to about 95%, or about 95% to about 100% Hoogsteen complementarity with the second nucleobase-bearing oligomer.

Selected Chemical Definitions

[0025] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March ’s Advanced Organic Chemistry, 5 ^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 ^rd Edition, Cambridge University Press, Cambridge, 1987.

[0026] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0027] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “Ci-Ce alkyl” is intended to encompass, Ci, C2, C3, C4, C5, Ce, C1-C6, C1-C ₅ , C1-C4, C1-C3, C1-C2, C2-C6, C ₂ -C ₅ , C2-C4, C2-C3, C3-C6, C ₃ -C ₅ , C3-C4, C4-C6, c ₄ - C5, and C5-C6 alkyl.

[0028] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention. [0029] As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 18 carbon atoms (“Ci-Cisalkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“Ci-Cualkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“Ci-Csalkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“Ci-Cvalkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Ci-Ce alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-C5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“Ci-C4alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-C3 alkyl”). In some embodiments, an alkyl group has

1 to 2 carbon atoms (“C1-C2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6alkyl”). Examples of Ci-C24alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tert-amyl (C5), n-hexyl (Ce), octyl (Cs), nonyl (C9), decyl (C10), undecyl (Cn), dodecyl (or lauryl) (C12), tridecyl (C13), tetradecyl (or myristyl) (C14), pentadecyl (C15), hexadecyl (or cetyl) (Cie), heptadecyl (C17), or octadecyl (or stearyl) (Cis), and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0030] As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms (“C2-Cisalkenyl”). In some embodiments, an alkenyl group has 2 to 12 carbon atoms (“C2-Ci2alkenyl”). In some embodiments, an alkenyl group has

2 to 8 carbon atoms (“C2-Csalkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-C7alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2- Ce alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-C4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-C3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carboncarbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). The one or more carbon double bonds can have cis or trans (or E or Z) geometry. Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2- butenyl (C4), butadienyl (C4), and the like. Examples of C2-C24alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (Ce), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (Cs), octatrienyl (Cs), nonenyl (C9), nonadienyl (C9), decenyl (C10), decadienyl (C10), undecenyl (Cn), undecadienyl (Cu), dodecenyl (C12), dodecadienyl (C12), tridecenyl (C13), tridecadienyl (C13), tetradecenyl (C14), tetradecadienyl (e.g., myristoleyl) (C14), pentadecenyl (C15), pentadecadienyl (C15), hexadecenyl (e.g., palmitoleyl) (Cie), hexadecadienyl (Cie), heptadecenyl (C17), heptadecadienyl (C17), octadecenyl (e.g., oleyl) (Cis), or octadecadienyl (e.g., linoleyl) (Cis), and the like. Each instance of an alkenyl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkenyl group is unsubstituted C2-10 alkenyl.

[0031] As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms (“C2-C18 alkynyl”). In some embodiments, an alkynyl group has 2 to 12 carbon atoms (“C2-C12 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2- C4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2- propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C2-6 alkynyl.

[0032] As used herein, the terms "heteroalkyl," “heteroalkenyl,” and “heteroalkynyl,” refer to a non-cyclic stable straight or branched alkyl, alkenyl, or alkynyl chains, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl, heteroalkenyl, or heteroalkynyl group. Exemplary heteroalkyl, heteroalkenyl, and heteroalkynyl groups include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH ₂ -N(CH ₃ )-CH3, -CH2-S-CH2-CH3, - CH ₂ -CH ₂ -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O)2-CH3, -CH=CH-O-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , - CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0033] The terms "alkylene," “alkenylene,” “alkynylene,” or “heteroalkylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described as, e.g., a Ci-Ce- membered alkylene, Ci-Ce-membered alkenylene, Ci-Ce-membered alkynylene, or Ci-Ce- membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety. In the case of heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R’- may represent both -C(O)2R’- and -R’C(O)2-. Each instance of an alkylene, alkenylene, alkynylene, or heteroalkylene group may be independently optionally substituted, z.e., unsubstituted (an “unsubstituted alkylene”) or substituted (a “substituted heteroalkylene) with one or more substituents.

[0034] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 it electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-Cu aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Ce aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a Ce-C 10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted Ce-Cu aryl. In certain embodiments, the aryl group is a substituted Ce-C 14 aryl.

[0035] As used herein, “cycloalkyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 7 ring carbon atoms (“C3-C7 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 7 ring carbon atoms (“C5-C7 cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4-C7-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (Ce), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. Exemplary C3-C7 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), and cycloheptatrienyl (C7), bicyclo[2.1.1]hexanyl (Ce), bicyclo[3.1.1]heptanyl (C7), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated. “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.

[0036] As used herein, the term “halo” refers to a fluorine, chlorine, bromine, or iodine radical (i.e., -F, -Cl, -Br, and -I, respectively).

[0037] As used herein, the term “heteroaryl,” refers to an aromatic heterocycle that comprises 1, 2, 3 or 4 heteroatoms selected, independently of the others, from nitrogen, sulfur and oxygen. As used herein, the term “heteroaryl” refers to a group that may be substituted or unsubstituted. A heteroaryl may be fused to one or two rings, such as a cycloalkyl, an aryl, or a heteroaryl ring. The point of attachment of a heteroaryl to a molecule may be on the heteroaryl, cycloalkyl, or aryl ring, and the heteroaryl group may be attached through carbon or a heteroatom. Examples of heteroaryl groups include imidazolyl, furyl, pyrrolyl, thienyl, thiazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, quinolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzisooxazolyl, benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, pyrazolyl, triazolyl, oxazolyl, tetrazolyl, benzimidazolyl, benzoisothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl, purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl or benzo(b)thienyl, each of which can be optionally substituted. Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.

[0038] As used herein, the term “heterocyclyl” refers to a radical of a heterocyclic ring system. Representative heterocyclyls include ring systems in which (i) every ring is non-aromatic and at least one ring comprises a heteroatom, e.g., tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is an aromatic carbon ring, e.g., 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and (iii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is aromatic and comprises a heteroatom, e.g., 3,4-dihydro-lH-pyrano[4,3-c]pyridine, and l,2,3,4-tetrahydro-2,6-naphthyridine. In certain embodiments, the heterocyclyl is a monocyclic or bicyclic ring, wherein each of said rings contains 3-7 ring atoms where 1, 2, 3, or 4 of said ring atoms are a heteroatom independently selected from N, O, and S. Each instance of a heterocyclyl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents.

[0039] As used herein, the term “hydroxy” refers to the radical -OH.

[0040] As used herein, the terms “carbonyl” and “oxo” each refer to the radical -C=O.

[0041] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Exemplary substituents on a substitutable atom of an “optionally substituted” group (such as an atom within a alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocyclyl or heteroaryl) may include, for example, deuterium, halogen, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, -OH, -O-alkyl, - O-alkenyl, O-alkynyl, O-aryl, O-heteroaryl, O-cycloalkyl, O-heterocyclyl, -NO2, -CN, -N3, =O, =S, -SH, -S-alkyl, -S(O)-alkyl, -S(O) ₂ -alkyl, -C(O)OH, -C(O)alkyl, -C(O)O-alkyl, -C(O)NH2, - C(O)NH-alkyl, -C(O)N(alkyl) ₂ , -P(O) ₂ -alkyl, -P(O)-(alkyl) ₂ , -OP(O)alkyl, -OP(O)(O- (alkyl)) ₂ ,or -Si(alkyl)s . In an embodiment, an optionally substituted group may be itself optionally substituted. The symbol “ as used herein refers to an attachment point to another moiety or functional group. For example, the -~^-may refer to an attachment point to a peptide nucleic acid backbone or the attachment point to another region or atom within a PNA intermediate. In some embodiments, denotes an attachment point to a PNA monomer or PNA oligomer.

[0042] As used herein, “hydrate” refers to a compound which is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R xEEO, wherein R is the compound and wherein x is a number greater than 0.

[0043] As used herein, a “pharmaceutically acceptable salt” refers to salts of the compounds that are prepared with acids or bases (e.g., relatively non-toxic acids or bases), depending on the particular substituents found on the compounds described herein. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of base addition salts (e.g., pharmaceutically acceptable base addition salts) include sodium, potassium, calcium, magnesium, ammonium, organic amino, or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of acid addition salts (e.g., pharmaceutically acceptable acid addition salts) include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts may be prepared by methods known to those skilled in the art. Other carriers (e.g., pharmaceutically acceptable carriers) known to those of skill in the art are suitable for the present invention.

[0044] As used herein, “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, dimethylsulfoxide, tetrahydrofuran, diethyl ether, and the like. The compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates.

[0045] The term “tautomer” as used herein refers to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of it electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest. [0046] Compounds described herein (e.g., PNA subunits and PNA oligomers) can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC), selective crystallization as chiral salts, or in the presence of chiral hosts, or from chiral solvents, as well as through enrichment using enzymes or chemical means including but not limited to dynamic kinetic resolution; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0047] As used herein, a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “5” form of the compound is substantially free from the “A” form of the compound and is, thus, in enantiomeric excess of the “A” form. In some embodiments, ‘substantially free’, refers to: (i) an aliquot of an “A” form compound that contains less than 2% “S” form; or (ii) an aliquot of an “S” form compound that contains less than 2% “A” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the single enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

[0048] Compounds described herein (e.g., PNA subunits and PNA oligomers) may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹ H, ² H (D or deuterium), and ³ H (T or tritium); C may be in any isotopic form, including ¹² C, ¹³ C, and ¹⁴ C; O may be in any isotopic form, including ¹⁶ O and ¹⁸ O; and the like.

Peptide Nucleic Acids

[0049] The present disclosure features PNA oligomers comprising at least one 2-aminopyridine nucleobase, as well as compositions and related methods of use and kits thereof. In some embodiments, the PNA oligomer is a tail-clamp peptide nucleic acid (tcPNA). A tcPNA may comprise a first region comprising a plurality of PNA subunits that participate in binding to the Hoogsteen face of a target sequence, wherein at least one PNA subunit comprises a 2- aminopyridine nucleobase. In an embodiment, the tcPNA further comprises a subregion, wherein the subregion consists of a plurality of PNA subunits that participate in binding to the Watson-Crick face of a target nucleic acid sequence, wherein the first region and second region are covalently linked through a linker (e.g., a polyethylene-glycol linker). In an embodiment, the second region further comprises another subregion, wherein the another subregion consists of a plurality of PNA subunits having complete Watson Crick complementarity with a tail target sequence, wherein the tail target sequence is a sequence contiguous with the target sequence and is not bound by the first region. In an embodiment, the tcPNA further comprises a positively charged region comprising positively charged amino acids (e.g., lysine residues) on at least one terminus of the tcPNA. In some embodiments, the tcPNA comprises one or more PNA subunits comprising a substituent at the gamma-position. In some embodiments, the tcPNA comprises one or more PNA subunits comprising a mini-PEG moiety at the gamma-position.

[0050] In some embodiments, the PNA oligomer comprises one or more linkers. Linkers, as contemplated herein, are divalent radicals that can link one region (e.g., the first, second, or third region) or subunit of the PNA oligomer to one or more other region(s) or subunit(s) of the PNA oligomer. In some embodiments, the first region is linked to the first positively charged region via a linker. In some embodiments, the second region is linked to the second positively charged region via a linker.

[0051] In some embodiments, the linker is C1-20 alkylene or C1-20 heteroalkylene.

[0052] In some embodiments, the linker is a residue of an omega-amino fatty acid, such as, for example, a residue of an omega-amino caproic acid. In some embodiments, the linker is a residue of a dicarboxylic acid. In some embodiments, the linker is a residue of oxalic acid or a residue of succinic acid. In some embodiments, the linker is a polyamine sequence. In some embodiments, the linker is a polyamide sequence. In some embodiments, the linker is non- cleavable. In some embodiments, the linkers comprises a polyethylene glycol group comprising 2-100 ethylene glycol residues.

[0053] In some embodiments, the first region is linked to the second region by the linker, wherein first linker comprises a polyalkylene glycol group.

[0054] In some embodiments, the linker is -[NH(CH2CH2O) _Z CH2C(O)] _W - or - [NHCH2CH2(OCH2CH2)ZC(O)] _W -, wherein each z is independently 1-100, and each w is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each z is independently 1-20. In some embodiments, each z is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each z is independently 1, 2, 3, 4, 5, or 6. In some embodiments, each w is independently 1, 2, 3, 4, or 5. In some embodiments, each w is 1. In some embodiments, each w is 2.

[0055] In some embodiments, the linker is -[NH(CH2CH2O)2CH2C(O)]2-. In some embodiments, the linker is -NH(CH2CH2O)3CH2C(O)-.

[0056] In some embodiments, the linker is independently -[NH(CH2CH2O) _Z CH2C(O)]2-, wherein each z is independently 1-100. In some embodiments, each z is independently 1-20. In some embodiments, each z is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each z is independently 1, 2, 3, 4, 5, or 6. In some embodiments, each w is independently 1, 2, 3, 4, or 5. In some embodiments, each w is 1. In some embodiments, each w is 2.

[0057] In some embodiments, the linker is independently selected from - NH(CH ₂ CH ₂ O)2CH ₂ C(O)-, -NH(CH2CH ₂ O) ₃ CH ₂ C(O)-, -NH(CH2CH ₂ O)4CH ₂ C(O)-, - NH(CH2CH ₂ O) ₅ CH ₂ C(O)-, -NH(CH ₂ CH ₂ O)6CH ₂ C(O)-, -[NH(CH ₂ CH ₂ O)2CH ₂ C(O)]2-, - [NH(CH2CH ₂ O) ₃ CH ₂ C(O)]2-, -[NH(CH2CH ₂ O)4CH ₂ C(O)]2-, -[NH(CH2CH ₂ O) ₅ CH ₂ C(O)]2-, and -[NH(CH ₂ CH ₂ O)6CH ₂ C(O)]2-.

[0058] In some embodiments, the linker is independently -[NHCH2CH2(OCH2CH2) _Z C(O)] _W -, wherein each z is independently 1-100. In some embodiments, each z is independently 1-20. In some embodiments, each z is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each z is independently 1, 2, 3, 4, 5, or 6. In some embodiments, each w is independently 1, 2, 3, 4, or 5. In some embodiments, each w is 1. In some embodiments, each w is 2.

[0059] In some embodiments, the linker is independently selected from - NHCH2CH ₂ OCH2CH ₂ C(O)-, -NHCH2CH2 (OCH ₂ CH ₂ ) ₂ C(O)-, -NHCH ₂ CH2(OCH ₂ CH2) ₃ C(O)-, -NHCH ₂ CH2(OCH ₂ CH2)4C(O)-, -NHCH ₂ CH2(OCH ₂ CH2)5C(O)-, and - NHCH ₂ CH2(OCH ₂ CH2)6C(O)-. PNA Oligomers Comprising 2 -Aminopyridine

[0060] Disclosed herein are PNA oligomers comprising at least one PNA subunit comprising a 2-aminopyridine nucleobase. A PNA subunit refers to a subunit within a PNA oligomer, and comprises a backbone moiety and, optionally, a nucleobase moiety that can form hydrogen bonds with the nucleobase of a target nucleic acid, e.g., as shown in FIG. 1. In an embodiment, a PNA subunit comprises a nucleobase and a backbone moiety. The nucleobase of the PNA subunit can form one or more hydrogen bonds with the nucleobase of a target nucleic acid sequence. The backbone moiety of the PNA subunit typically comprises a first terminus and a second terminus. In an embodiment, the first terminus is an amine terminus (i.e., N-terminus), and the second terminus is a carboxyl terminus (i.e., C-terminus), e.g., as shown in FIG. 1. The backbone moiety also comprises an atom to which the nucleobase is bound, typically through a spacer moiety.

[0061] 2-Aminopyridine is a cationic nucleobase that has been previously incorporated into PNA oligomers as a cytosine substitute. Earlier work by Rozners (see, e.g., U.S. Patent No. 10,260,089) provides evidence that PNA oligomers comprising a 2-aminopyridine nucleobase exhibit enhanced preference for binding to RNA over DNA hairpins. Only poor DNA binding was observed in earlier constructs. The present disclosure provides the surprising and unexpected result that incorporation of 2-aminopyridine into certain PNA oligomers (e.g., tailclamp PNA oligomers, or gamma-substituted tail-clamp PNA oligomers) provides improved DNA binding relative to analogous PNA oligomers that do not contain a 2-aminopyridine nucleobase (see, e.g., Examples provided herein). In particular, certain PNA oligomers (e.g., tail-clamp PNA oligomers, or gamma-substituted tail-clamp PNA oligomers) comprising at least one 2-aminopyridine nucleobase exhibit fast Hoogsteen binding to double-stranded DNA, as well as the formation of a stable PNA:DNA:PNA triplex structure. Incorporation of 2- aminopyridine into certain PNA oligomers (e.g., tail-clamp PNA oligomers, or gammasubstituted tail-clamp PNA oligomers) has further provided improved solubility and lower selfcomplementarity relative to analogous PNA oligomers that do not contain a 2-aminopyridine nucleobase. In addition, certain PNA oligomers (e.g., tail-clamp PNA oligomers, or gammasubstituted tail-clamp PNA oligomers) comprising at least one 2-aminopyridine nucleobase have exhibited enhanced ability to alter a DNA sequence (e.g., gene editing) compared to analogous PNA oligomers that do not comprise a 2-aminopyridine sequence.

[0062] In one aspect, the present disclosure features a PNA oligomer, e.g., a PNA oligomer which can form a triplex with a DNA strand, or a PNA clamp oligomer, e.g., a tail-clamp PNA oligomer (tcPNA), comprising: a) a first region comprising a plurality of PNA subunits having Hoogsteen complementarity with a target sequence, wherein at least one of the PNA subunits comprises a 2-aminopyridine nucleobase; and b) a second region comprising a plurality of PNA subunits having Watson Crick complementarity with the target sequence. In some embodiments, the PNA oligomer further comprises: a’) a first positively charged region comprising a positively charged amino acid subunit, e.g., a lysine subunit; c) a third region comprising a plurality of PNA subunits that participate in Watson Crick binding with a tail target sequence; or d) a second positively charged region comprising a positively charged amino acid subunit, e.g., a lysine subunit. In an embodiment, the PNA oligomer comprises a gamma modified PNA subunit, e.g., wherein the first region comprises a gamma modified PNA subunit. In an embodiment, the second region of the PNA oligomer comprises a gamma modified PNA subunit. In an embodiment, the another subregion of the PNA oligomer comprises a gamma modified PNA subunit. In an embodiment, the PNA oligomer comprises a gamma modified PNA subunit in the first region, the subregion, and the another subregion. In an embodiment, the PNA oligomer comprises is a PNA clamp oligomer (e.g., a PNA tail-clamp oligomer (tcPNA)). [0063] In another aspect, the present disclosure provides a peptide nucleic acid (PNA) oligomer, comprising a first region linked to a second region via a linker or a covalent bond, wherein: a) the second region comprises a plurality of PNA subunits having complete Watson-Crick complementarity with a target sequence, wherein at least one of the PNA subunits comprises a 2-aminopyridine nucleobase; and b) the first region comprises a plurality of PNA subunits having Hoogsteen complementarity with a subsequence of the target sequence. In some embodiments, the portion of the target sequence that is not the subsequence is contiguous. In some embodiments, a terminus of the subsequence is a terminus of the target sequence. In some embodiments, the PNA oligomer comprises a gamma modified PNA subunit. In some embodiments, the plurality of PNA subunits of the first region has complete Hoogsteen complementarity with the target sequence.

[0064] In another aspect, the present disclosure provides a peptide nucleic acid (PNA) oligomer, comprising a first region linked to a second region via a linker or a covalent bond, wherein: a) the first region comprises a plurality of PNA subunits having Hoogsteen complementarity with a target sequence, wherein at least one of the PNA subunits comprises a 2-aminopyridine nucleobase; and b) the second region comprises a subregion, wherein the subregion consists of a plurality of PNA subunits having complete Watson Crick complementarity with the target sequence. In some embodiments, the PNA oligomer comprises a gamma modified PNA subunit. In some embodiments, the plurality of PNA subunits of the first region has complete Hoogsteen complementarity with the target sequence.

[0065] In some embodiments, the PNA oligomer further comprises a first positively charged region linked to the first region by a second linker or covalent bond, wherein the first positively charged region comprises a positively charged amino acid subunit. In some embodiments, the PNA oligomer further comprises a second positively charged region linked to the second region via a third linker or covalent bond, wherein the second positively charged region comprises a positively charged amino acid subunit.

[0066] In some embodiments, the second region further comprises another subregion, wherein the another subregion consists of a plurality of PNA subunits having complete Watson Crick complementarity with a tail target sequence, wherein the tail target sequence is a sequence contiguous with the target sequence and is not bound by the first region.

[0067] In some embodiments, the first region comprises the gamma modified PNA subunit. In some embodiments, the second region comprises the gamma modified PNA subunit. In some embodiments, the subregion is contiguous with the another subregion. In some embodiments, the PNA oligomer comprises a gamma modified PNA subunit in the first region and the second region.

[0068] In some embodiments, the PNA oligomer comprises the first positively charged region. In some embodiments, the PNA oligomer comprises the first positively charged region and the second positively charged region. In some embodiments, the first positively charged region is linked to the first region via a covalent bond. In some embodiments, the second positively charged region is linked to the second region via a covalent bond.

[0069] In some embodiments, the first region comprises an abasic subunit. In some embodiments, the abasic subunit is -Gly-Gly-. In some embodiments, the abasic subunit has a position within the first region that corresponds to a position of a purine nucleobase of the target sequence.

[0070] In some embodiments, the PNA oligomer is a PNA clamp oligomer. In some embodiments, the PNA oligomer is a PNA tail-clamp oligomer (tcPNA).

[0071] In another aspect, the PNA oligomer comprises a PNA subunit of Formula (I-a): or a salt thereof, wherein P ¹ is a first terminus, e.g., an amine or a carboxyl terminus, which may participate in a covalent bond to the P ⁵ group of another PNA subunit within the PNA oligomer; P ⁵ is a second terminus, e.g., an amine or a carboxyl terminus, which may participate in a covalent bond to the P ¹ group of another PNA subunit within the PNA oligomer; each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen, deuterium, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, -OR ^A , cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkylene-heteroaryl, ’ /x UK / ,,

A A ^ OR ^{12 y} (IV-b), or the side chain of an optionally protected amino acid, wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, and heterocyclyl may be optionally substituted with one or more R ¹⁰ ; each of R ^9a and is R ^9b is independently hydrogen, C1-C12 alkyl, or an amine protecting group (e.g., Boc); each R ¹⁰ is independently halo, cyano, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, Ci-C 12 heteroalkyl, Ci- Ci2-haloalkyl, or -OR ^A1 ; R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl); y is 1, 2, 3, 4, or 5; X is N or CR ^b ; L is C1-C12 alkylene, C2-C12 alkenylene, C1-C12 heteroalkylene, cycloalkylene, or heterocyclylene, each of which is optionally substituted with one or more R ^B ; R ^b is hydrogen, deuterium, halo, or C1-C12 alkyl; each of R ^A and R ^A1 is independently hydrogen, deuterium, Ci-C 12 alkyl, C1-C12 heteroalkyl, C1-C12 haloalkyl, - N(R ^C )(R ^D ), or halo; each of R ^B , R ^c , and R ^D is independently hydrogen, halo, C1-C12 alkyl, or Ci- C12 heteroalkyl; and each independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C-terminus or N- terminus of a PNA oligomer. [0072] In some embodiments, P ¹ is an amine terminus (e.g., -NH2 or -NH-, wherein -NH- participates in a covalent bond to the P ⁵ group of another PNA subunit within the PNA oligomer). In some embodiments, P ⁵ is a carboxyl terminus (e.g., -C(O)OH, -C(O)CH3, - C(O)NH2, or -C(O)-, wherein -C(O)- participates in a covalent bond to the P ¹ group of another PNA subunit within the PNA oligomer). In some embodiments, P ¹ is an amine terminus and P ⁵ is a carboxyl terminus. In an embodiment, when P ¹ is an amine terminus, P ⁵ is a carboxyl terminus. In an embodiment, when P ⁵ is an amine terminus, P ¹ is a carboxyl terminus.

[0073] In some embodiments, X is CR ^b , wherein R ^b is hydrogen, deuterium, fluorine or alkyl. In some embodiments, X is N.

[0074] In some embodiments, L is alkylene or heteroalkylene, each of which is optionally substituted with one or more R ^B . In some embodiments, L is ethylene, propylene, or butylene, each of which is substituted with one R ^B . In some embodiments, L is heteroalkylene substituted with one R ^B . In some embodiments, R ^B is oxo.

[0075] In some embodiments, L is selected from 0 , In some embodiments, L is , In some embodiments, L is , with the carbonyl carbon linked to X.

In some embodiments, L is with the carbonyl carbon linked to X. In some embodiments, L is with the carbonyl carbon linked to X. In some embodiments,

O the carbonyl carbon linked to X.

[0076] In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen, Ci- Cuheteroalkyl, or Ci-Cu-haloalkyl. In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen. In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogenorCi-Cuheteroalkyl, e.g., a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG moiety comprising between 2 and 6 PEG units).

[0077] In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen or has the structure of Formula (IV-a) or (IV-b): (IV-a), (IV-b), wherein R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl), y is 1, 2, 3, 4, or 5, and denotes an attachment point to the PNA subunit. In some embodiments, R ¹² is hydrogen or methyl, and y is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and y is 1. In some embodiments, R ¹² is methyl or tert-butyl, and y is 1. In some embodiments, R ¹² is hydrogen and y is 1. In some embodiments, R ¹² is methyl and y is 1. In some embodiments, R ¹² is tert-butyl and y is 1.

[0078] In some embodiments, each of R ³ and R ⁴ has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl), y is 1, 2, 3, 4, or denotes an attachment point to the PNA subunit. In some embodiments, R ¹² is hydrogen or methyl, and y is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and y is 1. In some embodiments, R ¹² is methyl or tert-butyl, and y is 1. In some embodiments, R ¹² is hydrogen and y is 1. In some embodiments, R ¹² is methyl and y is 1. In some embodiments, R ¹² is tert-butyl and y is 1.

[0080] In some embodiments, R ³ has the structure of Formula (IV-a) or (IV-b), and R ⁴ is hydrogen. In some embodiments, R ³ has the structure of Formula (IV-a) or (IV-b), and each of R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen. In some embodiments, R ⁴ has the structure of Formula (IV-a) or (IV-b), and R ³ is hydrogen. In some embodiments, R ⁴ has the structure of Formula (IV-a) or (IV-b), and each of R ³ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen.

[0081] In some embodiments, each of R ^9a and R ^9b is hydrogen.

[0082] In some embodiments, the PNA subunit of Formula (I-a) is a structure of Formula (I-b): or a salt thereof, wherein R ² is hydrogen or Ci-Cualkyl; each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen, deuterium, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, Ci- Cuheteroalkyl, Ci-Ci2-haloalkyl, -OR ^A , cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl,

C1-C12 alkyl ene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, C1-C12 alkylene-heteroaryl, r the side chain of an optionally protected amino acid, wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, and heterocyclyl may be optionally substituted with one or more R ¹⁰ ; each of R ^9a and is R ^9b is independently hydrogen, C1-C12 alkyl, or an amine protecting group (e.g., Boc); each R ¹⁰ is independently halo, cyano, C1-C12 alkyl, C2-C 12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, or -OR ^A1 ; each R ¹² is independently hydrogen or alkyl; y is 1, 2, 3, 4, or 5; X is N or CR ^b ; L is C1-C12 alkylene, C2-C12 alkenylene, C1-C12 heteroalkylene, cycloalkylene, or heterocyclylene, each of which is optionally substituted with one or more R ^B ; R ^b is hydrogen, deuterium, halo, or Ci-Cualkyl; each of R ^A and R ^A1 is independently hydrogen, deuterium, Ci-C 12 alkyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, - N(R ^C )(R ^D ), or halo; each of R ^B , R ^c , and R ^D is independently hydrogen, halo, C1-C12 alkyl, or Ci- C12 heteroalkyl; and each independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C-terminus or N- terminus of a PNA oligomer. [0083] In some embodiments, X is CR ^b , wherein R ^b is hydrogen, deuterium, fluorine or alkyl. In some embodiments, X is N. [0084] In some embodiments, L is alkylene or heteroalkylene, each of which is optionally substituted with one or more R ^B . In some embodiments, L is ethylene, propylene, or butylene, each of which is substituted with one R ^B . In some embodiments, L is heteroalkylene substituted with one R ^B . In some embodiments, R ^B is oxo.

[0085] In some embodiments, L is selected from , In some embodiments, L is , In some embodiments, L is , with the carbonyl carbon linked to X.

O

In some embodiments, L the carbonyl carbon linked to X. In some

O embodiments, L the carbonyl carbon linked to X. In some embodiments,

O

L is with the carbonyl carbon linked to X.

[0086] In some embodiments, R ² is hydrogen.

[0087] In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen, Ci- Cuheteroalkyl, or Ci-Cu-haloalkyl. In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen. In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen or C1-C12 heteroalkyl, e.g., a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG moiety comprising between 2 and 6 PEG units).

[0088] In some embodiments, each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen or has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl), y is 1, 2, 3, 4, or denotes an attachment point to the PNA subunit. In some embodiments, R ¹² is hydrogen or methyl, and y is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and y is 1. In some embodiments, R ¹² is methyl or tert-butyl, and y is 1. In some embodiments, R ¹² is hydrogen and y is 1. In some embodiments, R ¹² is methyl and y is 1. In some embodiments, R ¹² is tert-butyl and y is 1.

[0090] In some embodiments, each of R ³ and R ⁴ has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl), y is 1, 2, 3, 4, or 5, and “•~w” denotes an attachment point to the PNA subunit. In some embodiments, R ¹² is hydrogen or methyl, and y is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and y is 1. In some embodiments, R ¹² is methyl or tert-butyl, and y is 1. In some embodiments, R ¹² is hydrogen and y is 1. In some embodiments, R ¹² is methyl and y is 1. In some embodiments, R ¹² is tert-butyl and y is 1.

[0092] In some embodiments, R ³ has the structure of Formula (IV-a) or (IV-b), and R ⁴ is hydrogen. In some embodiments, R ³ has the structure of Formula (IV-a) or (IV-b), and each of R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen. In some embodiments, R ⁴ has the structure of Formula (IV-a) or (IV-b), and R ³ is hydrogen. In some embodiments, R ⁴ has the structure of Formula (IV-a) or (IV-b), and each of R ³ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen.

[0093] In some embodiments, each of R ^9a and R ^9b is hydrogen.

[0094] In some embodiments, the PNA subunit of Formula (I-a) is structure of Formula (I-c): or a salt thereof, wherein R ² is hydrogen or C1-C12 alkyl; each of R ³ , R ⁴ , R ⁵ , and R ⁶ is independently hydrogen, deuterium, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, C1-C12 haloalkyl, -OR ^A , cycloalkyl, Ci-C 12 3-1 ky 1 ene ^_ c cl 0 I k 1 , heterocyclyl, Ci~

C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, C1-C12 alkyl ene-heteroaryl, r the side chain of an optionally protected amino acid, wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, and heterocyclyl may be optionally substituted with one or more R ¹⁰ ; each of R ^9a and is R ^9b is independently hydrogen, C1-C12 alkyl, or an amine protecting group (e.g., Boc); each R ¹⁰ is independently halo, cyano, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, C1-C12 haloalkyl, or -OR ^A1 ; each of R ^A and R ^A1 is independently hydrogen, deuterium, Ci-Cualkyl, Ci-C 12 heteroalkyl, Ci-Ci2-haloalkyl, -N(R ^C )(R ^D ), or halo; each of R ^c and R ^D is independently hydrogen, halo, C1-C12 alkyl, or Ci-C 12 heteroalkyl; each R ¹² is independently hydrogen or alkyl; y is 1, 2, 3, 4, or 5; and each “-~w” independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C- terminus or N- terminus of a PNA oligomer.

[0095] In some embodiments, R ² is hydrogen.

[0096] In some embodiments, each of R ³ , R ⁴ , R ⁵ , and R ⁶ is independently hydrogen, Ci- Cuheteroalkyl, or Ci-Ci2-haloalkyl. In some embodiments, each of R ³ , R ⁴ , R ⁵ , and R ⁶ is independently hydrogen. In some embodiments, each of R ³ , R ⁴ , R ⁵ , and R ⁶ is independently hydrogenorC 1 -C 12heteroalkyl, e.g., a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG moiety comprising between 2 and 6 PEG units).

[0097] In some embodiments, each of R ³ , R ⁴ , R ⁵ , and R ⁶ is independently hydrogen or has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl), y is 1, 2, 3, 4, or 5, and “-"w” denotes an attachment point to the PNA subunit. In some embodiments, R ¹² is hydrogen or methyl, and y is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and y is 1. In some embodiments, R ¹² is methyl or tert-butyl, and y is 1. In some embodiments, R ¹² is hydrogen and y is 1. In some embodiments, R ¹² is methyl and y is 1. In some embodiments, R ¹² is tert-butyl and y is 1.

[0098] In some embodiments, each of R ³ and R ⁴ has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl (e.g., C1-C4 alkyl, e.g., methyl, ethyl, isopropyl, tert-butyl), y is 1, 2, 3, denotes an attachment point to the PNA subunit. In some embodiments, R ¹² is hydrogen or methyl, and y is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and y is 1. In some embodiments, R ¹² is methyl or tert-butyl, and y is 1. In some embodiments, R ¹² is hydrogen and y is 1. In some embodiments, R ¹² is methyl and y is 1. In some embodiments, R ¹² is tert-butyl and y is 1.

[0099] In some embodiments, R ³ has the structure of Formula (IV-a) or (IV-b), and R ⁴ is hydrogen. In some embodiments, R ³ has the structure of Formula (IV-a) or (IV-b), and each of R ⁴ , R ⁵ , and R ⁶ is independently hydrogen. In some embodiments, R ⁴ has the structure of Formula (IV-a) or (IV-b), and R ³ is hydrogen. In some embodiments, R ⁴ has the structure of Formula (IV-a) or (IV-b), and each of R ³ , R ⁵ , and R ⁶ is independently hydrogen.

[00100] In some embodiments, each of R ^9a and R ^9b is hydrogen. [00101] In some embodiments, the PNA subunit of Formula (I-a) is a structure of Formula (I-d) or Formula (I-e): independently hydrogen, deuterium, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, C1-C12 haloalkyl, -OR ^A , cycloalkyl, Ci-C 12 alkylene-cycloalkyl, heterocyclyl, Ci- C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, C1-C12 alkyl ene-heteroaryl, or the side chain of an optionally protected amino acid, wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, and heterocyclyl may be optionally substituted with one or more R ¹⁰ ; each of R ^9a and is R ^9b is independently hydrogen, Ci-Cualkyl, or an amine protecting group (e.g., Boc); each R ¹⁰ is independently halo, cyano, C1-C12 alkyl, C2- C12 alkenyl, C2-C12 alkynyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, or -OR ^A1 ; R ¹² is hydrogen or Ci-Cualkyl; each of R ^A and R ^A1 is independently hydrogen, deuterium, Ci-Cualkyl, Ci- Cuheteroalkyl, Ci-Ci2-haloalkyl, -N(R ^C )(R ^D ), or halo; each of R ^c and R ^D is independently hydrogen, halo, Ci-Cualkyl, or Ci-Cuheteroalkyl; m is 1, 2, 3, 4, 5, 6, 7, or 8; and each “■«»” independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C-terminus or N- terminus of a PNA oligomer.

[00102] In some embodiments, R ² is hydrogen or methyl. In some embodiments, R ² is hydrogen.

[00103] In some embodiments, R ³ is hydrogen. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁶ is hydrogen. In some embodiments, each of R ³ , R ⁴ , R ⁵ and R ⁶ , if present, is independently hydrogen.

[00104] In some embodiments, each of R ^9a and R ^9b is hydrogen.

[00105] In some embodiments, R ¹² is hydrogen or methyl, and m is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and m is 1. In some embodiments, R ¹² is methyl or tert-butyl, and m is 1. In some embodiments, R ¹² is hydrogen and m is 1. In some embodiments, R ¹² is methyl and m is 1. In some embodiments, R ¹² is tert-butyl and m is 1.

[00106] In some embodiments, the PNA subunit of Formula (I-a) is a structure of Formula (I-f) or Formula (I-g): or a salt thereof, wherein R ² is hydrogen or C1-C12 alkyl; each of R ⁵ and R ⁶ is independently hydrogen, deuterium, C1-C12 alkyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, or the side chain of an optionally protected amino acid, wherein each alkyl, heteroalkyl, and haloalkyl may be optionally substituted with one or more R ¹⁰ ;each R ¹⁰ is independently halo, cyano, Ci- C12 alkyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, or -OR ^A1 ; R ¹² is hydrogen or Ci-Cualkyl; R ^A1 is independently hydrogen, deuterium, Ci-Cualkyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, - N(R ^C )(R ^D ), or halo; each of R ^c and R ^D is independently hydrogen, halo, Ci-Cualkyl, or Ci- Cuheteroalkyl; m is 1, 2, 3, 4, 5, 6, 7, or 8; and each “^w” independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C-terminus or N- terminus of a PNA oligomer.

[00107] In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁶ is hydrogen. In some embodiments, each of R ⁵ and R ⁶ is independently hydrogen.

[00108] In some embodiments, each of R ^9a and R ^9b is hydrogen.

[00109] In some embodiments, R ¹² is hydrogen or methyl, and m is 1. In some embodiments, R ¹² is hydrogen or tert-butyl, and m is 1. In some embodiments, R ¹² is methyl or tert-butyl, and m is 1. In some embodiments, R ¹² is hydrogen and m is 1. In some embodiments, R ¹² is methyl and m is 1. In some embodiments, R ¹² is tert-butyl and m is 1.

[00110] In any and all embodiments, each “ ” independently denotes an attachment point to an atom of the N-terminus of the PNA oligomer (e.g., a hydrogen), to an atom of the C-terminus of the PNA oligomer (e.g., -OH or -NH2), or to another PNA subunit. In some embodiments, one “ — ” is an attachment point to an atom of the C-terminus of the PNA oligomer and one“

” is an attachment point to another PNA subunit. In some embodiments, one ” is an attachment point to an atom of the N-terminus of the PNA oligomer and one “ ” is an attachment point to another PNA subunit. In some embodiments, both “ ” are attachment points to other PNA subunits. In some embodiments, one “ ” is an attachment point to a linker. In some embodiments, one ” is an attachment point to an amino acid. [00111] In some embodiments, the PNA subunit of Formula (I-a) is a structure of Formula (I-h): or a salt thereof, wherein each independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C-terminus or N- terminus of a PNA oligomer.

Formulations

[00112] Described herein are PNA oligomers comprising at least one PNA subunit comprising a 2-aminopyridine nucleobase. The PNA oligomers described herein may be formulated in nanoparticles, including lipid nanoparticles (LNPs) and synthetic polymer nanoparticles, (e.g., poly(lactic-co-glycolic acid (PLGA) nanoparticles)).

[00113] In some embodiments, a nanoparticle is an LNP and refers to a particle that comprises a lipid and one or more PNA oligomers. An LNP may further comprise one or more lipids, for example, at least one or more of an ionizable lipid, phospholipid, a sterol, or an alkylene glycol- containing lipid (e.g., a PEG-containing lipid), as well as a load component (e.g., a nucleic acid). [00114] In some embodiments, a nanoparticle is a synthetic polymer nanoparticle and refers to a particle that comprises a synthetic polymer (e.g., PLGA) and one or more PNA oligomers. A synthetic polymer nanoparticle may further comprise a synthetic polymer, or a plurality of synthetic polymers, for example, at least one or more of a non-naturally occurring polymer, including co-polymers, block polymers, block co-polymers, polymer mixtures, and polymer blends, as well as a load component (e.g., a nucleic acid).

Lipid Nanoparticles

[00115] The present disclosure features a lipid nanoparticle comprising one or more PNA oligomers and a lipid. Exemplary lipids include ionizable lipids, phospholipids, sterol lipids, alkylene glycol lipids (e.g., polyethylene glycol lipids), sphingolipids, glycerolipids, glycerophospholipids, prenol lipids, saccharolipids, fatty acids, and polyketides. In some embodiments, the LNP comprises a single type of lipid. In some embodiments, the LNP comprises a plurality of lipids. An LNP may comprise one or more of an ionizable lipid, a phospholipid, a sterol, or an alkylene glycol lipid (e.g., a polyethylene glycol lipid).

[00116] In an embodiment, the LNP comprises an ionizable lipid. An ionizable lipid is a lipid that comprises an ionizable moiety capable of bearing a charge (e.g., a positive charge e.g., a cationic lipid, or a negative charge, e.g., an anionic lipid) under certain conditions (e.g., at a certain pH range, e.g., under physiological conditions). An ionizable moiety may comprise an amine, carboxylic acid, hydroxyl, phenol, phosphate, sulfonyl, thiol, or a combination thereof. An ionizable lipid may be a cationic lipid or an anionic lipid. In addition to an ionizable moiety, an ionizable lipid may contain an alkyl or alkenyl group, e.g., greater than six carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons or more in length). Exemplary ionizable lipids include dilinoleylmethyl-4- dimethylaminobutyrate(DLin-MC3-DMA), 2, 2-dilinoleyl-4-dimethylamino-[l,3]-di oxolane (DLin-K-DMA), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-di oxolane (DLin-KC2-DMA), 2, 2-dilinoleyl-4-N-chloromethyl-7V,7V-dimethylamino-[l,3]-di oxolane (DLin-KC2-CIMDMA),

2, 2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]-di oxolane (DLin-KC3-DMA), 2,2-dilinoleyl-4- (4-dimethylaminobutyl)-[l,3]-di oxolane (DLin-KC4-DMA), l,2-dilinoleyloxy-3- dimethylaminopropane (D-Lin-DMA), l,2-dilinolenyloxy-dimethyl-3 -aminopropane (D-Len- DMA), (l,2-dilinoleoyl-3-dimethylaminopropane (D-Lin-DAP), 1,2-di oleyloxydimethylaminopropane (DODMA), l,2-distearyloxy-dimethyl-3-aminopropane (DSDMA), dioleoyl dimethyl-ammonium propane (DODAP), l,2-dimyristyloxy-propyl-3-dimethyl- hydroxy ethyl ammonium bromide (DMRIE), dimethyl-[2-(sperminecarboxamido)ethyl]-2,3- bis(dioleyloxy)-l-propaniminium or a salt thereof(DOSPA), 98N12-5, and C12-200. In some embodiments, the ionizable lipid comprises DLin-MC3-DMA, DLin-KC2-DMA, D-LinK- DMA, D-Lin-DAP, 98N12-5, C12-200, or DODMA. Additional ionizable lipids that may be included in an LNP described herein are disclosed in Jayaraman et al. (Angew. Chem. Int. Ed. 51 :8529-8533 (2012)), Semple et al. (Nature Biotechnol. 28: 172-176 (2010)), and US Patent Nos. 8,710,200 and 8,754,062, each of which is incorporated herein by reference in its entirety. [00117] In some embodiments, an LNP comprises an ionizable lipid having a structure of Formula (VI): or a pharmaceutically acceptable salt thereof, wherein Y is , each R ²² is independently alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is optionally substituted with R ^B ; each R ^B is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; n is an integer between 1 and 10 (inclusive); and denotes the attachment point. [00118] In some embodiments,

[00119] In some embodiments, each R ²² is independently alkyl (e.g., C2-C32 alkyl, C4-C28 alkyl, C8-C24 alkyl, C12-C22 alkyl, or C16-C20 alkyl). In some embodiments, each R ²² is independently alkenyl (e.g., C2-C32 alkenyl, C4-C28 alkenyl, C8-C24 alkenyl, C12-C22 alkenyl, or C16-C20 alkenyl). In some embodiments, each R ²² is independently C16-C20 alkenyl. In some embodiments, each R ²² is independently Cis alkenyl. In some embodiments, each R ²² is independently linoleyl (or cz ,cz -9,12-octadecadienyl). In some embodiments, each R ²² is the same. In some embodiments, each R ²² is different.

[00120] In some embodiments, n is an integer between 1 and 10, 1 and 8, 1 and 6, or 1 and 4 (inclusive). In some embodiments, n is 1, 2, 3, or 4. In some embodiments, n is 1, 2, or 3. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[00121] In some embodiments, the ionizable lipid is DLin-MC3-DMA. In some embodiments, the ionizable lipid is DLin-KC2-DMA. In some embodiments, the ionizable lipid is D-LinK- DMA. In some embodiments, the ionizable lipid is DLinDAP. In some embodiments, the ionizable lipid is 98N12-5. In some embodiments, the ionizable lipid is C12-200. In some embodiments, the ionizable lipid is DODMA.

[00122] An LNP may comprise an ionizable lipid at a concentration greater than about 0.1 mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration of greater than about 1 mol%, about 2mol%, about 4mol%, about 8mol%, about 20mol%, about 40mol%, about 50mol%, about 60mol%, about 80mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration of greater than about 20mol%, about 40mol%, or about 50mol%. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about lmol% to about 95mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about 2mol% to about 90mol%, about 4mol% to about 80mol%, about 10mol% to about 70mol%, about 20mol% to about 60mol%, about 40mol% to about 55mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about 20mol% to about 60mol%. In an embodiment, the LNP comprises an ionizable lipid at a concentration between about 40 mol% to about 55 mol%.

[00123] In an embodiment, the LNP comprises a phospholipid. A phospholipid is a lipid that comprises a phosphate group and at least one alkyl, alkenyl, or heteroalkyl chain. A phospholipid may be naturally occurring or non-naturally occurring (e.g., a synthetic phospholipid). A phospholipid may comprise an amine, amide, ester, carboxyl, choline, hydroxyl, acetal, ether, carbohydrate, sterol, or a glycerol. In some embodiments, a phospholipid may comprise a phosphocholine, phosphosphingolipid, or a plasmalogen. Exemplary phospholipids include l,2-dioleoyl- w-glycero-3 -phosphocholine (DOPC), 1,2- dipalmitoyl-sw-glycero-3 -phosphocholine (DPPC), l,2-dioleoyl- w-glycero-3- phosphoethanolamine (DOPE), l,2-distearoyl-sw-glycero-3-phosphocholine (DSPC),1,2- dilauroyl-sw-glycero-3-phosphocholine(DLPC), l,2-dimyristoyl-sw-glycero-3-phosphocholine (DMPC), l,2-distearoyl- w-glycero-3-phosphoethanolamine (DSPE), l-myristoyl-2-oleoyl- w- glycero-3 -phosphocholine (MOPC), l,2-diarachidonoyl- w-glycero-3 -phosphocholine (DAPC), l-palmitoyl-2-linoleoyl- w-glycero-3 -phosphatidylcholine (PLPC), l-palmitoyl-2-oleoyl- glycero-3 -phosphocholine (POPC), l-stearoyl-2-myristoyl-sw-glycero-3-phosphocholine (SMPC), l-palmitoyl-2-myristoyl-sw-glycero-3-phosphocholine (PMPC), bis(monoacylglycerol)phosphate (BMP), L-a-phosphatidylcholine, 1,2-Diheptadecanoyl-sw- glycero-3 -phosphorylcholine (DHDPC), and l-stearoyl-2-arachidonoyl-sw-glycero-3- phosphocholine (SAPC). Additional phospholipids that may be included in an LNP described herein are disclosed in Li, J. et al. (Asian J. Pharm. Sci. 10:81-98 (2015)), which is incorporated herein by reference in its entirety.

[00124] In some embodiments, an LNP comprises a phospholipid having a structure of Formula (VII):

O

R ²³ 0 °

^R2 ^°^^°-P-°^N(R ²⁵ ) _U

J OR ²⁴

O (VII), or a pharmaceutically acceptable salt thereof, wherein each R ²³ is independently alkyl, alkenyl, or heteroalkyl; wherein each alkyl, alkenyl, or heteroalkyl is optionally substituted with R ^c ; each R ²⁵ is independently hydrogen or alkyl; R ²⁴ is absent, hydrogen, or alkyl; each R ^c is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; m is an integer between 1 and 4 (inclusive); and u is 2 or 3.

[00125] In some embodiments, each R ²³ is independently alkyl (e.g., C2-C32 alkyl, C4-C28 alkyl, C8-C24 alkyl, C12-C22 alkyl, or C16-C20 alkyl). In some embodiments, each R ²³ is independently alkenyl (e.g., C2-C32 alkyl, C4-C28 alkenyl, C8-C24 alkenyl, C12-C22 alkenyl, or C16-C20 alkenyl). In some embodiments, each R ²³ is independently heteroalkyl (e.g., C4-C28heteroalkyl, Cs- C24heteroalkyl, Ci2-C22heteroalkyl, Ci6-C2oheteroalkyl). In some embodiments, each R ²³ is independently C16-C20 alkyl. In some embodiments, each R ²³ is independently C17 alkyl. In some embodiments, each R ²³ is independently heptadecyl. In some embodiments, each R ²³ is the same. In some embodiments, each R ²³ is different. In some embodiments, each R ²³ is optionally substituted with R ^c . In some embodiments, R ^c is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl.

[00126] In some embodiments, one of R ²⁵ is hydrogen. In some embodiments, one of R ²⁵ is alkyl. In some embodiments, one of R ²⁵ is methyl. In some embodiments, each R ²⁵ is independently alkyl. In some embodiments, each R ²⁵ is independently methyl. In some embodiments, each R ²⁵ is independently methyl and u is 2. In some embodiments, each R ²⁵ is independently methyl and u is 3.

[00127] In some embodiments, R ²⁴ is absent, and the oxygen to which it is attached carries a negative charge. In some embodiments, R ²⁴ is hydrogen.

[00128] In some embodiments, m is an integer between 1 and 10, 1 and 8, 1 and 6, 1 and 4. In some embodiments, m is 1, 2, 3, or 4. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3.

[00129] In some embodiments, the phospholipid is l,2-distearoyl-sw-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid isl,2-dioleoyl-sw-glycero-3-phosphocholine (DOPC). In some embodiments, the phospholipid isl,2-dipalmitoyl- w-glycero-3- phosphocholine (DPPC). In some embodiments, the phospholipid is l,2-dioleoyl-sw-glycero-3- phosphoethanolamine (DOPE).

[00130] An LNP may comprise a phospholipid at a concentration greater than about 0.1mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration of greater than about 0.5mol%, about lmol%, about 1.5mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 8mol%, about 10mol%, about 12mol%, about 15mol%, about 20mol%, about 50mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration of greater than about lmol%, about 5mol%, or about 10mol%. In an embodiment, the LNP comprises a phospholipid at a concentration between about 0.1mol% to about 50mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration between about 0.5mol% to about 40mol%, about lmol% to about 30mol%, about 5mol% to about 25mol%, about 10mol% to about 20mol%, about 10mol% to about 15mol%, or about 15mol% to about 20mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a phospholipid at a concentration between about 5mol% to about 25mol%. In an embodiment, the LNP comprises a phospholipid at a concentration between about 10mol% to 20mol%.

[00131] In an embodiment, the LNP comprises a sterol. A sterol is a lipid that comprises a polycyclic structure and an optionally a hydroxyl or ether substituent, and may be naturally occurring or non-naturally occurring (e.g., a synthetic sterol). Sterols may comprise no double bonds, a single double bond, or multiple double bonds. Sterols may further comprise an alkyl, alkenyl, halo, ester, ketone, hydroxyl, amine, polyether, carbohydrate, or cyclic moiety. An exemplary listing of sterols includes cholesterol, dehydroergosterol, ergosterol, campesterol, P- sitosterol, stigmasterol, lanosterol, dihydrolanosterol, desmosterol, brassicasterol, lathosterol, zymosterol, 7-dehydrodesmosterol, avenasterol, campestanol, lupeol, and cycloartenol. In some embodiments, the sterol comprises cholesterol, dehydroergosterol, ergosterol, campesterol, P- sitosterol, or stigmasterol. Additional sterols that may be included in an LNP described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46:839-862 (2005)), which is incorporated herein by reference in its entirety.

[00132] In some embodiments, an LNP comprises a sterol having a structure of Formula (VIII): pharmaceutically acceptable salt thereof, wherein

R ²⁶ is hydrogen, alkyl, heteroalkyl, or-C(O)R ^D , R ²⁷ is hydrogen, alkyl, or -OR ^E ; each of R ^D and R ^E is independently hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with alkyl, halo, or carbonyl; and each “ = ” is either a single or double bond, and wherein each carbon atom participating in the single or double bond is bound to 0, 1, or 2 hydrogens, valency permitting.

[00133] In some embodiments, R ²⁶ is hydrogen. In some embodiments, R ²⁶ is alkyl (e.g., C1-C4 alkyl, C4-C8 alkyl, Cs-Cu alkyl). In some embodiments, R ²⁶ is C(O)R ^D , wherein R ^D is alkyl (e.g., C1-C4 alkyl, C4-C8 alkyl, Cs-Cu alkyl) or heteroaryl (e.g., a nitrogen-containing heteroaryl). In some embodiments, R ²⁶ is heteroalkyl (e.g., Ci-C4heteroalkyl, C4-Csheteroalkyl, Cs-Cuheteroalkyl). In some embodiments, R ²⁶ is heteroalkyl (e.g., Ci-C4heteroalkyl, C4- Csheteroalkyl, Cs-Cuheteroalkyl) substituted with carbonyl.

[00134] In some embodiments, R ²⁷ is hydrogen. In some embodiments, R ²⁷ is alkyl (e.g., C1-C4 alkyl, C4-C8 alkyl, Cs-Cu alkyl).

[00135] In some embodiments, one of “ = ” is a single bond. In some embodiments, one of “ =” is a double bond. In some embodiments, two of are single bonds. In some embodiments, two of are double bonds. In some embodiments, each is a single bond. In some embodiments, each is a double bond. [00136] In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is dehydroergosterol. In some embodiments, the sterol is ergosterol. In some embodiments, the sterol is campesterol. In some embodiments, the sterol is P-sitosterol. In some embodiments, the sterol is stigmasterol. In some embodiments, the sterol is a corticosteroid, (e.g., corticosterone, hydrocortisone, cortisone, or aldosterone).

[00137] An LNP may comprise a sterol at a concentration greater than about 0. lmol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a sterol at a concentration greater than about 0.5mol%, about lmol%, about 5mol%, about 10mol%, about 15mol%, about 20mol%, about 25mol%, about 35mol%, about 40mol%, about 45mol%, about 50mol%, about 55mol%, about 60mol%, about 65mol%, or about 70mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a sterol at a concentration greater than about 10mol%, about 15mol%, about 20mol%, or about 25mol%. In an embodiment, the LNP comprises a sterol at a concentration between about lmol% to about 95mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a sterol at a concentration between about 5mol% to about 90mol%, about 10mol% to about 85mol%, about 20mol% to about 80mol%, about 20mol% to about 60mol%, about 20mol% to about 50mol%, or about 20mol% to 40mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises a sterol at a concentration between about 20mol% to about 50mol%. In an embodiment, the LNP comprises a sterol at a concentration between about 30mol% to about 60mol%.

[00138] In some embodiments, the LNP comprises an alkylene glycol-containing lipid. An alkylene glycol-containing lipid is a lipid that comprises at least one alkylene glycol moiety, for example, a methylene glycol or an ethylene glycol moiety. In some embodiments, the alkylene glycol-containing lipid comprises a polyethylene glycol (PEG). An alkylene glycol-containing lipid may be a PEG-containing lipid. A PEG-containing lipid may further comprise an amine, amide, ester, carboxyl, phosphate, choline, hydroxyl, acetal, ether, heterocycle, or carbohydrate. PEG-containing lipids may comprise at least one alkyl or alkenyl group, e.g., greater than six carbon atoms in length (e.g., greater than about 8 carbons, 10 carbons, 12 carbons, 14 carbons, 16 carbons, 18 carbons, 20 carbons or more in length), e.g., in addition to a PEG moiety. In an embodiment, a PEG-containing lipid comprises a PEG moiety comprising at least 20 PEG monomers, e.g., at least 30 PEG monomers, 40 PEG monomers, 45 PEG monomers, 50 PEG monomers, 100 PEG monomers, 200 PEG monomers, 300 PEG monomers, 500 PEG monomers, 1000 PEG monomers, or 2000 PEG monomers. Exemplary PEG-containing lipids include PEG-DMG (e.g, DMG-PEG2k), PEG-c-DOMG, PEG-DSG, PEG-DPG, PEG-DSPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, and PEG-DLPE. In some embodiments, the PEG-lipids include PEG-DMG (e.g., DMG-PEG2k), PEG-c-DOMG, PEG-DSG, and PEG-DPG. Additional PEG-lipids that may be included in an LNP described herein are disclosed in Fahy, E. et al. (J. Lipid. Res. 46:839-862 (2005) which is incorporated herein by reference in its entirety.

[00139] In some embodiments, an LNP comprises an alkylene glycol-containing lipid having a structure of Formula (IX): pharmaceutically acceptable salt thereof, wherein each R ²⁸ is independently alkyl, alkenyl, or heteroalkyl, each of which is optionally substituted with R ^F ; A is absent, O, CEE, C(O), or NH; E is absent, alkyl, or heteroalkyl, wherein alkyl or heteroalkyl is optionally substituted with carbonyl; each R ^F is independently alkyl, halo, hydroxy, amino, cycloalkyl, or heterocyclyl; and z is an integer between 10 and 200 (inclusive). [00140] In some embodiments, each R ²⁸ is independently alkyl. In some embodiments, each R ²⁸ is independently heteroalkyl. In some embodiments, each R ²⁸ is independently alkenyl. [00141] In some embodiments, A is O or NH. In some embodiments, A is CH2. In some embodiments, A is carbonyl. In some embodiments, A is absent.

[00142] In some embodiments, E is alkyl. In some embodiments, E is heteroalkyl. In some embodiments, both A and E are not absent. In some embodiments, A is absent. In some embodiments, E is absent. In some embodiments, either one of A or E is absent. In some embodiments, both A and E are independently absent.

[00143] In some embodiments, z is an integer between 10 and 200 (e.g., between 20 and 180, between 20 and 160, between 20 and 120, between 20 and 100, between 40 and 80, between 40 and 60, between 40 and 50). In some embodiments, z is 45.

[00144] In some embodiments, the PEG-lipid is PEG-DMG (e.g., DMG-PEG2k). In some embodiments, the PEG-lipid is PEG-c-DOMG. In some embodiments, the PEG-lipid is PEG- DSG. In some embodiments, the PEG-lipid is PEG-DPG.

[00145] An LNP may comprise an alkylene glycol-containing lipid at a concentration greater than about 0.1mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration of greater than about 0.5mol%, about lmol%, about 1.5mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 8mol%, about 10mol%, about 12mol%, about 15mol%, about 20mol%, about 50mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNPcomprises an alkylene glycol-containing lipid at a concentration of greater than about lmol%, about 4mol%, or about 6mol%. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 0. lmol% to about 50mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 0.5mol% to about 40mol%, about lmol% to about 35mol%, about 1.5mol% to about 30mol%, about 2mol% to about 25mol%, about 2.5mol% to about 20%, about 3mol% to about 15mol%, about 3.5mol% to about 10mol%, or about 4mol% to 9mol%, e.g., of the total lipid composition of the LNP. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 3.5mol% to about 10mol%. In an embodiment, the LNP comprises an alkylene glycol-containing lipid at a concentration between about 4mol% to 9mol%.

[00146] In some embodiments, the LNP comprises at least two types of lipids. In an embodiment, the LNP comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least three types of lipids. In an embodiment, the LNP comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid. In some embodiments, the LNP comprises at least four types of lipids. In an embodiment, the LNP comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid.

[00147] The LNP (e.g., as described herein) may comprise one or more of the following components: (i) an ionizable lipid at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); (ii) a phospholipid at a concentration between 0.1mol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%); (iii) a sterol at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); and (iv) a PEG-containing lipid at a concentration between about 0. lmol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%). In an embodiment, the LNP comprises one of

(i)-(iv). In an embodiment, the LNP comprises two of (i)-(iv). In an embodiment, the LNP comprises three of (i)-(iv). In an embodiment, the LNP comprises each of (i)-(iv). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (iv). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i),

(ii), and (iii). In some embodiments, the LNP comprises (i), (ii), and (iv). In some embodiments, the LNP comprises (ii), (iii), and (iv).

[00148] The LNP (e.g., as described herein) may comprise one or more of the following components: (i) DLin-MC3-DMA at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); (ii) DSPC at a concentration between 0.1mol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%); (iii) cholesterol at a concentration between about lmol% to about 95mol% (e.g. about 20mol% to about 80mol%); and (iv) DMG- PEG2k at a concentration between about 0.1mol% to about 50mol% (e.g. between about 2.5mol% to about 20mol%). In an embodiment, the LNP comprises two of (i)-(iv). In an embodiment, the LNP comprises three of (i)-(iv). In an embodiment, the LNP comprises each of (i)-(iv). In some embodiments, the LNP comprises (i) and (ii). In some embodiments, the LNP comprises (i) and (iii). In some embodiments, the LNP comprises (i) and (iv). In some embodiments, the LNP comprises (ii) and (iii). In some embodiments, the LNP comprises (ii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (iii) and (iv). In some embodiments, the LNP comprises (i), (ii), and (iii). In some embodiments, the LNP comprises (i), (ii), and (iv). In some embodiments, the LNP comprises (ii), (iii), and (iv).

[00149] In an embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1, 5:1, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1 : 1). In an embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 15:2. In an embodiment, the LNP comprises a ratio of ionizable lipid to phospholipid of about 5:1. In an embodiment, the LNP comprises a ratio of ionizable lipid to a sterol of about 10:1 to about 1:10 (e.g., 9:1, 8:1, 8:7, , 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In an embodiment, the LNP comprises a ratio of ionizable lipid to an alkylene-containing lipid of about 1:10 to about 10:1 (e.g., 1:9, 1:8, 7:8, 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In an embodiment, the LNP comprises a ratio of phospholipid to an alkylene- containing lipid of about 10:1 to about 1:10 (e.g., 9:1, 8:1, 8:7, , 7:1, 7:5, 7:3, 6:1, 6:5, 5:1, 5:3, 4:1, 4:3, 3:1, 2:1, 1:1, 1:2, 1:3, 3:4, 1:4, 3:5, 1:5, 4:5, 1:6, 5:6, 7:6, 7:8, or 8:9). In an embodiment, the LNP comprises a ratio of a sterol to an alkylene-containing lipid of about 50:1 to about 1:1 (e.g., 40:1, 32:3, 6:1, 7:1, 5:1, 24:1, 22:1, 20:1, 22:5, 24:5, 26:5, 10:3, 15:2, 16:7, 18:1, 3:1, 3:2, or 1:1).

[00150] An LNP (e.g., described herein) comprises two of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PEG-containing lipid). An LNP (e.g., described herein) comprises three of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PEG-containing lipid). An LNP (e.g., described herein) comprises each of an ionizable lipid, a phospholipid, a sterol, and an alkylene glycol-containing lipid (e.g., PEG-containing lipid). Synthetic Polymer Nanoparticles

[00151] The present disclosure features a synthetic polymer nanoparticle one or more PNA oligomers and a synthetic polymer. Exemplary synthetic polymers include polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(4-hydroxy-L- proline ester, other degradable polyesters, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly (caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), poly(amine-co-ester) polymers, or a combination of any two or more of the foregoing.

[00152] In some embodiments, the synthetic polymer comprises a structure of Formula (X): wherein each of R ²⁹ and R ³⁰ is independently hydrogen and alkyl; each of R ³¹ , R ³² , R ³³ , and R ³⁴ is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R ^G ; R ^G is hydrogen or alkyl; each of xa and xb is an integer between 0 and 100 (inclusive), wherein each of xa and xb cannot simultaneously be 0; and xc is an integer between 1 and 10,000 (inclusive).

[00153] In some embodiments, each of R ²⁹ and R ³⁰ is independently hydrogen. In some embodiments, each of R ²⁹ and R ³⁰ is independently alkyl. In some embodiments, one of R ³¹ and R ³² is hydrogen and the other of R ³¹ and R ³² is alkyl (e.g., methyl). In some embodiments, each of R ³³ and R ³⁴ is independently hydrogen.

[00154] In some embodiments, xa is an integer greater than 0 and xb is an integer greater than 0. In some embodiments, xb is 0. In some embodiments, xb is 1, 2, 3, 4, or 50 (inclusive), between 1 and 25, between 1 and 10, or between 1 and 5. In some embodiments, xb is 1. In some embodiments, xa is 0. In some embodiments, xa is 1, 2, 3, 4, or 50(inclusive), between 1 and 25, between 1 and 10, or between 1 and 5. In some embodiments, xa is 1.

[00155] In some embodiments, xc is an integer between 1 and 10,000 (inclusive), between 1 and 5,000 (inclusive), between 1 and 2,500 (inclusive), between 1 and 1,000 (inclusive), between 1 and 750 (inclusive), between 1 and 500 (inclusive), between 1 and 250 (inclusive), between 1 and 100 (inclusive), or between 1 and 50 (inclusive).

[00156] In some embodiments, the synthetic polymer having a structure of Formula (X) is selected from poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid(PGA), and polylactic acid (PLA). In some embodiments, the synthetic polymer having a structure of Formula (X) is PLGA. In some embodiments, the synthetic polymer having a structure of Formula (X) is PGA. In some embodiments, the synthetic polymer having a structure of Formula (X) is PLA. [00157] In some embodiments, the synthetic polymer may further comprise a polyethylene glycol moiety. For example, the synthetic polymer comprising a structure of Formula (X) may further comprise a PEG moiety. An exemplary synthetic polymer comprises mPEG-PLA. [00158] In an embodiment, a nanoparticle comprises a single type of synthetic polymer. In an embodiment, the nanoparticle comprises a plurality of synthetic polymers. For example, a nanoparticle of the present disclosure may comprise PLGA, or may comprise PLGA and a second synthetic polymer.

[00159] The amount of a synthetic polymer encapsulated and/or entrapped within the nanoparticle may vary depending on the identity of the synthetic polymer or plurality of synthetic polymer. For example, the amount of a synthetic polymer may be between 0.05% and 40% by weight of synthetic polymers to the total weight of the nanoparticle. In some embodiments, the amount of a synthetic polymer in the nanoparticle is greater than about 0.05%, e.g., greater than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, or 40% by weight of synthetic polymers to the total weight of the nanoparticle. In some embodiment, the amount of a synthetic polymer in the nanoparticle is between 0.5% and 20% by weight of synthetic polymers to the total weight of the nanoparticle, or between 1% and 10% by weight of a synthetic polymer to the total weight of the nanoparticle, or between 2% to 5% by weight of a synthetic polymer to the total weight of the nanoparticle.

[00160] In some embodiments, a nanoparticle or plurality of nanoparticles may further comprise an alkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG described herein). A polyalkylene glycol may be any size, for example, a PEG between 2 PEG subunits and 5,000 subunits. In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nanoparticles in the plurality comprise a PEG.

Load Components

[00161] In some embodiments, a nanoparticle (e.g., an LNP, or a synthetic polymer nanoparticle) further comprises a load component. In some embodiments, the load component is an additional biological component (e.g., a polymeric biological component), for example, a nucleic acid or polypeptide. In some embodiments, the load component is a nucleic acid. In some embodiments, the nucleic acid is double stranded. In some embodiments, the nucleic acid is single stranded. In some embodiments, the load component is an oligonucleotide. In some embodiments, the load component is a single stranded DNA. In some embodiments, the load component is a single stranded RNA. In some embodiments, the load component is a double stranded DNA. In some embodiments, the load component is a double stranded RNA. In some embodiments, the load component is an mRNA. In some embodiments, the load component is an siRNA. In some embodiments, the load component is an antisense oligomer (e.g., PNA, DNA, morpholinos (also known as PMOs, See: US Patents 5,142,047 and 5,185,444), pyrrolidine-amide oligonucleotide mimics (POMs - See: T. H. Samuel Tan et al., Org. Biomol. Chem., 5: 239-248 (2007), morpholinoglycine oligonucleotides (MGOs - See: Tatyana V. et al., Beilstein J Org Chem. 10: 1151-1158 (2014)), and methyl phosphonates.

[00162] In some embodiments, the load component is a nucleic acid (e.g., DNA) between 5 and 250 nucleotides in length. In some embodiments, the load component is a nucleic acid (e.g., DNA) between 10 and 200 nucleotides in length. In some embodiments, the load component is a nucleic acid (e.g., DNA) between 18 and 100 nucleotides in length). In some embodiments, the load component is a nucleic acid (e.g., DNA) between 20and 80 nucleotides in length. In some embodiments, the load component is a nucleic acid (e.g., DNA) between 25and 70 nucleotides in length. In some embodiments, the load component is a nucleic acid (e.g., DNA) between 35 and 65 nucleotides in length. In some embodiments, the load component is a nucleic acid (e.g., DNA) between 20 and 40 nucleotides in length. In some embodiments, the load component is a single stranded nucleic acid (e.g., DNA) between 20 and 70 nucleotides in length. In some embodiments, the load component is a double stranded nucleic acid (e.g., DNA), with each strand being independently between 20 and 70 nucleotides in length.

[00163] In some embodiments, the load component is a nucleic acid and comprises one or more phosphorothioate linkages at a terminus (e.g., the 5’ terminus and/or the 3’ terminus). In some embodiments, the load component is a nucleic acid and comprises one or more phosphorothioate linkages at an internucleotide linkage. In some embodiments, the load component comprises more than one phosphorothioate linkages (e.g., 2, 3, or 4) at each terminus, for example, at each of its 3’ and 5’ termini. In some embodiments, the nucleic acid comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate intemucleotide linkages.

[00164] In some embodiments, the load component comprises a nucleic acid having a sequence that is the same or the complement of a sequence to which the PNA oligomer, e.g., a clamp system, e.g., a tail clamp system, e.g., a PNA oligomer comprising a sequence of any one of Compound Nos. 1-21 as described herein, has Watson Crick complementarity. In some embodiments, a load component comprises a nucleic acid having a sequence which is the same or the complement of a sequence to which the PNA oligomer, e.g., a tcPNA, e.g., a PNA oligomer comprising a sequence of any one of Compound Nos. 1-21 as described herein, has Hoogsteen complementarity. In some embodiments, a load component comprises a nucleic acid having a sequence which is the same or the complement of a sequence that is within 1,000, 500, 200, 100, 75, 60, or 40 base pairs of a sequence to which the PNA oligomer, e.g., a tcPNA, e.g., a PNA oligomer comprising a sequence of any one of Compound Nos. 1-21 as described herein, has Watson Crick complementarity. In some embodiments, the load component comprises a nucleic acid having a sequence which is the same or the complement of a sequence that is within 1,000, 500, or 200 base pairs of a sequence to which the PNA oligomer, e.g., a tcPNA, e.g., a PNA oligomer comprising a sequence of any one of Compound Nos. 1-21 as described herein, has Hoogsteen complementarity.

[00165] In some embodiments, a nanoparticle (e.g., an LNP, or a synthetic polymer nanoparticle) comprises a PNA oligomer and a load component. In some embodiments, the ratio of PNA oligomer to load component is equal (ie. 1 : 1). In some embodiments, the ratio of PNA oligomer to load component is greater than 1 : 1, for example, about 1 : 1.1, about 1 : 1.2, about 1 : 1.3, about 1 : 1.5, about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 : 10, about 1 :20, about 1 :25, about 1 :50, about 1 :75, or about 1 : 100 PNA oligomer to load component. In some embodiments, the ratio of load component to PNA oligomer greater than 1 : 1, for example, about 1 : 1.1, about 1 : 1.2, about 1 : 1.3, about 1 : 1.5, about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 : 10, about 1 :20, about 1 :25, about 1 :50, about 1 :75, or about 1 : 100 load component to PNA oligomer. In an embodiment, the ratio of PNA oligomer to load component is about 1 : 1. In an embodiment, the ratio of PNA oligomer to load component is about 1 :2. In an embodiment, the ratio of PNA oligomer to load component is about 1 :5.

[00166] A nanoparticle (e.g., an LNP, or a synthetic polymer nanoparticle; e.g. PLGA) described herein comprises a nucleic acid mimic, for example, a PNA oligomer (e.g., a tcPNA), as well as related preparations and methods of making and using the same. In an embodiment, the PNA comprises a PNA oligomer. In an embodiment, the PNA comprises a tcPNA oligomer. In some embodiments, the PNA oligomer is a tcPNA oligomer disclosed herein.

[00167] In some embodiments, the PNA is a PNA oligomer comprising greater than 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 PNA subunits. In some embodiments, the PNA is a PNA oligomer comprising betweenlO to 25 PNA subunits. In some embodiments, the PNA is a PNA oligomer comprising between 20 to 35 PNA subunits. In some embodiments, the PNA is a PNA oligomer comprising between about 2 to 50 PNA subunits, e.g., between about 10 and 45, 15 and 40, 17 and 35, 18 and 30, and 25 and 38PNA subunits.

[00168] In some embodiments, the PNA oligomer is a tail-clamp PNA oligomer (tcPNA). In some embodiments, the PNA oligomer has a sequence shown in Table 1 herein. In some embodiments, the PNA oligomer comprises a 2-aminopyridine nucleobase and a trilysine sequence (i.e., KKK) on the N-terminus. In some embodiments, the PNA oligomer comprises a 2-aminopyridine nucleobase and a trilysine sequence (i.e., KKK) on the C-terminus. In some embodiments, the PNA oligomer comprises a 2-aminopyridine nucleobase and a trilysine sequence (i.e., KKK) on both the N-terminus and the C-terminus. In some embodiments, the PNA oligomer comprises a 2-aminopyridine nucleobase, a 2-thiouracil nucleobase, and a [Gly- Gly] sequence. In some embodiments, the PNA oligomer comprises a 2-aminopyridine nucleobase, a 2,6-diaminopurine nucleobase, and a [Gly-Gly] sequence. In some embodiments, the PNA oligomer comprises a 2-aminopyridine nucleobase, a 7-deazaguanine nucleobase, and a [Gly-Gly] sequence.

[00169] In some embodiments, the PNA oligomer has the sequence of Compound No. 1. In some embodiments, the PNA oligomer has the sequence of Compound No. 2. In some embodiments, the PNA oligomer has the sequence of Compound No. 3. In some embodiments, the PNA oligomer has the sequence of Compound No. 4. In some embodiments, the PNA oligomer has the sequence of Compound No. 5. In some embodiments, the PNA oligomer has the sequence of Compound No. 6. In some embodiments, the PNA oligomer has the sequence of Compound No. 7. In some embodiments, the PNA oligomer has the sequence of Compound No. 8. In some embodiments, the PNA oligomer has the sequence of Compound No. 9. In some embodiments, the PNA oligomer has the sequence of Compound No. 11. In some embodiments, the PNA oligomer has the sequence of Compound No. 12. In some embodiments, the PNA oligomer has the sequence of Compound No. 13. In some embodiments, the PNA oligomer has the sequence of Compound No. 14. In some embodiments, the PNA oligomer has the sequence of Compound No. 15. In some embodiments, the PNA oligomer has the sequence of Compound No. 16. In some embodiments, the PNA oligomer has the sequence of Compound No. 17. In some embodiments, the PNA oligomer has the sequence of Compound No. 18. In some embodiments, the PNA oligomer has the sequence of Compound No. 19. In some embodiments, the PNA oligomer has the sequence of Compound No. 20. In some embodiments, the PNA oligomer has the sequence of Compound No. 21.

[00170] A nanoparticle (e.g., an LNP, or a synthetic polymer nanoparticle) may comprise a single PNA oligomer or a plurality of PNA oligomers. In some embodiments, a nanoparticle comprises 1 PNA oligomer. In some embodiments, a nanoparticle comprises a plurality of PNA oligomers, for example, at least 2 PNAs, 3 PNAs, 4 PNAs, 5 PNAs, 6 PNAs, 7 PNAs, 8 PNAs, 9 PNAs, 10 PNAs, 15 PNAs, 20 PNAs, 25 PNAs, 30PNAs, 40 PNAs, 50 PNAs, 60 PNAs, 70 PNAs, 80 PNAs, 90 PNAs, 100 PNAs, 150 PNAs, 200 PNAs, 300 PNAs, 400 PNAs, 500 PNAs, 600 PNAs, 700 PNAs, 800 PNAs, 900 PNAs, or 1,000 PNAs. In some embodiments, a nanoparticle comprises 10-50 PNA oligomers. In some embodiments, a nanoparticle comprises 2-5 PNA oligomers. In some embodiments, a nanoparticle comprises 3-10 PNA oligomers. In some embodiments, a nanoparticle comprises 5-20 PNA oligomers. In some embodiments, a nanoparticle comprises 10-35 PNA oligomers. In some embodiments, a nanoparticle comprises 10-100 PNA oligomers. In some embodiments, a nanoparticle comprises between 100-1,000 PNA oligomers. In some embodiments, a nanoparticle comprises between 500-1,000 PNA oligomers.

[00171] The amount of a PNA (e.g., a PNA oligomer) encapsulated and/or entrapped within the nanoparticle (e.g., an LNP, or a synthetic nanoparticle) may vary depending on the identity of the PNA or plurality of PNAs. For example, the amount of PNA may be between 0.001% and 50% by weight of PNAs to the total weight of the nanoparticle. In some embodiments, the amount of PNA in the nanoparticle is greater than about 0.001%, e.g., greater than about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight of PNA to the total weight of the nanoparticle. In some embodiments, the amount of PNA oligomer in the nanoparticle is greater than about 0.001%, e.g., greater than about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight of PNA oligomer to the total weight of the nanoparticle. In some embodiments the amount of PNA oligomer in the nanoparticle is between 0.001% and 50% by weight of PNA oligomers to the total weight of the nanoparticle, or between 0.001% and 30% by weight of PNA oligomers to the total weight of the nanoparticle, or between 1% and 25% by weight of PNA oligomers to the total weight of the nanoparticle, or between 1% and 10% by weight of PNA oligomer to the total weight of the nanoparticle, or between 2% to 5% by weight of PNA oligomer to the total weight of the nanoparticle.

[00172] A nanoparticle (e.g., an LNP, or a synthetic polymer nanoparticle) described herein may comprise a single type of PNA (e.g., a single type of PNA oligomer, or a PNA oligomer of a single sequence), or may comprise multiple types of PNAs. In some embodiments, the nanoparticle comprises a single type of PNA. In some embodiments, the nanoparticle comprises a plurality of types of PNAs (e.g., a plurality of PNA oligomers).

[00173] A nanoparticle (e.g., an LNP, or a synthetic polymer nanoparticle) described herein (e.g., comprising an PNA, and optionally a load component) may have a certain ratio of components. For example, the LNP described herein may comprise a particular ratio of a lipid or a plurality of lipids to an PNA. In an embodiment, the ratio of a plurality of lipids to a PNA (e.g., a PNA oligomer) is between 100: 1 to 1 : 100 (e.g. about 75: 1 to 1 :75, about 60: 1 to 1 :60, 100: 1 to about 5: 1, 80: 1 to about 5: 1, 60: 1 to about 5: 1, or about 50: 1 to about 5: 1). In some embodiments, the ratio of a plurality of lipids to an PNA (e.g., a PNA oligomer) is about 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75:1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 28: 1, 26: 1, 24: 1, 25: 1, 22: 1, 20: 1, 18: 1, 16:1, 14: 1, 12: 1, 10: 1, 8: 1, 6: 1, 4: 1, 2: 1, 1 : 1, 1 :2, 1 :4, 1 :6, 1 :8, 1 : 10, 1 : 12, 1 : 14, 1 : 16, 1 : 18, 1 :20, 1 :22, 1 :24, 1 :25, 1 :26, 1 :28, 1 :30, 1 :35, 1 :40, 1 :45, 1:50, 1 :55, 1 :60, 1 :65, 1 :70, 1 :75, 1 :80. 1 :85, 1 :90, 1 :95, or 1 :100.

[00174] In some embodiments, an LNP described herein has a diameter between 5 and 500 nm, e.g., between 10 and 400 nm, 20 and 350 nm, 25 and 325nm, 30 and 300nm, 50 and 250 nm, 60 and 200 nm, 75 and 190 nm, 80 and 180 nm, 100 and 200 nm, 200 and 300 nm, and 150 and 250 nm. The diameter of an LNP may be determined by any method known in the art, for example, dynamic light scattering, transmission electron microscopy (TEM) or scanning electron microscopy (SEM). In some embodiments, an LNP has a diameter between 50 and 100 nm, between 70 and 100 nm, and between 80 and 100 nm. In an embodiment, an LNP has a diameter of about 90 nm. In some embodiments, an LNP described herein has a diameter greater than about 30 nm. In some embodiments, an LNP has a diameter greater than about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 225 nm, about 250 nm, about 275 nm or about 300 nm. In an embodiment, an LNP has a diameter greater than about 70 nm. In an embodiment, an LNP has a diameter greater than about 90 nm. In an embodiment, an LNP has a diameter greater than about 180 nm.

[00175] In some embodiments, a plurality of LNPs described herein has an average diameter greater than about 30 nm. In some embodiments, a plurality of LNPs has an average diameter greater than about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, or about 300 nm. In an embodiment, a plurality of LNPs has an average diameter greater than about 70 nm. In an embodiment, a plurality of LNPs has an average diameter greater than about 90 nm. In an embodiment, a plurality of LNPs has an average diameter greater than about 180 nm.

[00176] In some embodiments, a synthetic polymer nanoparticle or a plurality of nanoparticles have an average diameter between 5 and 500 nm, e.g., between 10 and 400 nm, 20 and 350 nm, 25 and 325 nm, 30 and 300 nm, 50 and 250 nm, 60 and 200 nm, 75 and 190 nm, 80 and 180 nm, 100 and 200 nm, 200 and 300 nm, and 150 and 250 nm. In some embodiments, a plurality of nanoparticles has an average diameter between 150 and 300 nm, between 35 and 100 nm, and between 75 and 220 nm.

[00177] In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nanoparticles of a plurality of nanoparticles have a diameter between 5 and 500 nm. The nanoparticles may range in diameter from about 10 to 400 nm, 20 to 300 nm, 25 to 250 nm, 30 to 150 nm, 35to 125nm, 40 to 100 nm, 80 to 180 nm, 100 to 200 nm, 200 to 300 nm, and 150 to 250 nm. In some embodiments, the nanoparticles may range in diameter from about 100 to 200 nm, 20 to 100 nm, 20 to 80 nm, and between 20 to 60 nm. [00178] In some embodiments, a nanoparticle or plurality of nanoparticles described herein has an average neutral to negative surface charge of less than -100 mV, for example, less than -90 mV, -80 mV, -70 mV, -60 mV, -50 mV, -40 mV, -30 mV, and -20 mV. In some embodiments, a nanoparticle or plurality of nanoparticles has an average surface charge of between -100 mV and 100 mV, between -75 mv and 0, or between -50 mv and -10 mv.

[00179] In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nanoparticles of a plurality of nanoparticles have an average neutral to negative surface charge of less than -100 mV. In some embodiments, at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the nanoparticles of a plurality of nanoparticles have an average neutral to positive surface charge of more than 1 mV. In some embodiments, a nanoparticle or plurality of nanoparticles has an average surface charge of between -100 mV and 100 mV, between -75 mV and 0, or between -50 mV and -10 mV.

Methods of Making Peptide Nucleic Acids

[00180] Described herein are methods for making peptide nucleic acid (PNA) oligomers comprising at least one 2-aminopyridine nucleobase. A PNA oligomer comprising at least one 2- aminopyridine nucleobase may be prepared through the stepwise addition of individual subunits, e.g., by reacting the amine of a first PNA subunit with a carboxylic acid of a second PNA subunit (e.g., an activated form of a carboxylic acid of a second subunit). In some embodiments, a PNA oligomer may be prepared by the stepwise addition of a first amino acid (e.g., lysine) to a second or subsequent amino acid (e.g., lysine). In some embodiments, a PNA oligomer may be prepared by the stepwise addition of a PNA subunit to an amino acid (e.g. a lysine). In some embodiments, a PNA oligomer may be prepared by the stepwise addition of an amino acid (e.g., a lysine) to a PNA subunit. A PNA oligomer may also be prepared through coupling smaller PNA oligomers comprising more than one subunit (e.g., through block synthesis; see, for example: US Patent No. 7,256,275).

[00181] A PNA oligomer may be synthesized in solution or on a solid support, or by using a combination of both techniques. PNA oligomers may be prepared using automated methods, for example, using an automated peptide synthesizer. A PNA oligomer may be prepared using a standard method known in the art; see, for example, Peptide Nucleic Acids, Protocols and Applications, 2nd Ed, Edited by Peter E. Nielsen, Horizon Bioscience, 2004; which is incorporated herein by reference in its entirety.

[00182] In an embodiment, the PNA oligomer is synthesized on a solid support. A solid support may be supplied in the form beads, and may be of different shapes (e.g. spherical beads) and sizes (e.g., 100 mesh, 150 mesh, 200 mesh, 250 mesh, 300 mesh, 350 mesh, 400 mesh, 450 mesh, 500 mesh). A solid support may comprise, for example, plastic, polymer, polystyrene, polyacrylate, polyacrylamide, or polyethyleneglycol. Polymers used in solid supports may be cross-linked (e.g., with 1-2% divinylbenzene), or uncrosslinked. A solid support may be a functionalized polymer (e.g., a Merrifield resin, Wang resin, brominated Wang resin, 4-(l ', 1 dimethyl-l'-hydroxypropyl)phenoxyacetyl (DHPP) resin, Kaiser resin, 4-hydroxymethyl- phenylacetamidomethyl (PAM) resin, benzhydrylamine (BHA) resin, 4-methylbenzhydrylamine (MBHA) resin, diphenyldiazomethane (PDDM) resin, TentaGelresin,4-(hydroxymethyl) phenoxyaceticacid (HMPA) resin, 4-(4-hydroxymethyl-3 -meth oxyphenoxy )butyric acid (HMPB) resin, 2-chlorotrityl resin, 4-carboxytrityl resin, Rink acid resin, Rink amide resin, peptide amide linker (PAL) resin, Sieber resin, 4-(hydroxymethyl)benzoylaminomethyl (HMBA) resin, 4-sulfamoylbenzoyl resin, or 4-(4-formyl-3-methoxyphenoxy)ethyl (FMP) resin). A solid support may comprise a functional group suitable for coupling to a subunit. In some embodiments, the functional group is an amine, a carboxylic acid, a halide, an oxime, a hydroxyl, a sulfamoyl, a hydrazine, or an aldehyde. In some embodiments, the functional group is an amine. Exemplary solid supports include Merrifield resin, Wang resin, MBHA resin, and Rink amide resin. In an embodiment, the PNA oligomer is synthesized on rink amide TentaGel resin (Rapp polymer, R28023).

[00183] In an embodiment, the PNA oligomer is formed by anchoring a first subunit onto a solid support. In some embodiments, the first subunit is an amino acid (e.g., lysine) or a PNA subunit. The first subunit may comprise one or more protecting groups, for example, a PNA subunit comprising a nucleobase that optionally comprises a protecting group, a PNA subunit with an activated carboxylatic acid, a PNA subunit with a protected amine, a PNA subunit with ana-side chain that is optionally protected, a PNA subunit with a P-side chain that is optionally protected, a PNA subunit with a y-side chain that is optionally protected, or any combination thereof. The first subunit may comprise a protecting group (PG) on the amino terminus, such as Fmoc or Boc. The anchoring of the first subunit to a solid support may further require use of a base (e.g., an organic base, e.g., diisopropylethylamine (DIPEA), triethylamine (TEA), collidine, pyridine, piperidine, methyldicyclohexylamine (MDCHA). In some embodiments, the anchoring of the first subunit to a solid support may further require an activating agent such asa carbodiimide (e.g., N,N’ -di cyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC)), benzotriazole (e.g., hydroxybenzotriazole (HOBt), l-hydroxy-7-azabenzotriazole (HO At), 3 -(di ethoxyphosphoryloxy)- 1,2, 3 -benzotriazin-4(3H)-one (DEPBT)), a phosphonium salt (e.g. (benzotri azol- l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP)), a uronium salt (e.g., hexafluorophosphate benzotriazole tetramethyl uronium(HBTU), 2-(lH-benzotriazole-l- yl)-l,l,3,3-tetramethylaminium tetrafluoroborate (TBTU), hexafluorophosphate azabenzotriazoletetramethyl uronium(HATU), O-(7-azabenzotriazol-l -yl)-N,N,N’N’- tetramethyl uroniumtetrafluorob orate (TATU), O-[(ethoxycarbonyl)cyanomethylenamino]- N,N,N’,N’ -tetra methyluronium tetrafluoroborate (TOTU)), or a fluoroformamidinium salt (e.g.,tetramethylfluoroformamidiniumhexafluorophosphate (TFFH), bis(tetramethylene)fluoroformamidiniumhexafluorophospnate(BT FFH)). The reaction that anchors the subunit to the solid support may be carried out in any appropriate solvent (e.g., dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), N- methylpyrrolidone (NMP), tetrahydrofuran (THF), dioxane, dichloromethane (DCM), or mixtures thereof. Generally, the solvent is an aprotic organic solvent. Once anchored to a solid support, the subunit may then be extended by one or more additional subunits (e.g. PNA subunit, amino acid, linker, label, solubility enhancer, etc.) to form an oligomer (e.g., a PNA oligomer). [00184] Synthesis of a PNA oligomer on a solid support typically entails repetition of a cycle of steps to extend the growing PNA oligomer. In general, each cycle comprises three steps, deprotection, coupling and capping.

[00185] In some embodiments, the first step of each cycle involves cleavage of a protecting group (PG) such as Fmoc or Boc from the terminus (e.g., the N-terminus) of the solid-supported PNA using a suitable reagent (i.e. deprotection). In some embodiments, deprotection of the PG is achieved with an organic base (e.g., piperidine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), DIPEA or collidine) or an acid (e.g., TFA, trifluoromethanesulfonic acid (TFMSA), hydrochloric acid, or hydrofluoric acid).

[00186] In some embodiments, the second step of the cycle (i.e. the coupling step) involves contacting the solid-supported PNA with a second or subsequent subunit(s) dissolved in a solvent (e.g., DMF, NMP, or a mixture of solvents). The second step of the cycle may also involve activation of the free carboxylic acid of the second or subsequent subunit(s). Activation may require use of a base (e.g., DIPEA, TEA, collidine, pyridine, or piperidine). In some embodiments, the carboxylic acid of the first subunit is activated with an activating agent such as a carbodiimide (e.g., DCC, DIC), benzotriazole (e.g., HOBt, HO At, DEPBT), a phosphonium salt (e g. BOP, PyBOP, PyBrop) or uronium salt (e.g, HBTU, TBTU, HATU, TATU, TOTU), or a fluoroformamidinium salt (e.g., TFFH, BTFFH). Once activated, the carboxyl group may then react with the PG-free terminus of the solid-supported PNA to form a peptide bond and extend the PNA oligomer.

[00187] In some embodiments, an excess of the first subunit is used. In some embodiments, the ratio of the first subunit to the second subunit is about 1 : 1, about 1 : 1.1, about 1 : 1.2, about 1 : 1.3, about 1 : 1.4, about 1 : 1.5; about 1.1.75; about 1 :2, about 1 :2.5, about 1 :3, 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, or about 1 : 10. In some embodiments, an excess of the second or subsequent subunit(s) is used. In some embodiments, the ratio of the second or subsequent subunit(s) to the first subunit is about 1 : 1, about 1 : 1.1, about 1 : 1.2, about 1 : 1.3, about 1 : 1.4, about 1 : 1.5; about 1.1.75; about 1 :2, about 1 :2.5, about 1 :3, 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, or about 1 : 10.

[00188] The third step of the cycle may entail capping. Capping is used to terminate elongation of any particular oligomer that has not undergone elongation during the coupling step. In this way, oligomers possessing a deleted subunit are not created and can be easily purified away from the desired product - post synthesis.

[00189] In some embodiments, the subunit is a PNA subunit, an amino acid (e.g., lysine), a linker a label, or a solubility enhancer (See for Example: US Patent Nos. 6,326,479 & 6,770,442). In some embodiments, the linker is a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a C6-C20 PEG linker (e.g., a PEG2 or PEG3 linker). In some embodiments, the subunit is a C12-PEG linker. In some embodiments, a PNA oligomer comprises one type of subunit. In some embodiments a PNA oligomer comprises more than one type of subunit. In some embodiments, a PNA oligomer comprises all types of subunits (e.g. PNA subunits, amino acids, linkers, labels, solubility enhancers and the like).

[00190] In some embodiments, the solid-supported PNA is washed with an appropriate solvent (e.g., dimethylformamide, dimethylacetamide, dimethylsulfoxide, A-methylpyrrolidone, tetrahydrofuran, dioxane, dichloromethane, or mixtures thereof) and filtered between each coupling step. In some embodiments, each coupling step is also carried out in one or more of these solvents. In some embodiments, the solid-supported PNA oligomer is extended through multiple cycles of the above described steps. These steps may be carried out manually, or by using the semi-automated or fully-automated instruments discussed above, or any combination of these methods.

[00191] Once the desired subunits and other components are added to the PNA oligomer, the synthesis may be terminated, or the PNA oligomer may be modified further, for example, through acetylation or other end-capping methods. In some embodiments, the PNA oligomer is subjected to selective deprotection of the PNA side chains or PNA nucleobases. In some embodiments, the PNA oligomer is fully deprotected before cleavage. In some embodiments, the fully deprotected PNA oligomer is modified prior to cleavage.

[00192] In some embodiments, a final step involving cleavage of the PNA oligomer from the solid support is carried out. This step may involve treatment of the solid-supported PNA oligomer with an acid, base, nucleophile, phenol (e.g. meta cresol), thiol, or photolysis, or combination of two or more of the foregoing. For example, a PNA oligomer may be cleaved from the solid support through incubation with an acid (e.g., trifluoroacetic acid, hydrofluoric acid, trifluoromethanesulfonic acid, trimethylsilyl trifluoromethanesulfonate, hydrobromic acid) or in some cases an alcohol (e.g., hexafluoroisopropanol). In some embodiments, cleavage of the PNA oligomer from the solid support may also effect removal of the protecting groups from the PNA side chains and/or the PNA nucleobases. In some embodiments, scavengers such as water, sulfides, thiols, phenols, and silanes may be used in the final cleavage step to prevent side-reactions or racemization of any chiral centers in the PNA oligomer. In some embodiments, residual acid or other reagents or side-products from the cleavage step may be removed through trituration, filtration, dialysis, chromatography, or other purification methods. [00193] A PNA oligomer may be synthesized using solution phase synthesis. Many of the same methods outlined above for solid-phase peptide synthesis apply to solution phase peptide synthesis. The use of protecting group manipulations, activating agents, and the sequential addition of PNA units to the growing oligomer are similarly applied. One distinction is that solution phase peptide synthesis does not use a solid support (e.g., a resin or beads).

Subsequently, each step of the repeating cycle to grow the PNA oligomer may require purifying the oligomer from the reaction mixture using techniques such as extraction, trituration, column chromatography, HPLC, or other common purification techniques. As no solid support is used in solution phase peptide synthesis, no cleavage from a resin is required; however there may be one or more steps required to remove at least one or all protecting groups from the PNA oligomer. Furthermore, in the absence of a solid-support, one or both termini (e.g., the carboxyl terminus and the amine termini) of the PNA oligomer may also be protected during PNA oligomer preparation, and removal of the protecting group(s) or modification to introduce an end-cap or other modification of the PNA oligomer may be required.

[00194] A PNA oligomer may be synthesized using a combination of solid phase and solution phase methods.

[00195] In some embodiments the PNA oligomer is purified after synthesis. Exemplary methods of purification include silica gel chromatography, high performance liquid chromatography (HPLC), extraction, and/or trituration. For example, a PNA oligomer may be purified on a C18 reverse phase column using a solvent system (e.g., 1% TFA, acetonitrile and water). In some embodiments, an isocratic elution is used. In some embodiments, a gradient elution is used.

[00196] In some embodiments, the PNA oligomer is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% pure. Purity of a PNA oligomer may be determined by any known method in the art, for example, through HPLC analysis.

[00197] After synthesis, the PNA oligomer may be characterized to confirm the identity of the PNA sequence. Characterization methods include mass spectrometry (including liquid chromatography mass spectrometry (LCMS), nuclear magnetic resonance (NMR) spectroscopy, Raman spectroscopy, infrared spectroscopy, HPLC, fluorimetry, X-ray crystallography.

[00198] In some embodiments, the individual subunits in the oligomerization reaction are vacuum dried prior to use in the reaction. In some embodiments, the individual subunits in the oligomerization reaction are freshly distilled over a drying agent (e.g., with CaH2 or K2CO3), purified, recrystallized, or dried prior to use in the reaction. In some embodiments, the individual subunits in the oligomerization reaction are synthesized prior to use. In some embodiments, the individual subunits in the oligomerization reaction are commercially available.

Methods of Making Nanoparticles

[00199] Described herein are methods for producing a nanoparticle that comprises PNA oligomers comprising a 2-aminopyridine nucleobase and optionally other components (e.g., nucleic acids). In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the nanoparticle is a nanoparticle comprising a synthetic polymer (e.g., a nanoparticle comprising PLGA). A method of forming such nanoparticles may require a double emulsion process, single emulsion process or a process involving mixing premade solutions to effect nanoprecipitation.

Lipid Nanoparticles (LNPs)

[00200] The method of making an LNP comprising a PNA oligomer may entail mixing a first solution with a second solution. In some embodiments, the first solution comprises a lipid or a plurality of lipids and a PNA oligomer, e.g., a tcPNA oligomer, in a solvent. The solvent may be any water miscible solvent (e.g., ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane or tetrahydrofuran). In some embodiments, the first solution comprises a small percentage of water. The first solution may comprise up to at least 60% by volume of water, e.g., up to at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume of water. In an embodiment, the first solution comprises between about 0.05% and 60% by volume of water, e.g., between about 0.05% and 50%, about0.05% and 40%, or about 5% and 20% by volume of water.

[00201] The first solution may comprise a single type of PNA oligomer or a plurality of PNA oligomers, e.g., of different PNA sequences. In an embodiment, the first solution comprises a single type of PNA oligomer (e.g., a tcPNA oligomer). In an embodiment, the first solution comprises a plurality of PNA oligomers (e.g., tcPNA oligomers), wherein the PNAs comprise different sequences and bind to different target nucleic acid sequences.

[00202] In some embodiments, the first solution comprises a single type of lipid, for example, an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the first solution comprises a plurality of lipids. In some embodiments, the plurality comprises an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid. In some embodiments, the plurality of lipids comprise cholesterol, l,2-distearoyl-sw-glycero-3-phosphocholine (DSPC),l,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene20 00 (DMG-PEG2k), anddilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA). The plurality of lipids may exist in any ratio. In an embodiment, the plurality of lipids comprises an ionizable lipid, a phospholipid, a sterol, or a PEG-containing lipid of the above lipids in a particular ratio (e.g., a ratio described herein).

[00203] In some embodiments, the second solution is water. In some embodiments, the second solution is an aqueous buffer. The second solution may comprise a load component, e.g., a nucleic acid (e.g., a single-stranded DNA). In some embodiments, the nucleic acid is a DNA oligomer (e.g. a donor DNA). The second solution may comprise a small percentage of water- miscible organic solvent. The second solution may comprise up to at least 60% by volume of at least one water miscible organic solvent, e.g., up to at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume of at least one organic solvent (e.g., a water miscible organic solvent). In an embodiment, the second solution comprises between about 0.05% and 60% by volume of organic solvent, e.g., between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume of organic solvent(e.g., a water miscible organic solvent). The aqueous buffer solution may be an aqueous solution of citrate buffer. In some embodiments, the aqueous buffer solution is a citrate buffer solution with a pH between 4-6 (e.g., a pH of about 4, about 5, or about 6). In an embodiment, the aqueous buffer solution is a citrate buffer solution with a pH of about 6. [00204] In some embodiments, the solution comprising a mixture of the first and second solutions comprising the LNP suspension can be diluted. In some embodiments, the pH of the solution comprising a mixture of the first and second solutions comprising the LNP suspension can be adjusted. Dilution or adjustment of the pH of the LNP suspension may be achieved with the addition of water, acid, base or aqueous buffer. In some embodiments, no dilution or adjustment of the pH of the LNP suspension is carried out. In some embodiments, both dilution and adjustment of the pH of the LNP suspension is carried out.

[00205] In some embodiments, excess reagents, solvents, free PNA or free nucleic acid maybe removed from the LNP suspension by tangential flow filtration (TFF) (e.g., diafiltration). The organic solvent (e.g., ethanol) and buffer may also be removed from the LNP suspension with TFF. In some embodiments, the LNP suspension is subjected to dialysis and not TFF. In some embodiments, the LNP suspension is subjected to TFF and not dialysis. In some embodiments, the LNP suspension is subjected to both dialysis and TFF.

[00206] In one aspect, the present disclosure features a method comprising treating a sample of LNPs comprising PNAs and optionally nucleic acids, with a fluid comprising a detergent (e.g., Triton X-100) for a period of time suitable to degrade the lipid layer and thereby release the encapsulated and/or entrapped PNA(s) and optionally nucleic acid(s). In an embodiment, the method further comprises analyzing the sample for the presence, absence, and/or amount of the released PNA(s) and optionally nucleic acid(s).

[00207] In some embodiments, the present disclosure features a method of manufacturing, or evaluating, an LNP or preparation of LNPs comprising providing a preparation of LNPs described herein, and acquiring, directly or indirectly, a value for a preparation parameter. In an embodiment, the method further comprises making the preparation of LNPs by a method described herein. In an embodiment, the method further comprises evaluating the value for the preparation parameter, e.g., by comparing it with a standard or reference value. In an embodiment, wherein responsive to the evaluation, the method further comprises selecting a course of action, and optionally, performing the action. For example, the method may comprise providing a preparation of LNPs comprising a PNA, acquiring a value for a preparation parameter (e.g., average particle size), evaluating the preparation the value of the preparation parameter by comparing it with a standard or reference value, selecting a course of action (e.g., selecting to administer the preparation of LNPs to a subject), and performing the action (administering the preparation of LNPs to a subject). Synthetic Polymer Nanoparticles

[00208] The method of making a synthetic polymer nanoparticle comprising a PNA oligomer may entail mixing a first solution with a second solution. In some embodiments, the first solution comprises a PNA oligomer (e.g. a tcPNA oligomer) and synthetic polymer (e.g., PLGA) or mixture of synthetic polymers in a solvent. The solvent may be any water-miscible solvent (e.g., ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dioxane, tetrahydrofuran). In some embodiments, the first solution comprises a small percentage of water. The first solution may comprise up to at least 60% by volume of at water, e.g., up to at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume of water. In an embodiment, the first solution comprises between about 0.05% and 60% by volume water, e.g., between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume water.

[00209] The first solution may comprise a single type of PNA oligomer or a plurality of PNA oligomers, e.g., of different PNA sequences. In an embodiment, the first solution comprises a single type of PNA oligomer, e.g., a tcPNA oligomer. In an embodiment, the first solution comprises a plurality of PNA oligomers, e.g., tcPNA oligomer, wherein the PNAs comprise different sequences and bind to different target nucleic acid sequences.

[00210] In some embodiments, the synthetic polymer is polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(4-hydroxy-L-proline ester), a polyester, a polyanhydride, a poly(ortho)ester, a polyurethane, a poly(butyric acid), poly(valeric acid), poly(caprolactone), a poly(hydroxyalkanoate), a poly(lactide-co-caprolactone), a poly(amine-co- ester) polymer, or a combination of any two or more thereof. In some embodiments, the synthetic polymer is PLGA.

[00211] In some embodiments the second solution is water. In some embodiments, the second solution is an aqueous buffer. The second solution may comprise a load component, e.g., a nucleic acid (e.g., a single-stranded DNA). In some embodiments, the nucleic acid is a DNA oligomer (e.g., a donor DNA). The second solution may comprise a small percentage of water miscible organic solvent. The second solution may comprise up to at least 60% by volume of at least one water miscible organic solvent, e.g., up to at least about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume of at least one organic solvent (e.g., a water miscible organic solvent). In an embodiment, the second solution comprises between about 0.05% and 60% by volume organic solvent, e.g., between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume organic solvent (e.g., a water miscible organic solvent). In an embodiment, the aqueous buffer solution is a citrate buffer. The aqueous buffer solution may be an aqueous solution with a pH between 3-9 (e.g., a pH between about 3-8, about 3-7, about 3-6, about 3-5, about 3-6, about 4-8, about 4- 7, about 4-6, about 4-5, about 5-8, about 5-7, about 5-6, about 6-8, about 6-7, about 7-8, about 8- 9). In an embodiment, the aqueous buffer solution has a pH of about 3. In an embodiment, the aqueous buffer solution has a pH of about 4. In an embodiment, the aqueous buffer solution has a pH of about 5. In an embodiment, the aqueous buffer solution has a pH of about 6. In an embodiment, the aqueous buffer solution has a pH of about 7. In an embodiment, the aqueous buffer solution has a pH of about 8. In an embodiment, the aqueous buffer solution has a pH of about 9.

[00212] In some embodiments, the process involves mixing the above solutions to produce nanoparticles that encapsulate the PNA oligomer and, optionally, a nucleic acid load component. The process may further require dilution (e.g., with water or a buffer). In some embodiments, the process involves the introduction of a surface stabilizer (e.g., trehalose, sucrose, or cyclodextrin). The process may involve diafiltration to remove excess reagents, nonencapsulated PNA oligomers or DNA, solvents, or buffers from the nanoparticles. In some embodiments, dialysis is used to remove excess reagents, non-encapsulated PNA oligomers or DNA, solvents, or buffers from the nanoparticles. The process may further require sterilization of the nanoparticles, for example by filtration through a filter of a select pore size (e.g., 0.2 pM) to remove microbes. The method may additionally feature the addition of cryoprotectants, excipients, or other components. The method may require transferring the nanoparticles to containers suitable for distribution and use for administration. The nanoparticles may be stored at low temperatures, such as at 0 °C, -20 °C, -80°C lower.

[00213] In some embodiments, the loading of PNA oligomer and other components in the nanoparticle are analyzed through many methods. In some embodiments, analysis involves digesting nanoparticles by treatment with ammonia or dimethylsulfoxide (DMSO) to release encapsulated PNA oligomers and any other encapsulated components (e.g., DNA). The amount of total PNA and DNA in the digest can then be determined by spectroscopic methods (e.g., UV absorbance), HPLC, or other means (e.g., OliGreen/RiboGreen methods). In some embodiments, nanoparticles are analyzed by scanning electron microscopy (SEM) techniques. For example, nanoparticles may be coated in platinum and imaged using SEM to determine size and morphology of the nanoparticles.

Gene Targeting Compositions and Methods of Treatment

[00214] Described herein are PNA oligomers comprising a PNA subunit comprising a 2- aminopyridine nucleobase and compositions thereof, as well as methods of using the same to alter a target nucleic acid sequence. The methods of using the PNA oligomers comprising a 2- aminopyridinenucleobase may be performed in vitro (e.g., in an in vitro cell system) or in vivo (e.g., in a subject, e.g., a human subject). In some embodiments, the method is performed in an in vitro cell free system. In some embodiments, the method is performed in a cell. The cell may be a cultured cell, e.g., a cell from a cell line, or may be a cell derived from a subject. In some embodiments, the method is performed in vivo, e.g., in a subject. The subject may be a mammal (e.g., a mouse, other non-human primate or a human).

[00215] The target nucleic acid sequence used in the described methods may be single-stranded or double-stranded. In some embodiments, altering a target nucleic acid sequence comprises altering a target double-stranded nucleic acid sequence. Altering a target double-stranded nucleic acid sequence may comprise one or more of: a) altering the state of association of the two strands of a target double-stranded nucleic acid sequence; b) altering the helical structure of a target double-stranded nucleic acid sequence; c) altering the topology in a strand of a doublestranded nucleic acid sequence, for example, by introducing a kink or bend in a strand of the target double-stranded nucleic acid sequence; d) recruiting a nucleic acid-modifying protein (e.g., enzyme), for example, a member of the nucleotide excision repair pathway, to a target double stranded nucleic acid. Exemplary members of the nucleotide excision repair pathway include XPA, RPA, XPF, and XPG, or a functional variant or fragment thereof; e) cleaving a strand of a target double stranded nucleic acid; or f) altering the sequence of a target double stranded nucleic acid. In some embodiments, the sequence of a target double stranded nucleic acid is altered to the sequence of a template nucleic acid. In some embodiments, the sequence of a target double stranded nucleic acid is altered from a mutant or disorder-associated sequence (e.g., allele) to a non-mutant or non-disease associated sequence (e.g., allele) a subject having a disease, disorder, or condition.

[00216] In some embodiments, altering a nucleic acid comprises two of (a)-(f). In some embodiments, altering a nucleic acid comprises three of (a)-(f). In some embodiments, altering a nucleic acid comprises four of (a)-(f). In some embodiments, altering a nucleic acid comprises five of (a)-(f). In some embodiments, altering a nucleic acid comprises each of (a)-(f). In some embodiments, altering a nucleic acid comprises (a). In some embodiments, altering a nucleic acid comprises (b). In some embodiments, altering a nucleic acid comprises (c). In some embodiments, altering a nucleic acid comprises (d). In some embodiments, altering a nucleic acid comprises (e). In some embodiments, altering a nucleic acid comprises (f).

[00217] The PNA oligomer comprising one or more 2-aminopyridine nucleobases may promote a particular effect in a target nucleic acid sequence. For example, the PNA oligomer may bind a target nucleic acid sequence, which may provide a decrease in the melting point (Tm) of the target nucleic acid sequence (e.g., a decrease of about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, or more), may promote melting or dissociation of the strands of the target nucleic acid sequence (e.g., a melting or dissociation of about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, or more of the strands of the target sequence), may cleave the target nucleic acid sequence (e.g., effect cleavage in about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, or more target nucleic acid sequences), may edit the target nucleic acid sequence (e.g., edit about 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, or more of the strands of the target sequence).

[00218] In some embodiments, the PNA oligomer comprising one or more 2-aminopyridine nucleobases may induce gene modification in at least one target allele to occur at frequency of at least 0.1, 0.2. 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% of target cells. In some embodiments, gene modification occurs in at least one target allele at a frequency of about 0.1-25%, or 0.5-25%, or 1-25% 2- 25%, or 3-25%, or 4-25% or 5-25% or 6-25%, or 7-25%, or 8-25%, or 9-25%, or 10-25%, 11- 25%, or 12-25%, or 13%-25% or 14%-25% or 15-25%, or 2-20%, or 3-20%, or 4-20% or 5-20% or 6-20%, or 7-20%, or 8-20%, or 9-20%, or 10-20%, 11-20%, or 12-20%, or 13%-20% or 14%- 20% or 15-20%, 2-15%, or 3-15%, or 4-15% or 5-15% or 6-15%, or 7-15%, or 8-15%, or 9- 15%, or 10-15%, 11-15%, or 12-15%, or 13%-15% or 14%-15%.

[00219] In some embodiments, a PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is administered at a particular dosage, e.g., a therapeutically effective dosage. Exemplary dosages may be expressed in mg/kg of the subject, and may be, for example, 0.1 mg/kg to about 1,000 mg/kg, or 0.5 mg/kg to about 1,000 mg/kg, or 1 mg/kg to about 1,000 mg/kg, or about 10 mg/kg to about 500 mg/kg, or about 20 mg/kg to about 500 mg/kg per dose, or 20 mg/kg to about 100 mg/kg per dose, or 25 mg/kg to about 75 mg/kg per dose, or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mg/kg per dose.

[00220] In some embodiments, the PNA oligomer comprising one or more 2-aminopyridine nucleobases alters a target nucleic acid sequence with little to no off-target effects. In some embodiments, off-target modification of a nucleic acid is undetectable using routine analysis, e.g., nucleic acid sequencing. In some embodiments, off-target modification of a nucleic acid occurs at a frequency of 0-1%, or 0-0.1%, or 0-0.01%, or 0-0.001%, or 0-0.0001%, or 0- 0000.1%, or 0-0.000001%. In some embodiments, off-target modification of a nucleic acid occurs at a frequency that is about 10 ² , 10 ³ , 10 ⁴ , or 10 ⁵ -fold lower than at the target nucleic acid sequence.

Methods of Treatment

[00221] The PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof (e.g. a nanoparticle) may further be used in a method to prevent and/or treat a subject having a particular disease, disorder or condition. In an embodiment, the method comprises administering to a subject a PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof (e.g., a nanoparticle comprising the PNA oligomer). In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases is administered to the subject in a therapeutically effective amount, e.g., a dosage sufficient to prevent, treat, or inhibit a symptom of a disease, disorder or condition. In an embodiment, the disease, disorder, or condition is a human genetic disease, for example, in which at least one addition, deletion or mutation is present in an allele compared to a non-disease control. Exemplary diseases, disorders, or conditions that may be prevented or treated with the PNA oligomers and compositions thereof described herein include cystic fibrosis, hemophilia, a globinopathy (e.g., sickle cell anemia, beta-thalassemia), xeroderma pigmentosum, a lysosomal storage disease, or a cancer (e.g., a cancer related to PD-1). In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is used in a method to treat cystic fibrosis in a subject. In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is used in a method to treat hemophilia in a subject. In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is used in a method to treat a globinopathy (e.g., sickle cell anemia, beta-thalassemia) in a subject. In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is used in a method to treat xeroderma pigmentosum in a subject. In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is used in a method to treat a lysosomal storage disease in a subject. In an embodiment, the PNA oligomer comprising one or more 2-aminopyridine nucleobases or a composition thereof is used in a method to treat a cancer in a subject.

NUMBERED EMBODIMENTS

[00222] 1. A peptide nucleic acid (PNA) oligomer, comprising a first region linked to a second region via a linker or a covalent bond, wherein: a) the first region comprises a plurality of PNA subunits having Hoogsteen complementarity with a target sequence, wherein at least one of the PNA subunits comprises a 2-aminopyridine nucleobase; and b) the second region comprises a subregion, wherein the subregion consists of a plurality of PNA subunits having complete Watson Crick complementarity with the target sequence. [00223] 2. The PNA oligomer of embodiment 1, wherein the PNA oligomer comprises a gamma modified PNA subunit. [00224] 3. The PNA oligomer of embodiment 2, wherein the first region comprises the gamma modified PNA subunit.

[00225] 4. The PNA oligomer of embodiment 2, wherein the second region comprises the gamma modified PNA subunit.

[00226] 5. The PNA oligomer of embodiment 2, wherein the PNA oligomer comprises a gamma modified PNA subunit in the first region and the second region.

[00227] 6. The PNA oligomer of any one of embodiments 1-5, wherein the first region comprises an abasic subunit.

[00228] 7. The PNA oligomer of embodiment 6, wherein the abasic subunit is -Gly-Gly-.

[00229] 8. The PNA oligomer of embodiment 6 or embodiment 7, wherein the abasic subunit has a position within the first region that corresponds to a position of a purine nucleobase of the target sequence.

[00230] 9. The PNA oligomer of any one of embodiments 1-8, further comprising: a first positively charged region linked to the first region by a second linker or covalent bond, wherein the first positively charged region comprises a positively charged amino acid subunit; or a second positively charged region linked to the second region via a third linker or covalent bond, wherein the second positively charged region comprises a positively charged amino acid subunit.

[00231] 10. The PNA oligomer of embodiment 9, comprising: the first positively charged region.

[00232] 11. The PNA oligomer of embodiment 9, comprising: the first positively charged region and the second positively charged region.

[00233] 12. The PNA oligomer of any one of embodiments 9-11, wherein the first positively charged region is linked to the first region via a covalent bond.

[00234] 13. The PNA oligomer of any one of embodiments 9-12, wherein the second positively charged region is linked to the second region via a covalent bond.

[00235] 14. The PNA oligomer of any one of embodiments 1-13, wherein the second region further comprises another subregion, wherein the another subregion consists of a plurality of PNA subunits having complete Watson Crick complementarity with a tail target sequence, wherein the tail target sequence is a sequence contiguous with the target sequence and is not bound by the first region.

[00236] 15. The PNA oligomer of embodiment 14, wherein the subregion is contiguous with the another subregion.

[00237] 16. The PNA oligomer of any one of embodiments 1-15, wherein the PNA oligomer is a PNA clamp oligomer.

[00238] 17. The PNA oligomer of any one of embodiments 1-16, wherein the PNA oligomer is a PNA tail-clamp oligomer (tcPNA).

[00239] 18. The PNA oligomer of any one of embodiments 1-17, wherein the first region is linked to the second region by the linker, wherein linker comprises a polyalkylene glycol group. [00240] 19. The PNA oligomer of any one of embodiments 1-18, wherein the linker is - [NH(CH2CH ₂ O)zCH ₂ C(O)]w- or -[NHCH ₂ CH2(OCH ₂ CH2)zC(O)]w-, wherein each z is independently 1-100, and each w is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[00241] 20. The PNA oligomer of embodiment 19, wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[00242] 21. The PNA oligomer of embodiment 19 or embodiment 20, wherein w is 1 or 2.

[00243] 22. The PNA oligomer of any one of embodiments 1-21, wherein the linker is - [NH(CH2CH2O)2CH2C(O)]2-.

[00244] 23. The PNA oligomer of any one of embodiments 1-21, wherein the linker is - NH(CH2CH2O)3CH2C(O)-.

[00245] 24. The PNA oligomer of any one of embodiments 1-23, wherein the plurality of PNA subunits of the first region has complete Hoogsteen complementarity with the target sequence.

[00246] 25. The PNA oligomer of any one of embodiments 1-24, wherein the PNA oligomer comprises a PNA subunit of Formula (I-b): or a salt thereof, wherein:

R ² is hydrogen or Ci-Cualkyl; each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen, deuterium, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, Ci-Cuheteroalkyl, Ci-Ci2-haloalkyl, -OR ^A , cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, -x AO. /A

^A O ^ ^ OR ¹² heteroaryl, C1-C12 alkylene-heteroaryl, ^y (IV-a), or the side chain of an optionally protected amino acid, wherein each alkyl, alkylene, alkenyl, alkenylene, alkynyl, heteroalkyl, haloalkyl, cycloalkyl, and heterocyclyl may be optionally substituted with one or more R ¹⁰ ; each of R ^9a and is R ^9b is independently hydrogen, Ci-Cualkyl, or an amine protecting group (e.g., Boc); each R ¹⁰ is independently halo, cyano, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, Ci- C12 heteroalkyl, Ci-Ci2-haloalkyl, or -OR ^A1 ; each R ¹² is independently hydrogen or alkyl;

X is N or CR ^b ;

L is Ci-Cualkylene, C2-Ci2alkenylene, Ci-C 12 heteroalkylene, cycloalkylene, or heterocyclylene, each of which is optionally substituted with one or more R ^B ;

R ^b is hydrogen, deuterium, halo, or Ci-CAalkyl; each of R ^A and R ^A1 is independently hydrogen, deuterium, C1-C12 alkyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, -N(R ^C )(R ^D ), or halo; each of R ^B , R ^c , and R ^D is independently hydrogen, halo, C1-C12 alkyl, or C1-C12 heteroalkyl; y is 1, 2, 3, 4, or 5; and each “ ” independently denotes an attachment point to a PNA subunit, a protecting group, a linker, an amino acid, or the C-terminus or N-terminus of a PNA oligomer.

[00247] 26. The PNA oligomer of embodiment 25, wherein each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen or C1-C12 heteroalkyl.

[00248] 27. The PNA oligomer of embodiment 25 or embodiment 26, wherein each of R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen or has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl; y is 1, 2, 3, 4, or 5; and “«w” denotes an attachment point to the PNA subunit.

[00249] 28. The PNA oligomer of any one of embodiments 25-27, wherein one of R ³ and R ⁴ has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl; y is 1, 2, 3, 4, or 5; each of R ⁵ , R ⁶ , R ⁷ , and R ⁸ is independently hydrogen, and “ ” denotes an attachment point to the PNA subunit.

[00250] 29. The PNA oligomer of any one of embodiments 25-28, wherein R is hydrogen, methyl, or tert-butyl and y is 1.

[00251] 30. The PNA oligomer of any one of embodiments 25-29, wherein X is N.

[00252] 31. The PNA oligomer of any one of embodiments 25-30, wherein R ² is hydrogen. [00253] 32. The PNA oligomer of any one of embodiments 25-31, wherein L is alkylene or heteroalkylene, each of which is optionally substituted with one or more R ^B .

[00254] 33. The PNA oligomer of any one of embodiments 25-32, wherein L is selected from

[00255] 34. The PNA oligomer of any one of embodiments 25-33, wherein R ² is hydrogen, one of R ³ and R ⁴ has the structure of Formula (IV-a) or (IV-b): wherein R ¹² is hydrogen or alkyl; y is 1, 2, 3, 4, or 5; and the other of R ³ and R ⁴ is hydrogen.

[00256] 35. The PNA oligomer of any one of embodiments 25-34, wherein each of R ^9a and R ^9b is hydrogen.

[00257] 36. The PNA oligomer of any one of embodiments 1-35, wherein the target sequence is a target nucleic acid sequence.

[00258] 37. The PNA oligomer of embodiment 36, wherein the target nucleic acid sequence comprises a guanine nucleobase.

[00259] 38. The PNA oligomer of embodiment 37, wherein the target nucleic acid sequence comprises a guanine nucleobase at a site corresponding to the PNA subunit comprising the 2- aminopyridine nucleobase in the PNA oligomer.

[00260] 39. The PNA oligomer of any one of embodiments 1-38, wherein the PNA oligomer forms a PNA-DNA-PNA triplex with a target DNA strand.

[00261] 40. The PNA oligomer of any one of embodiments 1-39, formulated as a nanoparticle.

[00262] 41. The PNA oligomer of embodiment 40, wherein the nanoparticle is a lipid nanoparticle or a synthetic polymer nanoparticle.

[00263] 42. The PNA oligomer of embodiment 40 or embodiment 41, wherein the nanoparticle further comprises a load component that is encapsulated or entrapped within the nanoparticle.

[00264] 43. The PNA oligomer of embodiment 42, wherein the load component comprises a nucleic acid.

[00265] 44. The PNA oligomer of embodiment 43, wherein the nucleic acid comprises DNA.

[00266] 45. The PNA oligomer of embodiment 43 or embodiment 44, wherein the nucleic acid comprises no more than 20 to 100 nucleotides.

[00267] 46. The PNA oligomer of any one of embodiments 43-45, wherein the nucleic acid comprises a phosphorothioate linkage. [00268] 47. A preparation of peptide nucleic acid (PNA) oligomers, comprising a property of any one of embodiments 1-46.

[00269] 48. A therapeutically acceptable composition comprising a PNA oligomer of any one of embodiments 1-46.

[00270] 49. A therapeutically acceptable composition comprising: a PNA oligomer of any of embodiments 1-46 disposed in a nanoparticle.

[00271] 50. A composition comprising a PNA oligomer of any of embodiments 1-39 and a load component.

[00272] 51. The composition of embodiment 50, wherein the load component is a nucleic acid.

[00273] 52. The composition of embodiment 51, wherein the load component comprises DNA.

[00274] 53. The composition of embodiment 51 or embodiment 52, wherein the nucleic acid comprises no more than 20 to 100 nucleotides.

[00275] 54. The composition of any one of embodiments 51-53, wherein the nucleic acid comprises a phosphorothioate linkage.

[00276] 55. The composition of any one of embodiments 50-54, wherein the composition is a therapeutically acceptable composition.

[00277] 56. A kit comprising a PNA oligomer of any of embodiments 1-39 and a load component.

[00278] 57. The kit of embodiment 56, wherein the load component comprises a nucleic acid. [00279] 58. The kit of embodiment 57, wherein the nucleic acid comprises no more than 20 to 100 nucleotides.

[00280] 59. The kit of embodiment 57 or embodiment 58, wherein the nucleic acid comprises a phosphorothioate linkage.

[00281] 60. The kit of any one of embodiments 56-59, wherein the PNA oligomer is disposed in a first container and the load component is disposed in a second container.

[00282] 61. The kit of any one of embodiments 56-60, wherein the PNA oligomer and the load component are disposed in a container.

[00283] 62. A method of altering the structure of a target nucleic acid, the method comprising administering to the subject a peptide nucleic acid (PNA) oligomer of any one of embodiments 1-44. [00284] 63. The method of embodiment 62, wherein altering the structure of a target nucleic acid comprises forming a complex between the PNA oligomer and the target nucleic acid.

[00285] 64. The method of embodiment 62 or embodiment 63, wherein altering the structure of a target nucleic acid comprises cleaving the target nucleic acid.

[00286] 65. The method of any one of embodiments 62-64, wherein altering the structure of a target nucleic acid comprises altering the sequence of the target nucleic acid.

[00287] 66. The method of any one of embodiments 62-65, wherein the method comprises an in vitro method.

[00288] 67. The method of any one of embodiments 62-65, wherein the method comprises an in vivo method.

[00289] 68. The method of any one of embodiments 62-65, wherein the method comprises an ex vivo method.

[00290] 69. A method of treating a disease in a subject, the method comprising administering to the subject a peptide nucleic acid (PNA) oligomer of any one of embodiments 1-46.

[00291] 70. The method of embodiment 69, wherein altering the structure of a target nucleic acid comprises forming a complex between the PNA oligomer and the target nucleic acid.

[00292] 71. The method of embodiment 69 or embodiment 70, wherein altering the structure of a target nucleic acid comprises cleaving the target nucleic acid.

[00293] 72. The method of any one of embodiments 69-71, wherein altering the structure of a target nucleic acid comprises altering the sequence of the target nucleic acid.

[00294] 73. The method of any one of embodiments 69-71, wherein the method comprises an in vitro method.

[00295] 74. The method of any one of embodiments 69-71, wherein the method comprises an in vivo method.

[00296] 75. The method of any one of embodiments 69-71, wherein the method comprises an ex vivo method.

[00297] 76. The method of any one of embodiments 69-75, wherein the disease comprises a blood disorder.

[00298] 77. The method of embodiment 76, wherein the blood disorder is a red blood cell disorder.

EXAMPLES

[00299] In order that the disclosure described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the PNA oligomers, compositions, methods, and kits provided herein and are not to be construed in any way as limiting their scope.

[00300] The PNA oligomers and compositions thereof provided herein can be prepared from readily available starting materials using modifications to the specific synthetic protocols set forth below in combination with what would be well known to those of skill in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.

[00301] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in Greene et al., Protecting Groups in Organic Synthesis, Fourth Edition, Wiley, New York, 2011, and references cited therein, each of which are incorporated herein by reference in their entirety.

[00302] Exemplary PNA intermediates and compositions thereof may be prepared using any of the strategies described below.

Example 1: Synthesis of “M-monomer”; a.k.a Fmoc-aeg-M(boc)-OH

Step 1: Synthesis of 1 -(tert-butyl) 3-ethyl 2-(6-nitropyridin-3-yl)malonate (7)

[00303] In an oven-dried 500 mL round bottom flask was suspended 9.37 g of sodium hydride (NaH) (234 mmol, 57-63% oil dispersion) in dry N,N’ -dimethylformamide (DMF) under argon. The suspension was chilled in an ice-bath, and 44.4 mL of tert-butyl ethyl malonate (234 mmol) was added dropwise over 10 minutes. The reaction mixture was stirred for 1 hour after which 38.71 g of 5-bromo-2-nitropyridine (6; 191 mmol) was added and the reaction was stirred at room temperature overnight. The reaction was quenched by the careful addition of water, the solvent was evaporated, and the residue re-dissolved in ethyl acetate (EtOAc) and the organic solution washed with water and brine. The EtOAc layer was dried over anhydrous MgSO4 and concentrated by rotary evaporation. The resulting slurry was dissolved in a minimal amount of EtOAc and kept overnight. The precipitate which formed was collected by vacuum filtration to afford after drying 58.35 g of crude solid product (7; 98% yield) which was used in the next step without further purification.

Step 2: Synthesis of ethyl 2-(6-nitropyridin-3-yl)acetate (8)

[00304] To a stirring solution of 8.17 g of 1 -(tert-butyl) 3-ethyl 2-(6-nitropyri din-3 -yl) malonate (7; 26 mmol) in 50 mL of dry dichloromethane (DCM) in a round-bottom flask in an ice-bath was added 8.17 mL of trifluoracetic acid (TFA) dropwise. The resulting solution was heated to reflux with heat set at 90°C for 16 hours. The reaction was cooled and concentrated before being diluted with ice-cold water. A solution of 5% (w/v) sodium bicarbonate was added to neutralize excess acid and then the water mixture was extracted with EtOAc. The resulting EtOAc solution was concentrated in vacuo and to the resulting oil was added a minimal amount of EtOAc and this mixture was allowed to crystallize at room temperature overnight. The precipitate which is an impurity was removed by filtration and the filtrate was then purified by flash chromatography using a gradient of 0 to 100% 0 to 100% EtOAc into Hexanes to yield 5.33 g oil (8; 96% yield).

Step 3: Synthesis of ethyl 2-(6-aminopyridin-3-yl)acetate (9)

[00305] Ethyl 2-(6-nitropyri din-3 -yl) acetate, 5.33 g (8; 25 mmol), 33.6 g of ammonium chloride (628 mmol) and 16.50 g of zinc dust (252 mmol) were placed in a 500 mL round bottom flask. MeOEkEEO (175 mL of 2: 1, (v/v)) was added, and the mixture was stirred at room temperature for 1 hour. Reaction progress was monitored by TLC and LCMS. After the reaction was complete, the mixture was diluted with EtOAc and filtered through Celite. The filter cake was washed with water and the filtrate extracted with EtOAc. The organic layers were combined and dried over anhydrous MgSO4, filtered and evaporated to give 3.96 g (9; 87% yield) of product, which was used in the next step without further purification.

Step 4: Synthesis of ethyl 2-(6-((tert-butoxycarbonyl)amino)pyridin-3-yl)acetate (10)

[00306] To 9.94 g of ethyl 2-(6-aminopyri din-3 -yl) acetate (9; 55 mmol) and 14.47 g of di-tert- butyl dicarbonate (66 mmol) was added 147 mL of tert-butyl alcohol under argon. Tri ethylamine (9.25 mL, 66 mmol) was added, and the reaction was stirred for 3 hours at 50 ⁰ C. After confirming that the reaction was complete by TLC and LCMS, the solution was concentrated by rotary evaporation and the residue was dissolved in EtOAc. The ethyl acetate solution was then washed with water and brine, dried over granular anhydrous MgSCU, and concentrated then purified by flash chromatography using a gradient of 0 to 100% 0 to 100% EtOAc into Hexanes to yield 8.34 g (10; 54% yield) as a solid.

Step 5: Synthesis of 2-(6-((tert-butoxycarbonyl)amino)pyridin-3-yl)acetic acid (11)

[00307] Ethyl 2-(6-((tert-butoxycarbonyl) amino) pyridin-3-yl) acetate (10; 10.97 g, 39 mmol) in a 500 mL round bottom flask was dissolved 150 mL of acetonitrile: ethanol: water in 2:2: 1 (v/v/v) ratio. Some heat was applied to dissolve all the ester. The solution was then placed on ice-bath for 20 min. Then was added a solution of 16.42 g of LiOH monohydrate (391 mmol) dissolved in 156 mL of water (2.5M solution). The reaction mixture was briskly stirred for 10 minutes and quenched by the rapid addition of 195.6 mL of 2M HC1. The pH was brought down to ~5 using saturated KHSO4 upon which the product precipitated out. The mixture was filtered to obtain 8.29 g of a pure solid (11; 84% yield).

Notes:

1. When the initially dissolved starting material is placed in the ice-bath, it may precipitate out of solution but in a form that allows the de-esterification by LiOH to proceed.

2. Upon acidification, not all product precipitates out of solution, about 10% is left in solution and this can be recovered by extraction into DCM.

3. Lowering the pH below 4 leads to product going back into solution due to protonation of the pyridine nitrogen. Thus, pH of about 5 is optimal for precipitation and collection of product. Step 6: Synthesis of 2-iodoethyl N-(2-((((9H-jluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-(2-

( 6-( tert-butoxycarbonyl)amino)pyridin-3-yl)acetyl)glycinate (13)

[00308] Ethyl 2-(6-((tert-butoxycarbonyl) amino) pyridin-3-yl) acetate 8.29 g (11; 33 mmol), 25.02 grams of hexafluorophosphate azabenzotriazoletetramethyl uranium (HATU) (66 mmol), and 18.25 g Fmoc protected iodoethyl ester backbone tosyl salt (12; 27 mmol - purchased from a custom synthesis vendor using the procedures described in WO 2018/175927, published 27 September 2018) were dissolved in dry DMF under argon. 11.46 mL of N’N diisopropylethylamine (DIPEA) (66 mmol) was added and the reaction was stirred for an hour. After TLC/LCMS confirmed reaction was complete, the DMF was removed by rotary evaporation. The resulting oil was dissolved in DCM and extracted with saturated sodium bicarbonate, followed by water and brine. The organic layer was dried over granular anhydrous MgSCU then concentrated and the product was purified by flash chromatography using a gradient of 0 to 100% EtOAc into Hexanes to yield 18.94 g of product (13; 95% yield).

Step 7: Synthesis ofN-(2-((((9H-jluoren-9-yl)methoxy)carbonyl)amino)ethyl)-N-( 2-(6-((tert- butoxycarbonyl)amino)pyridin-3-yl)acetyl)glycine (14)

[00309] TXE Buffer was made by combining 50 mmol KH2PO4, 25 mmol of ethylenediaminetetraacetic acid (EDTA) and 25 mmol of ethylenediaminetetraacetic acid zinc disodium salt hydrate (EDTA-ZnH2O) in 150 mL of deionized water and 50 mL of glacial acetic acid (HO Ac). This mixture was permitted to stir overnight after which lOOmL of tetrahydrofuran (THF) was added and after 30 minutes of additional stirring, the solids were removed by filtration and the resulting filtrate was used as TXE Buffer. Fmoc M iodoethyl ester, 16.95 g (13; 23 mmol) was dissolved in a mixture of 161 mL of TXE buffer and 161 mL of THF. It was placed on a salt-bath for 30 minutes. Also placed a water bottle and saturated KH2PO4 on ice. When the reaction was cold, 23 mL of HO Ac, 23 mL of H2O and 23 mL of saturated KH2PO4 were added to the reaction in that order (this is roughly 1 mL of each reagent per milli mole of monomer). Then 5.07 g of Zn dust (77.5 mmol, a third of total Zn required) was added. Stirred for 20 minutes then added the above liquid reagents in the order listed with another 5.07 g of Zn dust. Stirred for another 20 minutes and added the liquid reagents in the prescribed order a third time and a final addition of 5.07 g of Zn dust then stirred the reaction for an addition hour monitoring the reaction progress by TLC and LCMS (always keeping the reaction cold in a salt/ice bath). After the reaction was complete, the mixture was filtered through Celite and washed with a solution of THF:H2O (4: 1; v/v) containing a couple of drops of acetic acid. The resulting solution was concentrated in vacuo to remove all the THF until the solution began to freeze on the roto-evaporator (no heat added to the flask via water bath). At this point, lOOmL of water was added, the aqueous layer was extracted three times with 150 mL aliquots of DCM. The DCM fractions were then combined and washed twice with 50mL (a total of 100 mL) of extraction buffer (Extraction Buffer is 1g KH2PO4 and 0.5g KHSO4 per 10 mL of deionized water). The organic layer was then dried over anhydrous MgSCU (granular), filtered, and evaporated. The crude compound was purified by flash chromatography in 0 to 100% EtOAc into Hexanes to yield 12.56 g, (93% yield 14). Further chromatographic purification using a gradient of MeOH into DCM gave product that was greater than 98% pure.

Notes:

1. Use Zn Powder average 4-7 micron 97.5% powder or flakes. Using granular Zn, for instance Zn powder -140 +325 mesh with 99.9% (metal basis) leads to very little hydrolysis of iodoethyl group.

2. The final product can be dissolved in a minimum volume of DCM and precipitated by dropwise addition to a briskly stirring solution of hexanes or hexanes/di ethyl ether (generally in a ratio of about 5/1 to 10/1). After allowing to stir for 2 hours, the product can be collected by vacuum filtration and vacuum dried to obtain an easily weighable powder.

Example 2. Synthesis of PNA Oligomers

[00310] PNA Monomers'. Classic Fmoc-protected PNA monomers (monomers having an unsubstituted 2-aminoethylglycine backbone) were purchased from commercial sources and/or prepared by a vendor on a custom synthesis basis. Fmoc gamma miniPEG monomers were prepared by a vendor on a custom synthesis basis by generally following published procedures. Fmoc-protected gamma miniPEG PNA monomers could be prepared according to the general methods described in Sahu et al. J. Org. Chem. (2011) 76:5614-5627 using a protected serinol intermediate. Identity, purity and chiral purity (if applicable) were confirmed for all PNA monomers after receipt using 'H-NMR (proton nuclear magnetic resonance) and LCMS (liquid chromatography mass spectrometry) of the PNA monomers and/or PNA oligomers prepared therefrom.

[00311] Linkers: Fmoc protected PEG2PEG2 and PEG3 linkers were purchased from commercial sources such as PurePEG and used without further analysis.

[00312] Amino Acids: Amino acids (e.g., N-alpha-Fmoc-N-epsilon-Fmoc-L-lysine and N-alpha- Fmoc-N-epsilon-boc-L-lysine) were purchased from commercial sources such as Chem Impex International, Bachem and Matrix Innovations and used without any analysis. For PNA oligomers comprising a Gly-Gly PNA subunit (a.k.a. a “Gly-Gly bridge”) in the Hoogsteen segment of the oligomer, a single coupling of the commercially available dimer Fmoc-Gly-Gly- OH was used instead of two back-to-back couplings of the amino acid Fmoc-Gly-OH; however, a PNA oligomer comprising a Gly-Gly bridge may be prepared using any method known in the art.

[00313] General Procedure for Synthesis of PNA Oligomers: All PNA oligomers were synthesized on an Intavis MultiPepRSi automated peptide synthesizer using Fmoc solid phase peptide synthesis protocol using rink amide TentaGel resin (Rapp polymer, R28023) as the solid support. The synthesis protocol comprised three synthetic steps (in addition to washing steps) wherein each of the steps was repeated for each new PNA monomer, linker, amino acid or other building block (e.g., synthon) until the polyamide was completely assembled. Specifically, a single synthetic cycle comprised: 1) deprotection of the N-terminal Fmoc group; 2) coupling of a new monomer, linker, amino acid or synthon to the growing polyamide; and 3) capping of the unreacted amino groups. Between each step in the cycle, the resin was washed extensively with N,N’ -dimethylformamide (DMF) to remove unreacted reagents and other unwanted impurities and side products of the reaction.

[00314] Protocol for Small Scale Synthesis: Approximately 45 mg (5.8 pmol) rink amide TentaGel resin was placed in the reaction column of the Intavis and treated with 800 pL di chloromethane (DCM) for 15 minutes (min) to swell the resin prior to initiation of the PNA oligomer synthesis. The resin was then treated twice with 600 pL of 20% (v/v) piperidine/DMF for 5 min each to remove/deprotect the Fmoc group. After five washes with DMF, approximately 45 pmol of a PNA monomer, linker, amino acid (e.g., lysine) or other synthon (as applicable based on the sequence of the PNA oligomer to be prepared) was delivered to the resin from a solution comprising PNA monomer, linker, amino acid or synthon dissolved in dry DMF. To the resin was also delivered a mixture of N,N’-diisopropylethylamine (DIEA; approximately 56 pmol) dissolved in dry N-methyl pyrrolidone (NMP) and approximately 42 pmol of 1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridi nium 3-oxid hexafluorophosphate (HATU) in dry DMF. Also, 2,6-lutidine can be added to the DIEA solution in a ratio of approximately 1/1.5 DIEA/2.6-lutidine (v/v). Once all the reagents were delivered for the reaction column, the resin was agitated for 30 min. The reaction mixture was then drained from the reaction vessel and the resin was washed extensively with DMF. The capping step was then performed by treating the resin with 600 pL of capping solution (5% acetic anhydride and 6% lutidine in DMF (v/v)) while agitating the resin for 5 min. These three steps were repeated sequentially for each new PNA monomer, linker, amino acid or other building block until the PNA oligomer was completely assembled. However, for bis-Fmoc PNA oligomers the protocol was modified so that the final Fmoc deprotection step was eliminated so that the PNA oligomer remained Fmoc-ON. Hence, after the final capping step, the instrument did not execute a final step that removed the terminal Fmoc moiety but rather, the resin was simply washed extensively with DCM and then dried. For all bis-Fmoc PNA oligomers, the final coupling was performed using N-alpha-Fmoc-N-epsilon-Fmoc-L-lysine.

[00315] PNA oligomer synthesis could be scaled using adaptations of the above described protocol wherein volumes, reagent amounts and reaction times were altered, with some optimization, to produce similar results as are obtained with the 5.8 pmol scale synthesis. Other representative scales included 15 pmol and 52 pmol.

[00316] Protocol for Small Scale Synthesis: Approximately 45 mg (5.8 pmol) rink amide TentaGel resin was placed in the reaction column of the Intavis and treated with 800 pL di chloromethane (DCM) for 15 minutes to swell the resin prior to initiation of the PNA oligomer synthesis. The resin was then treated twice with 600 pL of 20% (v/v) piperidine/DMF for 5 min each to remove/deprotect the Fmoc group. After five washes with DMF, approximately 45 pmol of a PNA monomer, linker, amino acid (e.g., lysine) or other synthon (as applicable based on the sequence of the PNA oligomer to be prepared) was delivered to the resin from a solution comprising PNA monomer, linker, amino acid or synthon dissolved in dry DMF. To the resin was also delivered a mixture of N,N’ -diisopropylethylamine (DIEA; approximately 56 pmol) dissolved in dry N-methyl pyrrolidone (NMP) and approximately 42 pmol of 1- [bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridi nium 3-oxid hexafluorophosphate (HATU) in dry DMF. Also, 2,6-lutidine can be added to the DIEA solution in a ratio of approximately 1/1.5 DIEA/2.6-lutidine (v/v). Once all the reagents were delivered for the reaction column, the resin was agitated for 30 min. The reaction mixture was then drained from the reaction vessel and the resin was washed extensively with DMF. The capping step was then performed by treating the resin with 600 pL of capping solution (5% acetic anhydride and 6% lutidine in DMF (v/v)) while agitating the resin for 5 min. These three steps were repeated sequentially for each new PNA monomer, linker, amino acid or other building block until the PNA oligomer was completely assembled.

[00317] PNA oligomer synthesis could be scaled using adaptations of the above described protocol wherein volumes, reagent amounts and reaction times were altered, with some optimization, to produce similar results as are obtained with the 5.8 pmol scale synthesis. Other representative scales included 15 pmol and 52 pmol.

[00318] General Protocol for Cleavage and Deprotection of PNA Oligomers: Crude PNA oligomers were obtained by treating the dried resin with 1 mL of cleavage mixture containing, trifluoracetic acid (TFA), m-cresol, water, and thioanisole (in a ratio of 95/2/2/1 : v/v/v/v) for 2 hours at room temperature. The sample was then filtered to remove the resin and the filtrate was subsequently treated with cold diethyl ether to cause precipitation of the PNA oligomer. After repeated suspension and pelleting of the PNA oligomer with cold diethyl ether, the crude PNA oligomer was dissolved in approximately 2 mL of a 1/1 (v/v) water/acetonitrile mixture. The crude PNA oligomer was then obtained for purification by lyophilization of this solution. PNA oligomers could also be purified with preparative HPLC, and subsequently lyophilized.

[00319] Table 1 below provides exemplary sequences of PNA oligomers that have been prepared using the methodology described above. The terms “Compound No.”, “PNA NO.”, and “PNA-X” are used interchangeably throughout the disclosure, and refer to the same entity (e.g., Compound No. 1 is the same entity as PNA NO: 1 or PNA-1).

Table 1. Exemplary PNA Oligomers

Legend: each letter corresponds to the nucleobase in the sequence (e.g., t = thymine; j = pseudoisocytosine, c = cytosine; a = adenine; g = guanine; si = 2-thiouracil; dl = 2,6- diaminopurine; dg = 7-deazaguanine; m = 2-aminopyridine); a lower-case letter indicates use of a classic (i.e. an unsubstituted aminoethylglycine) PNA monomer subunit; an upper-case letter indicates a right-handed gamma miniPEG (-CEE- OCEECEE^-OEI) substituted aminoethylglycine PNA monomer subunit was used (Sahu et al.); K is the amino acid L-lysine; Gly is the amino acid glycine; PEG3 is -NH^EECEEO^CEE O)-; PEG2PEG2 is - [NH(CH2CH2O)2CH2C(O)]2-. All PNA oligomers are illustrated in the N-terminal to C- terminal direction. The -NH2 at the C-terminus of the PNA oligomer indicates a C-terminal amide group.

Example 3. Visualization of DNA Binding and Strand Invasion

[00320] To visualize the level of Hoogsteen binding and DNA sequence invasion, PNA NOs.18- 21 were mixed with a complementary double stranded template DNA sequence at a ratio of 15: 1 PNA:DNA in a pH 7.4 buffer containing 50 mM potassium chloride. After incubation at 37 °C for 18 hours, samples were subjected to acrylamide gel electrophoresis using an automated Agilent TapeStation. As shown in FIG 2A, PNA NOs. 18-21 demonstrated formation of higher molecular weight species indicating DNA binding, compared to the control which was the DNA template alone. Notably, PNA NO. 20 which contains 2-aminopyridine (M) nucleobase in combination with thiouracil (si) and other modified nucleobases exhibits strong and rapid binding to the dsDNA template. To determine whether the PNAs could form stable strand invasion complexes the samples were denatured by heating to 95 °C and then cooled slowly to room temperature. FIG. 2B shows that each of the PNAs NOs 18-21 form stable, higher molecular weight species relative to the control, suggesting formation of stable strand invasion products.

Example 4. Preparation of Nanoparticles

Double Emulsion

[00321] Nanoparticles (e.g., PLGA nanoparticles) comprising a peptide nucleic acid (e.g., a PNA oligomer) and/or a nucleic acid may be prepared using a double-emulsion method as previously described (see, e.g., McNeer, et al., Molecular Therapy (2011) 19: 172-180, and Bahai et al. Nat. Commun. (2016) 7: 13304), which are incorporated herein by reference in their entirety. For example, nanoparticles comprising PNA oligomers and optionally a loading component (e.g., DNA) were prepared as follows.

[00322] A series of four solutions were prepared, including i) a mixture of DNA and PNA oligomer in aqueous buffer solution, ii) a solution of synthetic polymer (e.g. PLGA) in a water immiscible organic solvent (e.g. dichloromethane), iii) an aqueous solution containing polyvinyl alcohol (PVA) at a high concentration (5%), and iv) another aqueous solution of PVA at a lower concentration (0.3%). The aqueous solution containing DNA and PNA (i) was then added dropwise to the mixture of synthetic polymer (ii), with vortexing and sonication applied as necessary to form an emulsion. The emulsion was then added to the concentrated PVA solution (iii), and the resulting mixture was then transferred to the dilute PVA solution (iv), to produce the nanoparticles.

[00323] The nanoparticles as formed can be extracted for analysis/quality control. Otherwise, the solution can be transferred to a centrifuge tube and spun down, and then decanted to afford the nanoparticles. In order to remove any residual solvent, PVA and/or buffer, the nanoparticles can be put through one or more cycles of washing by resuspension in an aqueous wash solution, followed by centrifugation to re-pellet the nanoparticles. When washed to a level of purity that is desired, the nanoparticles can be resuspended in a solution, and if desired, the batch can be split into portions by aliquoting into separate containers (e.g. an Eppendorf tube). Whether or not split into portions, the nanoparticles can then be prepared for long term storage by lyophilization.

Example 5. Characterization of Nanoparticles

[00324] Nanoparticles prepared as outlined in Example 4 were characterized to interrogate size, polydispersity, and zeta potential using a Zetasizer Nano ZSP (Malvern Panalytical).

Nanoparticle samples were first diluted 50-fold in deionized water, then 1.0-1.5mL was transferred to a disposable cuvette. After taking a size measurement reading in the instrument, 900 pL of the diluted sample was transferred from the cuvette into the folded capillary cell to determine the zeta-potential.

Example 6. Protocol for Droplet Digital PCR (ddPCR)

[00325] Droplet digital PCR (ddPCR) is performed with Bio-Rad QX200 using primers and probes as described below. ddPCR is a quantitative PCR method useful for the detection and measuring the amount of rare genetic variant in a DNA sample. This is achieved by partitioning DNA molecules in a sample, mixed with PCR reagents, into nanoliter-sized droplets formed in a water-oil emulsion. These individual droplets function as an individual PCR sample reaction. For the quantification of the amount of rare genetic variant in a DNA sample, the number of droplets without DNA, droplets positive for rare variant allele, and droplets positive for WT allele are measured fluorescently by the ddPCR reader, and the amount of rare variant allele is measured based on the Poisson distribution and the number of these droplets.

[00326] The concentration of double-stranded DNA extracted from cells treated with nanoparticles was measured fluorometrically by Qubit Fluorometer with double stranded DNA (dsDNA) High Sensitivity (HS) Assay Kit before using digital droplet PCR (ddPCR) to evaluate activity. Primer sequences are as follows: primer-forward (5 ’-C ACC AACTTCATCCACGTTC AC-3’); primer-reverse (5’-TCTATTGCTTACATTTGCTTCTGACA-3’).

[00327] Probes are designed with 5’ Dye and 3’ minor groove binder non-fluorescent quencher (MGBNFQ): mutant (VIC®), (5’-CAGACTTCTCCACAGGA-3’); wildtype (fluorescein amidite; FAM) (5’-CAGACTTCTCCTCAGGA-3’). PCR was performed under the following conditions: 95 °C, 10 min; x40 [94 °C, 30 s; 54.8 °C, 4 min ramp 2°C/s]; 98 °C, 10 min; 4 °C forever.

EQUIVALENTS AND SCOPE

[00328] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[00329] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, Figures, or Examples but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.