SULFUR-CONTAINING COMPOUNDS AND COMPOSITIONS THEREOF FOR DELIVERY OF NUCLEIC ACIDS

BACKGROUND OF THE INVENTION

[001] There has been a long-felt but unmet need in the art for compositions and methods for delivering nucleic acids to cells and for genetically modifying cells in vivo, ex vivo and in vitro. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors, including AAVs, can cause acute toxicity and harmful side-effects in patients. The present disclosure provides improved compositions, methods and kits for the delivery of nucleic acids to various types of cells, including T-cells and hepatocytes, in vivo, ex vivo and in vitro. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods of using the same. These lipid nanoparticle compositions and methods allow for the delivery of nucleic acids to cells with high efficiency and low toxicity. Thus, the compositions and methods of the present disclosure have wide applicability to a diverse number of fields, including gene therapy and the production of cell-based therapeutics.

SUMMARY OF THE INVENTION

[002] In some aspects, provided are novel lipid nanoparticles (“LNPs”) comprising a novel compound. In one aspect, the novel compound is a compound of Formulas (I)-(IV).

[003] In some aspects, provided are pharmaceutical compositions, comprising a composition of the present disclosure and at least one pharmaceutically-acceptable excipient or diluent.

[004] In some aspects, provided are methods of delivering at least one nucleic acid to at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure.

[005] In some aspects, provided are methods of genetically modifying at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure. [006] In some aspects, provided are methods of treating at least one disease or disorder in a subject in need thereof comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.

[007] In some aspects, provided are methods of delivering at least one nucleic acid to at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure.

[008] In some aspects, provided are cells modified according to methods of the present disclosure.

[009] Any of the aspects and/or embodiments described herein can be combined with any other aspect and/or embodiment described herein.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0011] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure provides novel compounds, novel lipid nanoparticle compositions (LNPs) comprising the novel compounds, methods for preparing the LNPs, and methods for using same. In a non-limiting example, the compositions and methods of the present limiting disclosure can be used gene delivery and cell-based therapeutics. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to liver cells, in vivo, ex vivo or in vitro, for the treatment of certain diseases and disorders, including, but not limited to liver disorders. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to T-cells, in vivo, ex vivo or in vitro, for the treatment of certain disorders, including, but not limited to cancer. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to primary, unactivated T-cells, in vivo, ex vivo or in vitro, for the treatment of certain diseases and disorders, including, but not limited to cancer. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid for the purpose of vaccination. In a non-limiting example, the compositions and methods of the present disclosure can be broadly used to deliver a nucleic acid to induce the expression of a secreted therapeutic protein.

Compositions of the Present Disclosure

[0013] The present disclosure provides a composition comprising at least one lipid nanoparticle comprising a compound of the present disclosure and at least one nucleic acid molecule. In some aspects, a lipid nanoparticle can further comprise at least one structural lipid. In some aspects, a lipid nanoparticle can further comprise at least one phospholipid. In some aspects, a lipid nanoparticle can further comprise at least one PEGylated lipid. In a non-limiting example, the compositions and methods of the present disclosure can be used for gene delivery.

Compounds

[0014] In some embodiments, the present disclosure provides compounds of Formula (I):

A is substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic; each R ₁ is independently hydrogen, substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic, wherein at least one occurrence of R ₁ is hydrogen; each of B, C, D is independently hydrogen, substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic, or- (CHR ₆ )CH(SXLY)R ₆ ;

X is S or CH2;

L is substituted or unsubstituted, branched or unbranched C1-6 aliphatic, substituted or unsubstituted, branched or unbranched C1-6 heteroaliphatic; eachR ₆ is independently hydrogen, substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic, wherein at least one occurrence of R ₆ is hydrogen; or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, one of B, C, and D is -(CHR6)CH(SXLY)R6.. In some embodiments, two of B, C, and D are -(CHR6)CH(SXLY)R6. In some embodiments, each of B, C, and D is independently -(CHR6)CH(SXLY)R6. [0016] In certain embodiments when one of B, C, and D is -(CHR6)CH(SXLY)R6, the other two variables are both the same. For example, in certain embodiments when B is - (CHR6)CH(SXLY)R6, C and D are both the same. In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXLY)R6, the other two variables are different. For example, in certain embodiments when B is -(CHR ₆ )CH(SXLY)R6, C and D are different.

[0017] In some embodiments, two of B, C and D are the same. In some embodiments, B, C and D are all the same. In some embodiments, B, C and D are all different.

[0018] In some embodiments, X is S and Y is -OH.

[0019] In some embodiments, X is S. In some embodiments, X is CH2. In certain of these embodiments, Y is -OH.

[0020] In some embodiments, L is unsubstituted, branched or unbranched C1-6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0021] In certain embodiments when one of B, C, and D is -(CHR ₆ )CH(SXLY)R ₆ , each of the two occurrences of R ₁ and R ₆ that is not hydrogen is the same. In certain embodiments when one of B, C, and D is -(CHR ₆ )CH(SXLY)R ₆ , each of the two occurrences of R ₁ and R ₆ that is not hydrogen is different.

[0022] In certain embodiments when two of B, C, and D are -(CHR6)CH(SXLY)R6, each of the three occurrences of R ₁ and R ₆ that is not hydrogen is the same. In certain embodiments when two of B, C, and D are -(CHR ₆ )CH(SXLY)R ₆ , two of the three occurrences of R ₁ and R ₆ that are not hydrogen are the same. In certain embodiments when two of B, C, and D are - (CHR ₆ )CH(SXLY)R ₆ , each of the three occurrences of R ₁ and R ₆ that is not hydrogen is different.

[0023] In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C1-15 alkyl. In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C1-10 alkyl. In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C1-5 alkyl.

[0024] In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C1-15 aliphatic. In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C1- 10 aliphatic. In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C1-5 aliphatic. [0025] In some embodiments, R ₁ is or

[0026] In some embodiments, R ₁ is substituted or unsubstituted, branched or unbranched C 1-15 alkenyl. In some embodiments, R ₁ is

or

[0027] In some embodiments, R ₁ is

or

[0028] In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched C 1-15 alkyl. In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched C1-10 alkyl. In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched C1-5 alkyl.

[0029] In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched C 1-15 aliphatic. In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched Cl- 10 aliphatic. In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched C1-5 aliphatic.

[0030] In some embodiments, R ₆ is or

[0031] In some embodiments, R ₆ is substituted or unsubstituted, branched or unbranched C1-15 alkenyl. In some embodiments, R ₆ is

[0032] In some embodiments, R ₆ is or

[0033] In some embodiments, IS

[0034] In some embodiments, In some embodiments, is

[0035] In some embodiments, is substituted or unsubstituted, branched or unbranched aliphatic. In some embodiments, is substituted or unsubstituted, branched or unbranched alkyl. In some embodiments, is substituted or unsubstituted, branched or unbranched heteroaliphatic.

[0036] In some embodiments when one of B, C, and D is methyl, X is S and Y is -OH. In certain of these embodiments, R ₁ is C12H25. In certain of these embodiments, R ₆ is C12H25. In certain of these embodiments, L is unsubstituted C2 alkyl. In certain of these embodiments, is C3 alkyl. [0037] In some embodiments, the present disclosure provides compounds of the formula:

[0038] In some embodiments, the present disclosure provides compounds of Formula (II): substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic; each of R1, R2, R3, and R4 is independently hydrogen, substituted or unsubstituted, branched or unbranched C1-15 aliphatic, or substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic, wherein at least one occurrence of R ₁ is hydrogen, at least one occurrence of R2 is hydrogen, at least one occurrence of R3 is hydrogen, and at least one occurrence of R4 is hydrogen;

X is S or CH2;

L is substituted or unsubstituted, branched or unbranched C1-6 aliphatic, substituted or unsubstituted, branched or unbranched C1-6 heteroaliphatic; or a pharmaceutically acceptable salt thereof.

[0039] In some embodiments, X is S and Y is -OH.

[0040] In some embodiments, X is S. In some embodiments, X is CH2. In certain of these embodiments, Y is -OH.

[0041] In some embodiments, L is unsubstituted, branched or unbranched C1-6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0042] In some embodiments, each of R1, R2, R3, and R4 is independently substituted or unsubstituted, branched or unbranched C1-15 alkyl. In some embodiments, each of R ₁ , R ₁ , R3, and R4 is independently substituted or unsubstituted, branched or unbranched C 1-10 alkyl. In some embodiments, each of R1, R2, R3, and R4 is independently substituted or unsubstituted, branched or unbranched Cl-5 alkyl.

[0043] In some embodiments, each of R1, R2, R3, and R4 is independently substituted or unsubstituted, branched or unbranched C1-15 aliphatic. In some embodiments, each of R ₁ , R2, R3, and R4 is independently substituted or unsubstituted, branched or unbranched C1-10 aliphatic. In some embodiments, each of R1, R2, R3, and R4 is independently substituted or unsubstituted, branched or unbranched Cl-5 aliphatic.

[0044] In some embodiments, each of R1, R2, R3, and R4 is independently:

or

[0045] In some embodiments, each of R ₁ , Rz, R3, and R» is independently substituted or unsubstituted, branched or unbranched C1-15 alkenyl. In some embodiments, each of R ₁ , Rz, R3, and R* is independently:

or

[0046] In some embodiments, each of R1, R2, R3, and R4 is independently:

or

[0047] In some embodiments, each of R1, R2, R3, and R4 is independently unsubstituted, branched or unbranched C1-15 heteroaliphatic.

[0048] In some embodiments, each of R1, R2, R3, and R4 is independently C10H21.

[0049] In some embodiments, R1, R2, R3, and R4 are all the same. In some embodiments, two of R1, R2, R3, and R4 are the same. In some embodiments, three of R1, R2, R3, and R4 are the same. In some embodiments, R1, R2, R3, and R4 are all different. [0050] In some embodiments

[0051] In some embodiments . In some embodiments, is

[0052] In some embodiments, is substituted or unsubstituted, branched or unbranched aliphatic. In some embodiments, is substituted or unsubstituted, branched or unbranched alkyl. In some embodiments, is substituted or unsubstituted, branched or unbranched heteroaliphatic.

[0053] In some embodiments, the present disclosure provides compounds of the formula:

[0054] In some embodiments, the present disclosure provides compounds of Formula (in): wherein: each of Ra and Rb is independently or

X is S or CH2;

L is substituted or unsubstituted, branched or unbranched C1-6 aliphatic, substituted or unsubstituted, branched or unbranched C1-6 heteroaliphatic;

Rz is substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic; x is an integer between 1 and 10, inclusive; y is an integer between 1 and 10, inclusive; each Ry and Rz is independently or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, Ra is and Rb is [0056] In some embodiments, Ra is In some embodiments, Ra is

. In some embodiments, Ra is

[0057] In some embodiments, Rb is In some embodiments, Rb is In some embodiments, Rb is

[0058] In some embodiments, x is 1 and y is 2.

[0059] In some embodiments, X is S and ¥ is -OH.

[0060] In some embodiments, X is S. In some embodiments, X is CH ₂ . In certain of these embodiments, ¥ is -OH.

[0061] In some embodiments, L is unsubstituted, branched or unbranched Cl -6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0062] In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C 1-15 alkyl. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-10 alkyl. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-5 alkyl.

[0063] In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C 1-15 aliphatic. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched Cl- 10 aliphatic. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-5 aliphatic.

[0064] In some embodiments, R ₇ is C10H21.

[0065] In some embodiments, Ra is

[0066] In some embodiments, Rb is

[0067] In some embodiments, the present disclosure provides compounds of the formula:

[0068] In some embodiments, the present disclosure provides compounds of Formula (IV):

wherein: each of R ₆ and Rf is independently x is an integer between 1 and 10, inclusive;

L is substituted or unsubstituted, branched or unbranched C1-6 aliphatic, substituted or unsubstituted, branched or unbranched C1-6 heteroaliphatic; R ₇ is substituted or unsubstituted, branched or unbranched C1-15 aliphatic, substituted or unsubstituted, branched or unbranched C1-15 heteroaliphatic; or a pharmaceutically acceptable salt thereof. [0069] In some embodiments, R ₆ is

[0070] In some embodiments, Rf is

[0071] In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3.

[0072] In some embodiments, X is S and Y is -OH.

[0073] In some embodiments, X is S. In some embodiments, X is CH ₂ . In certain of these embodiments, Y is -OH.

[0074] In some embodiments, L is unsubstituted, branched or unbranched Cl -6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0075] In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-15 alkyl. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-10 alkyl. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-5 alkyl.

[0076] In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C 1-15 aliphatic. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1- 10 aliphatic. In some embodiments, R ₇ is substituted or unsubstituted, branched or unbranched C1-5 aliphatic.

[0077] It will be understood that the compounds of any one of the Formulae disclosed herein and any pharmaceutically acceptable salts thereof, comprise stereoisomers, mixtures of stereoisomers, polymorphs of all isomeric forms of said compounds.

[0078] It will be understood that compounds disclosed herein may be presented without specified configuration (e.g., without specified stereochemistry). Such presentation intends to encompass all available isomers, tautomers, regioisomers, and stereoisomers of the compound. In some embodiments, the presentation of a compound herein without specified configuration intends to refer to each of the available isomers, tautomers, regioisomers, and stereoisomers of the compound, or any mixture thereof.

[0079] It is to be understood that the compounds of any Formula described herein include the compounds themselves, as well as their salts, and their solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a substituted compound disclosed herein. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).

General Methods for Preparation of Compounds of Formulas (I) - (IV) of the Present Disclosure

[0080] The inventive compounds may be prepared by any method known in the art The compounds may be prepared from commercially available starting materials, such as amines and thioepoxide compounds. The compounds may also be prepared by total synthesis from commercially available starting materials.

[0081] In general, the first step in preparation of compounds of Formulas (I)-(IV) of the present disclosure is the selection of an appropriate amine to prepare the “A” amine core precursor. Non-limiting examples of amines that may be selected to prepare the “A” amine core precursor include:

[0082] In some embodiments, the next step in the preparation of a compound of Formulas (I)-(IV) is reaction of the amine with a terminal thioepoxide to form the precursor compound. The following is an exemplary reaction scheme between a primary amine and a thioepoxide to form the precursor compound by ring opening of the thioepoxide and attachment to the amine: wherein R is an aliphatic group, as defined herein.

[0083] In some embodiments, one equivalent of an amine is reacted with one equivalent of a thioepoxide compound. In other embodiments, one equivalent of an amine is reacted with one, two, three, four, five or more equivalents of a thioepoxide compound. The amount of thioepoxide compound may be limited to prevent the functionalization of all amino groups. The resulting compounds may contain secondary amino groups and/or primary amino groups, which may be further functionalized with a different terminal thioepoxide or different electrophile, for example. Such further functionalization of the amines may result in compounds with different thioepoxide derived tails.

[0084] In some embodiments, the amine and the thioepoxide will react at the unsubstituted carbon of the thioepoxide to result in a compound:

[0085] In some embodiments, the amine and the thioepoxide will react at the substituted carbon of the thioepoxide to result in a compound:

[0086] Thioepoxide compounds that are useful in the present invention include any thioepoxide compounds that are racemic or stereoisomers thereof, all of varying chain lengths and with functional groups having varying degrees of saturation. In certain embodiments, the thioexpoxide is stereochemically pure (e.g., enantiomerically pure). In certain embodiments, the thioepoxide contains one or more chiral centers. In certain embodiments, the thioepoxide compounds are of the formula:

[0087] In some embodiments, reaction of the amine with a terminal thioepoxide generates a compound with a thiol moiety linked to the carbon in the beta position (“beta carbon”) to the amino group.

[0088] In certain embodiments, the preparation of a compound of Formulas (I)-(IV) comprises reaction of the amine with a terminal thioepoxide under conditions to form the “A” amine precursor compound with a disulfide linked to the beta carbon of the amino group. For example, the amine can be reacted with a thioepoxide compound in the presence of methanethiosulfonate to form the precursor compound with a disulfide-linked methyl group linked to the beta carbon of the amino group.

[0089] In some embodiments, the disulfide can then be treated with a reducing agent to form a free thiol.

[0090] In some embodiments, the final step in the preparation of a compound of Formulas (I)-(IV) comprises further functionalization of the thiol moiety. For example, the amine precursor compound can be treated with 2-(2-(Pyridin-2-yl)disulfanyl)ethanol to add a disulfide-linked hydroxyl group to the amine precursor compound at the beta carbon of the amino group. In another example, the amine precursor compound can be treated with an acrylate or acrylamide to add a sulfur-linked ester or sulfur-linked amide group to the amine precursor compound at the beta carbon of the amino group.

[0091] In some embodiments, the present disclosure provides methods comprising the step of reacting or more equivalents of an amine of one of the formula:

with a thioepoxide-containing compound of the formula: under conditions to form a compound of one of the Formulas (I)-(IV):

wherein A, B, C, D, X, L, Y, R ₁ , R2, R3, R4, Ra, Rb, R ₆ and Rf are as defined herein.

[0092] In some embodiments, the present disclosure provides a method comprising the steps of reacting one or more equivalents of an amine of the formula: with a

thioepoxide-containing compound of the formula under conditions to form a compound of the formula

(b) reducing the compound of the formula: to produce a compound of the formula:

(c) reacting a compound of the formula: with a compound of the formula: to produce a compound of the formula: wherein A, R ₁ , L, Y, B, C, and D are as described herein.

[0093] In some embodiments, the present disclosure provides a method comprising the steps of reacting one or more equivalents of an amine of the formula: with a thioepoxide-containing compound of the formula under conditions to form a compound of the formula:

(b) reducing the compound of the formula: to produce a compound of the formula:

(c) reacting a compound of the formula: with a compound of the formula: to produce a compound of the formula: wherein R ₇ , L and Y are as described herein.

[0094] In some embodiments, the preparation of a compound of Formulas (I)-(IV) comprises reaction of the amine with an aldehyde to form the amine precursor compound. The following is an exemplary reaction scheme between an amine and a terminal aldehyde to form the precursor compound by reductive amination: wherein R is an aliphatic group, as defined herein, and Z is hydrogen or -SR.

[0095] In some embodiments, reaction of the amine with a terminal aldehyde generates a compound with a thiol moiety linked to the beta carbon of the amino group.

[0096] In some embodiments, the preparation of a compound of Formulas (I)-(IV) comprises reaction of the amine with a terminal aldehyde under conditions to form the “A” amine precursor compound with a disulfide linked to the beta carbon of the amino group. For example, the amine can be reacted with an aldehyde compound comprising a methyl disulfide group in the beta position to the aldehyde to form the precursor compound with a methyl disulfide linked to the beta carbon of the amino group.

[0097] In some embodiments, the disulfide can then be treated with a reducing agent to form a free thiol.

[0098] In some embodiments, the final step in the preparation of a compound of Formulas (I)-(IV) comprises further functionalization of the thiol moiety. For example, the amine precursor compound can be treated with 2-(2-(Pyridin-2-yl)disulfanyl)ethanol to add a disulfide-linked hydroxyl group to the amine precursor compound at the beta carbon of the amino group. In another example, the amine precursor compound can be treated with an acrylate or acrylamide to add a sulfur-linked ester or sulfur-linked amide group to the amine precursor compound at the beta carbon of the amino group.

[0099] In some embodiments, the present disclosure provides a method comprising the steps of:

(a) preparing an aldehyde of the formula: ; (b) condensing the aldehyde of the formula: with an amine of the formula to produce a compound of [00100] In some embodiments, one equivalent of an amine is reacted with one equivalent of an aldehyde compound. In other embodiments, one equivalent of an amine is reacted with one, two, three, four, five or more equivalents of an aldehyde compound. The amount of aldehyde compound may be limited to prevent the functionalization of all amino groups. The resulting compounds may contain secondary amino groups and/or primary amino groups, which may be further functionalized with a different aldehyde or different electrophile, for example. Such further functionalization of the amines may result in compounds with tails derived from different aldehyde compounds.

[00101] In some embodiments, the preparation of a compound of Formulas (I)-(IV) comprises the selection or formation of an aminoalcohol precursor compound. For example, an exemplary amine can be reacted with an epoxide to form an aminoalcohol precursor compound by ring opening of the epoxide. In some embodiments, the next step in the preparation of a compound of Formulas (I)-(IV3 comprises further functionalization of the aminoalcohol moiety. For example, the amine precursor compound can be treated with 2 -mercaptoethanol to add a sulfur- linked ethylalcohol group to the precursor compound at the beta carbon of the amino group.

LNP COMPONENTS

[00102] In some aspects, an LNP of the present disclosure can comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5% or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% of at least one compound of the present disclosure by moles. In some aspects, the at least one compound is at least one compound of Formulas (I)-(IV), as described herein.

[00103] In some aspects, an LNP of the present disclosure can comprise about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5% or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% of at least one compound of the present disclosure by moles. In some aspects, the at least one compound is at least one compound of Formulas (I)-(TV), as described herein.

[00104] In some aspects, an LNP can further comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5% or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% of at least one structural lipid by moles.

[00105] In some aspects, an LNP can further comprise at least about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about

22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about

37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about

52.5%, or about 55%, or about 57.5% or about 60%, or about 62.5%, or about 65%, or about

67.5%, or about 70% of at least one structural lipid by moles.

[00106] In some aspects, an LNP can further comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5% or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% of at least one phospholipid by moles.

[00107] In some aspects, an LNP can further comprise at least about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5% or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% of at least one phospholipid by moles.

[00108] In some aspects, an LNP can further comprise at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1.0%, or at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10% PEGylated lipid by moles.

A, Structural Lipid

[00109] In some aspects, a structural lipid can be a steroid. In some aspects, a structural lipid can be a sterol. In some aspects, a structural lipid can comprise cholesterol. In some aspects, a structural lipid can comprise ergosterol. In some aspects, a structural lipid can be a phytosterol.

B. Phospholipid

[00110] As used herein, the term “phospholipid” is used in its broadest sent to refer to any amphiphilic molecule that comprises a polar (hydrophilic) headgroup comprising phosphate and two hydrophobic fatty acid chains. In some aspects, a phospholipid can comprise dioleoylphosphatidylethanolamine (DOPE). In some aspects, a phospholipid can comprise 1,2- Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some aspects, a phospholipid can comprise DDPC (l,2-Didecanoyl-sn-glycero-3 -phosphocholine), DEPA-NA (l,2-Dierucoyl-sn-glycero-3- phosphate (Sodium Salt)), DEPC (l,2-Dierucoyl-sn-glycero-3-phosphocholine), DEPE (1,2- Dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-Dierucoyl-sn-glycero-

3 [Phospho-rac-(l -glycerol) (Sodium Salt)), DLOPC (l,2-Dilinoleoyl-sn-glycero-3- phosphocholine), DLPA-NA (l,2-Dilauroyl-sn-glycero-3-phosphate (Sodium Salt)), DLPC (1,2- Dilauroyl-sn-glycero-3-phosphocholine), DLPE (l,2-Dilauroyl-sn-glycero-3- phosphoethanolamine), DDLLPPGG--NNAA ( 1 ,2-Dilauroyl-sn-glycero-3 [Phospho-rac-(l -glycerol) (Sodium Salt)), DLPG-NH4 (l,2-Dilauroyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Ammonium Salt)), DLPS-NA (l^-Dilauroyl-sn-glycero-3-phosphoserine (Sodium Salt)), DMPA-NA (1,2- Dimyristoyl-sn-glycero-3-phosphate (Sodium Salt)), DMPC (l,2-Dimyristoyl-sn-glycero-3- phosphocholine), DMPE (l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2- Dimyristoyl-sn-glycero-3[Phospho-rac-(l-glycerol) (Sodium Salt)), DMPG-NH4 (1,2- Dimyristoyl-sn-glycero-3[Phospho-rac-(l -glycerol) (Ammonium Salt)), DMPG-NH4/NA (1,2- Dimyristoyl-sn-glycero-3[Phospho-rac-(l-glycerol) (Sodium/Ammonium Salt)), DMPS-NA(1,2- Dimyristoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DOPA-NA (l,2-Dioleoyl-sn-glycero-3- phosphate (Sodium Salt)), DOPC (l,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine), DOPG-NA (l,2-Dioleoyl-sn-glycero-3[Phospho- rac-(l -glycerol) (Sodium Salt)), DOPS-NA (l,2-Dioleoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DPPA-NA (l,2-Dipalmitoyl-sn-glycero-3-phosphate (Sodium Salt)), DPPC (1,2- Dipalmitoyl-sn-glycero-3-phosphocholine), DPPE (l,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine), DPPG-NA ( 1 ,2-Dipalmitoyl-sn-glycero-3 [Phospho-rac-( 1 -glycerol)

(Sodium Salt)), DDPPPPGG--NNHH44 (l,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(l-glycerol) (Ammonium Salt)), DPPS-NA (l,2-Dipalmitoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DSPA-NA (l,2-Distearoyl-sn-glycero-3-phosphate (Sodium Salt)), DSPC (1,2-Distearoyl-sn- glycero-3-phosphocholine), DSPE (l,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DSPG- NA (l,2-Distearoyl-sn-glycero-3[Phospho-rac-(l-glycerol) (Sodium Salt)), DSPG-NH4 (1,2- Distearoyl-sn-glycero-3[Phospho-rac-(l-glycerol) (Ammonium Salt)), DSPS-NA(l,2-Distearoyl- sn-glycero-3-phosphoserine (Sodium Salt)), EPC (Egg-PC), HEPC (Hydrogenated Egg PC), HSPC (Hydrogenated Soy PC), LYSOPC MYRISTIC (l-Myristoyl-sn-glycero-3- phosphocholine), LYSOPC PALMITIC (l-Palmitoyl-sn-glycero-3-phosphocholine), LYSOPC STEARIC (l-Stearoyl-sn-glycero-3 -phosphocholine), Milk Sphingomyelin (MPPC; 1-Myristoyl- 2-palmitoyl-sn-glycero 3-phosphocholine), MSPC (l-Myristoyl-2-stearoyl-sn-glycero-3- phosphocholine), PMPC (l-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), POPC (1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (l-Palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine), POPG-NA (l-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(l-glycerol)] (Sodium Salt)), PSPC (l-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (1-Stearoyl- 2-myristoyl-sn-glycero-3-phosphocholine), SOPC (l-Stearoyl-2-oleoyl-sn-glycero-3- phosphocholine), SPPC (l-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or any combination thereof.

C. PEGylated Lipid

[00111] As used herein, the term “PEGylated lipid” is used to refer to any lipid that is modified (e.g. covalently linked to) at least one polyethylene glycol molecule. In some aspects, a PEGylated lipid can comprise l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, hereafter referred to as DMG-PEG2000.

LNP Compositions [00112] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, and at least one structural lipid.

[00113] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, and at least one PEGylated lipid.

[00114] In some aspects, the at least one structural lipid is a mixture of two structural lipids.

[00115] In some aspects, the at least one PEGylated lipid is a mixture of two PEGylated lipids.

[00116] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, at least one structural lipid, at least one PEGylated lipid or any combination thereof.

[00117] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, and at least one PEGylated lipid.

[00118] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, at least one PEGylated lipid or any combination thereof.

[00119] In some aspects, a lipid nanoparticle can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid and at least one PEGylated lipid.

[00120] In some aspects, the at least one compound of the present disclosure is a compound of Formulas (I)-(TV).

[00121] In some aspects, the at least one compound of the present disclosure is a mixture of two or more compounds of Formulas (I)-(IV).

[00122] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 54% of at least one compound of Formulas (I)-(IV) by moles, about 35% of at least one structural lipid by moles, about 10% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 44% to about 64% of at least one compound of Formulas (0-(IV) by moles, about 25% to about 45% of at least one structural lipid by moles, about 0.1% to about 20% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 49% to about 59% of at least one compound of Formulas (I)-(IV) by moles, about 30% to about 40% of at least one structural lipid by moles, about 5% to about 15% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles.

[00123] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 43.3% of at least one compound of Formulas (I)-(IV) by moles, about 43.3% of at least one structural lipid by moles, about 12% of at least one phospholipid by moles, and about 1.5% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 33.3% to about 53.3% of at least one compound of Formulas (T)-(TV) by moles, about 33.3% to about 53.3% of at least one structural lipid by moles, about 2% to about 22% of at least one phospholipid by moles, and about 0.1% to about 11.5% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 38.3% to about 48.3% of at least one compound of Formulas (I)-(IV) by moles, about 38.3% to about 48.3% of at least one structural lipid by moles, about 7% to about 17% of at least one phospholipid by moles, and about 0.5% to about 6.5% of at least one PEGylated lipid by moles.

[00124] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 33.5% of at least one compound of Formulas (I)-(IV) by moles, about 33.5% of at least one structural lipid by moles, about 32% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 23.5% to about 43.5% of at least one compound of Formulas (I)- (IV) by moles, about 23.5% to about 43.5% of at least one structural lipid by moles, about 22% to about 42% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 28.5% to about 38.5% of at least one compound of Formulas (I)-(IV) by moles, about 28.5% to about 38.5% of at least one structural lipid by moles, about 27% to about 37% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles. [00125] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 49.6% of at least one compound of Formulas (I)-(IV) by moles, about 39.9% of at least one structural lipid by moles, about 9.5% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 39.6% to about 59.6% of at least one compound of Formulas (I)-(IV) by moles, about 29.9% to about 49.9% of at least one structural lipid by moles, about 0.1% to about 19.5% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 44.6% to about 54.6% of at least one compound of Formulas (I)-(IV) by moles, about 34.9% to about 44.9% of at least one structural lipid by moles, about 4.5% to about 14.5% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles.

[00126] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 52.1% of at least one compound of Formulas (I)-(IV) by moles, about 45% of at least one structural lipid by moles, about 1.9% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 42.1% to about 62.1% of at least one compound of Formulas (I)-(TV) by moles, about 35% to about 55% of at least one structural lipid by moles, about 0.1% to about 11.9% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 47.1% to about 57.1% of at least one compound of Formulas (I)-(IV) by moles, about 40% to about 50% of at least one structural lipid by moles, about 0.5% to about 6.9% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles.

[00127] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 60% of at least one compound of Formulas (I)-(IV) by moles, about 30% of at least one structural lipid by moles, about 9% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 50% to about 70% of at least one compound of Formulas (I)-(IV) by moles, about 20% to about 40% of at least one structural lipid by moles, about 0.1% to about 19% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 55% to about 65% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 35% of at least one structural lipid by moles, about 4% to about 14% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles.

[00128] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 34% to about 60% of at least one compound of Formulas (I)-(IV) by moles, about 30% to about 60% of at least one structural lipid by moles, about 5% to about 11.9% of at least one phospholipid by moles, and about 1% to about 2% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 24% to about 70% of at least one compound of Formulas (T)-(IV) by moles, about 20% to about 70% of at least one structural lipid by moles, about 0.1% to about 21.9% of at least one phospholipid by moles, and about 0.1% to about 12% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 29% to about 65% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 65% of at least one structural lipid by moles, about 1% to about 16.9% of at least one phospholipid by moles, and about 0.5% to about 7% of at least one PEGylated lipid by moles.

[00129] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise about 49.6% to about 60% of at least one compound of Formulas (I)-(IV) by moles, about 30% to about 45% of at least one structural lipid by moles, about 0.2% to about 9.5% of at least one phospholipid by moles, and about 1% to about 1.5% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 39.6% to about 70% of at least one compound of Formulas (I)-(IV) by moles, about 20% to about 55% of at least one structural lipid by moles, about 0.1% to about 19.5% of at least one phospholipid by moles, and about 0.1% to about 11.5% of at least one PEGylated lipid by moles. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 44.6% to about 65% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 50% of at least one structural lipid by moles, about 0.1% to about 14.5% of at least one phospholipid by moles, and about 0.5% to about 6.5% of at least one PEGylated lipid by moles.

[00130] In some aspects, the compound of Formulas (I)-(IV) comprised in the LNP composition is one of COMPOUNDS 3, 6, 9, 12-16, and 19. [00131] In some aspects of the preceding LNPs, the structural lipid can be cholesterol. In some aspects of the preceding LNPs, the phospholipid can be DOPE. In some aspects of the preceding LNPs, the phospholipid can be DSPC. In some aspects of the preceding LNPs, the phospholipid can be DOPC. In some aspects of the preceding LNPs, the PEGylated lipid can be DMG-PEG2000.

[00132] In some aspects of the preceding LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPE and the PEGylated lipid can be DMG-PEG2000.

[00133] In some aspects of the preceding LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPC and the PEGylated lipid can be DMG-PEG2000.

[00134] In some aspects of the preceding LNPs, the at least one nucleic acid molecule is a DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid.

[00135] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one nucleic acid molecule. In some aspects, a lipid nanoparticle can comprise a plurality of nucleic acid molecules. In some aspects, the at least one nucleic acid molecule or the plurality of nucleic acid molecules can be formulated in a lipid nanoparticle.

[00136] In some aspects, a lipid nanoparticle can comprise lipid and nucleic acid at a specified ratio (weight/weight).

[00137] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1 or about 30:1 to about 40: 1, or about 35: 1 to about 45:1, or about 40:1 to about 50: 1, or about 45: 1 to about 55: 1, or about 50: 1 to about 60: 1, or about 55: 1 to about 65: 1, or about 60: 1 to about 70: 1, or about 65:1 to about 75: 1, or about 70: 1 to about 80: 1, or about 75: 1 to about 85: 1, or about 80: 1 to about 90: 1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or about 115:1 to about 125:1, or about 120:1 to about 130:1, or about 125:1 to about 135:1, or about 130:1 to about 140:1, or about 135:1 to about 145:1, or about 140:1 to about 150:1, lipidmucleic acid, weight/weight. [00138] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 5:1, or about 10: 1, or about 15:1, or about 20: 1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1, lipidmucleic acid, weight/weight.

[00139] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise lipid and nucleic acid at a ratio of about 10: 1, or about 25: 1, or about 40: 1, lipidmucleic acid, weight/weight.

[00140] In some aspects of the preceding LNPs, the at least one nucleic acid molecule is an RNA molecule. In some aspects, the RNA molecule is an mRNA molecule. In some aspects, the mRNA molecule further comprises a 5’-CAP.

[00141] Thus, the present disclosure provides a lipid nanoparticle comprising about 54% of at least one compound of Formulas (I)-(IV) by moles, about 35% of at least one structural lipid by moles, about 10% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising about 44% to about 64% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 45% of at least one structural lipid by moles, about 0.1% to about 20% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising about 49% to about 59% of at least one compound of Formulas (I)-(IV) by moles, about 30% to about 40% of at least one structural lipid by moles, about 5% to about 15% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the mRNA molecule further comprises a 5’-CAP. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 90:1 to about 110:1 (w/w), or about 95:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100:1 (w/w). [00142] In some aspects, a lipid nanoparticle is provided comprising about 43.3% of at least one compound of Formulas (I)-(IV) by moles, about 43.3% of at least one structural lipid by moles, about 12% of at least one phospholipid by moles, and about 1.5% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising about 33.3% to about 53.3% of at least one compound of Formulas (I)-(IV) by moles, about 33.3% to about 53.3% of at least one structural lipid by moles, about 2% to about 22% of at least one phospholipid by moles, and about 0.1% to about 11.5% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising about 38.3% to about 48.3% of at least one compound of Formulas (I)-(IV) by moles, about 38.3% to about 48.3% of at least one structural lipid by moles, about 7% to about 17% of at least one phospholipid by moles, and about 0.5% to about 6.5% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the mRNA molecule further comprises a 5’-CAP. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 90: 1 to about 110: 1 (w/w), or about 95:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100:1 (w/w).

[00143] In some aspects, a lipid nanoparticle is provided comprising about 33.5% of at least one compound of Formulas (I)-(IV) by moles, about 33.5% of at least one structural lipid by moles, about 32% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising about 23.5% to about 43.5% of at least one compound of Formulas (I)-(IV) by moles, about 23.5% to about 43.5% of at least one structural lipid by moles, about 22% to about 42% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising about 28.5% to about 38.5% of at least one compound of Formulas (I)-(IV) by moles, about 28.5% to about 38.5% of at least one structural lipid by moles, about 27% to about 37% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g. mRNA molecule). In some aspects, the mRNA molecule further comprises a 5’-CAP. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 30:1 to about 50:1 (w/w) or about 35:1 to about 45:1 (w/w). In some aspects, the ratio oflipid to nucleic acid in the nanoparticle can be about 40:1 (w/w).

[00144] In some aspects, the nucleic acid molecule is a DNA molecule. Thus, the present disclosure provides a lipid nanoparticle comprising about 49.6% of at least one compound of Formulas (I)-(IV) by moles, about 39.9% of at least one structural lipid by moles, about 9.5% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 39.6% to about 59.6% of at least one compound of Formulas (I)-(IV) by moles, about 29.9% to about 49.9% of at least one structural lipid by moles, about 0.1% to about 19.5% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule, hi some aspects, the present disclosure provides a lipid nanoparticle comprising about 44.6% to about 54.6% of at least one compound of Formulas (I)- (IV) by moles, about 34.9% to about 44.9% of at least one structural lipid by moles, about 4.5% to about 14.5% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 110: 1 to about 130: 1 (w/w), or about 115: 1 to about 125:1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 120:1 (w/w).

[00145] In some aspects, a lipid particle is provided comprising about 52.1% of at least one compound of Formulas (I)-(IV) by moles, about 45% of at least one structural lipid by moles, about 1.9% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 42.1% to about 62.1% of at least one compound of Formulas (I)-(IV) by moles, about 35% to about 55% of at least one structural lipid by moles, about 0.1% to about 11.9% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 47.1% to about 57.1% of at least one compound of Formulas (I)-(IV) at least one compound of Formulas (I)-(IV) by moles, about 40% to about 50% of at least one structural lipid by moles, about 0.5% to about 6.9% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 90: 1 to about 110: 1 (w/w), or about 95: 1 to about 105: 1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100: 1 (w/w).

[00146] In some aspects, a lipid particle is provided comprising about 60% of at least one compound of Formulas (I)-(IV) by moles, about 30% of at least one structural lipid by moles, about 9% of at least one phospholipid by moles, and about 1% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 50% to about 70% of at least one compound of Formulas (I)-(IV) by moles, about 20% to about 40% of at least one structural lipid by moles, about 0.1% to about 19% of at least one phospholipid by moles, and about 0.1% to about 11% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 55% to about 65% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 35% of at least one structural lipid by moles, about 4% to about 14% of at least one phospholipid by moles, and about 0.5% to about 6% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 110:1 to about 130:1 (w/w), or about 115:1 to about 125:1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 120:1 (w/w).

[00147] In some aspects, a lipid particle is provided comprising about 34% to about 60% of at least one compound of Formulas (I)-(IV) by moles, about 30% to about 60% of at least one structural lipid by moles, about 5% to about 11.9% of at least one phospholipid by moles, and about 1% to about 2% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 24% to about 70% of at least one compound of Formulas (0-(IV) by moles, about 20% to about 70% of at least one structural lipid by moles, about 0.1% to about 21.9% of at least one phospholipid by moles, and about 0.1% to about 12% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 29% to about 65% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 65% of at least one structural lipid by moles, about 1% to about 16.9% of at least one phospholipid by moles, and about 0.5% to about 7% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 70: 1 to about 130: 1 (w/w), or about 75: 1 to about 125: 1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 80:1 (w/w) to about 120:1 (w/w).

[00148] In some aspects, a lipid particle is provided comprising about 49.6% to about 60% of at least one compound of Formulas (I)-(IV) by moles, about 30% to about 45% of at least one structural lipid by moles, about 0.2% to about 9.5% of at least one phospholipid by moles, and about 1% to about 1.5% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 39.6% to about 70% of at least one compound of Formulas (I)-(TV) by moles, about 20% to about 55% of at least one structural lipid by moles, about 0.1% to about 19.5% of at least one phospholipid by moles, and about 0.1% to about 11.5% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising about 44.6% to about 65% of at least one compound of Formulas (I)-(IV) by moles, about 25% to about 50% of at least one structural lipid by moles, about 0.1% to about 14.5% of at least one phospholipid by moles, and about 0.5% to about 6.5% of at least one PEGylated lipid by moles, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA molecule is a DoggyBone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 70:1 to about 130:1 (w/w), or about 75:1 to about 125:1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 80: 1 (w/w) to about 120: 1 (w/w).

[00149] In some aspects, the compound of Formulas (I)-(IV) comprised in the LNP composition is one of COMPOUNDS 3, 6, 9, 12-16, and 19.

[00150] In some aspects of the preceding LNPs, the structural lipid can be cholesterol. In some aspects of the preceding LNPs, the phospholipid can be DOPE. In some aspects of the preceding LNPs, the phospholipid can be DSPC. In some aspects of the preceding LNPs, the phospholipid can be DOPC. In some aspects of the preceding LNPs, the PEGylated lipid can be DMG-PEG2000.

[00151] In some aspects of the preceding LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPE and the PEGylated lipid can be DMG-PEG2000.

[00152] In some aspects of the preceding LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPC and the PEGylated lipid can be DMG-PEG2000.

Pharmaceutical Compositions of the Present Disclosure

[00153] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the present disclosure and at least one second nanoparticle of the present disclosure, wherein the at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, wherein the at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon. In some aspects, the at least one nucleic acid molecule encoding at least one transposase can be an RNA molecule (e.g. mRNA molecule) and the at least one nucleic acid molecule encoding at least one transposon can be a DNA molecule (e.g. a DoggyBone DNA molecule or a DNA nanoplasmid).

[00154] In some aspects, the present disclosure provides a composition comprising at least one cell that has been contacted by at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using any method of the present disclosure.

[00155] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been contacted by at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been genetically modified using any method of the present disclosure.

Methods of the Present Disclosure

[00156] The present disclosure provides a method of delivering at least one nucleic acid to at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of delivering at least one nucleic acid to at least one cell comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[00157] In all methods, compositions and kits of the present disclosure, an at least one cell can be a liver cell. A liver cell can include, but is not limited to, a hepatocyte, a hepatic stellate cell, Kupffer cell or a liver sinusoidal endothelial cell. In all methods, compositions and kits of the present disclosure, an at least one cell can be a T-cell. A T-cell can be a resting T-cell, an activated T-cell, stem memory T cells (TSCM cells), central memory T cells (TCM), or stem cell-like T cells.

[00158] In some aspects of any methods of the present disclosure, a cell can be in vivo, ex vivo or in vitro. In some aspects, any of the methods of the present disclosure can be applied in vivo, ex vivo or in vitro.

[00159] The present disclosure provides a method of genetically modifying at least one cell comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of genetically modifying at least one cell comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[00160] In some aspects, genetically modifying a cell can comprise delivering at least one exogenous nucleic acid to the cell such that the cell expresses at least one protein that the cell otherwise would not normally express, or such that the at least one cell expresses at least one protein at a level that is higher than the level that the cell would otherwise normally express the at least one protein, or such that the cell expresses at least one protein at a level that is lower than the level that the cell would otherwise normally express. In some aspects, genetically modifying a cell can comprise delivering at least one exogenous nucleic to the cell such that at least one exogenous nucleic acid is integrated into the genome of the at least one cell.

[00161] In all methods of the present disclosure, T-cells can be activated prior to, concurrently with, or after contacting the T-cells with at least one composition or at least one nanoparticle of the present disclosure. In some aspects, T-cells can be activated using standard techniques known in the art, including, but not limited to, contacting the T-cells with CD3/CD28/CD2 activator solution, anti-CD3 antibody beads, anti-CD28 antibody bead, anti-CD2 antibody beads, anti-CD3 and anti-CD28 antibody beads, tetrameric antibody complexes that bind CD3, CD28 and CD2 cell surface ligands, or any combination thereof.

[00162] In some aspects, T-cells can be activated at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 10 hours, or at least 11 hours, or at least 12 hours, or at least 13 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 17 hours, or at least 18 hours, or at least 19 hours, or at least 20 hours, or at least 21 hours, or at least 22 hours, or at least 23 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 60 hours, or at least 72 hours prior to be contacted with at least one composition or nanoparticle of the present disclosure.

[00163] In some aspects, T-cells can be activated at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 10 hours, or at least 11 hours, or at least 12 hours, or at least 13 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 17 hours, or at least 18 hours, or at least 19 hours, or at least 20 hours, or at least 21 hours, or at least 22 hours, or at least 23 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 60 hours, or at least 72 hours after being contacted with at least one composition or nanoparticle of the present disclosure. [00164] In some aspects of the preceding methods, step c) can be performed at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 10 hours, or at least 11 hours, or at least 12 hours, or at least 13 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 17 hours, or at least 18 hours, or at least 19 hours, or at least 20 hours, or at least 21 hours, or at least 22 hours, or at least 23 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 60 hours, or at least 72 hours after step b).

[00165] In some aspects of the preceding methods, step a) can be performed at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 10 hours, or at least 11 hours, or at least 12 hours, or at least 13 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 17 hours, or at least 18 hours, or at least 19 hours, or at least 20 hours, or at least 21 hours, or at least 22 hours, or at least 23 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 60 hours, or at least 72 hours prior to step b).

[00166] In some aspects, the methods of the present disclosure can yield a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cell in the plurality express at least one protein that was encoded in at least one nucleic acid that was delivered to the plurality of cells via a nanoparticle of the present disclosure.

[00167] In some aspects, the methods of the present disclosure can yield a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells in the plurality are stem memory T cells. [00168] In some aspects, the methods of the present disclosure can yield a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells are express one or more cell-surface marker(s) of a stem memory T cell (TSCM) or a TscM-like cell and wherein the one or more cell-surface markers) comprises CD62L and CD45RA.

[00169] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[00170] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[00171] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of cells, wherein the cells have been contacted by at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein. The present disclosure provides a method of treating at least one disease in a subj ect, the method comprising administering at least one therapeutically effective amount of cells, wherein the cells have been genetically modified using the compositions and/or methods of the present disclosure.

[00172] In some aspects, the at least one disease can be a malignant disease, including, but not limited to, cancer. In some aspects, the at least one disease can be a metabolic liver disorder (MLD). In some aspects, the at least one disease can be a urea cycle disorder (UCD). An MLD and/or UCD can include, but is not limited to, N-Acetylglutamate Synthetase (NAGS) Deficiency, Carbamoylphosphate Synthetase I Deficiency (CPSI Deficiency), Ornithine Transcarbamylase (OTC) Deficiency, Argininosuccinate Synthetase Deficiency (ASSD) (Citrullinemia I), Citrin Deficiency (Citrullinemia II), Argininosuccinate Lyase Deficiency (Argininosuccinic Aciduria), Arginase Deficiency (Hyperargininemia), Ornithine Translocase Deficiency (HHH Syndrome), methylmalonic addemia (MMA) or any combination thereof.

[00173] In some aspects, the at least one disease can be hemophilia A.

[00174] Accordingly, the present disclosure provides a method of treating hemophilia A in a subject in need thereof comprising administering to the subject at least one composition comprising at least one lipid nanoparticle of the present disclosure, wherein the lipid nanoparticle comprises a nucleic acid encoding a FVIII polypeptide.

[00175] The present disclosure provides a method of treating Ornithine Transcarbamylase (OTC) Deficiency in a subject in need thereof comprising administering to the subject at least one composition comprising at least one lipid nanoparticle of the present disclosure, wherein the lipid nanoparticle comprises a nucleic acid encoding an ornithine transcarbamylase (OTC) polypeptide.

[00176] The present disclosure provides a method of treating methylmalonic acidemia (MMA) in a subject in need thereof comprising administering to the subject at least one composition comprising at least one lipid nanoparticle of the present disclosure, wherein the lipid nanoparticle comprises a nucleic acid encoding a methylmalonyl-CoA mutase (MUT1) polypeptide.

Nucleic Acid Molecules

[00177] In some aspects, a nucleic acid molecule can be an RNA molecule. Thus, in some aspects, a lipid nanoparticle can comprise at least one RNA molecule. The at least one RNA molecule can be encapsulated within the lipid nanoparticle. In some aspects, an RNA molecule can be an mRNA molecule. In some aspects, a lipid nanoparticle can comprise at least one mRNA molecule. The mRNA molecule can be encapsulated within the lipid nanoparticle.

[00178] In some aspects, a nucleic acid molecule can be a synthetic nucleic add molecule. In some aspects, a nucleic acid molecule can be a non-naturally occurring nucleic add molecule. In some aspects, a non-naturally occurring nucleic acid molecule can comprise at least one non- naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non- naturally occurring nucleotide known in the art. In some aspects, a nucldc acid molecule can be a modified nucleic acid molecule. In some aspects, a modified nucleic acid molecule can comprise at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[00179] In some aspects, an mRNA molecule can be capped using any method and/or capping moiety known in the art. An mRNA molecule can be capped with m7G(5’)ppp(5’)G moiety. A m7G(5’)ppp(5’)G moiety is also referred to herein as a “CapO”. An mRNA molecule can be capped with a CleanCap® moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. A CleanCap® moiety can comprise a m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. An mRNA molecule can be capped with an anti-reverse cap analog (ARC A®) moiety. An ARCA® moiety can comprise a m7(3’-O- methyl)G(5’)ppp(5’)G moiety. An mRNA molecule can be capped with a CleanCap® 3’OMe moiety (CleanCap®+ARCA®).

[00180] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[00181] The at least one modified nucleic acid can comprise 5-methoxyuridine (5moU). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in an mRNA molecule are 5-methoxyuridine bases. In some aspects, all of the uridine bases in an mRNA molecule are 5-methoxyuridine bases. Without wishing to be bound by theory, 5-methoxyuridine can improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy - Nucleic Acids, 2018, 12, 530- 542).

[00182] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[00183] The at least one modified nucleic acid can comprise N1- methylpseudouridine (me ¹ ψ). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in an mRNA N1- methylpseudouridine bases. In some aspects, all of the uridine bases in an mRNA molecule areN1- methylpseudouridine bases. Without wishing to be bound by theory, Ni- methylpseudouridine can improve protein expression (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853).

[00184] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[00185] The at least one modified nucleic acid can comprise pseudouridine (T). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in an mRNA pseudouridine bases. In some aspects, all of the uridine bases in an mRNA molecule are pseudouridine bases. Without wishing to be bound by theory, pseudouridine can improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy - Nucleic Acids, 2018, 12, 530-542).

[00186] In some aspects, an mRNA molecule can comprise at least one modified nucleic acid.

[00187] The at least one modified nucleic acid can comprise 5-methylcytidine (5-MeC). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the cytidine bases in an mRNA 5- MeC bases. In some aspects, all of the cytidine bases in an mRNA molecule are 5-MeC bases.

[00188] In some aspects, a nucleic acid molecule can comprise a DNA molecule. Thus, in some aspects, a lipid nanoparticle can comprise a DNA molecule. In some aspects, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid or DNA nanoplasmid. Thus, in some aspects, a lipid nanoparticle can comprise a circular DNA molecule. In some aspects, a lipid nanoparticle can comprise a Doggybone DNA molecule. In some aspects, a lipid nanoparticle can comprise a DNA plasmid. In some aspects, a lipid nanoparticle can comprise a DNA nanoplasmid. In some aspects, a DNA molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid or a linearized DNA nanoplasmid.

[00189] A DNA plasmid or DNA nanoplasmid can comprise can be at least about 0.25 kb, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb in length.

[00190] In some aspects, a nucleic acid molecule formulated in a lipid nanoparticle of the present disclosure can comprise at least one transgene sequence. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one transposase. In some aspects, a transgene sequence can comprise a nucleotide sequence encoding at least one transposon. In some aspects, a transposon can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transposon can comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, wherein the at least one therapeutic protein is operatively linked to the at least one promoter sequence. [00191] In some aspects, a therapeutic protein can be an ornithine transcarbamylase (OTC) polypeptide, a methylmalonyl-CoA mutase (MUT1) polypeptide, a chimeric antigen receptor, or a Factor VIII (FVIII) polypeptide.

[00192] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform. In some aspects, the microfluidic-mixing platform can be a non-turbulent microfluidic mixing platform.

[00193] In some aspects, a microfluidic-mixing platform can produce the lipid nanoparticles of the present invention by combining a miscible solvent phase comprising the lipid components of the nanoparticle and an aqueous phase comprising the lipid nanoparticle cargo (e.g. nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some aspects, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow for immediate mixing of the two phases. As the two phases move under laminar flow in a microfluidic channel, microscopic features in the channel can allow for controlled, homogenous mixing to produce the lipid nanoparticles of the present disclosure.

[00194] In some aspects, the microfluidic-mixing platform can include, but are not limited to the NanoAssemblr® Spark (Precision NanoSystems), the NanoAssemblr® Ignite™ (Precision NanoSystems), the NanoAssemblr® Benchtop (Precision NanoSystems), the NanoAssemblr® Blaze (Precision NanoSystems) or the NanoAssemblr® GMP System (Precision NanoSystems).

[00195] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform, wherein the microfluidic mixing platform mixes at a rate of at least about 2.5 ml/min, or at least about 5 ml/min, or at least about 7.5 ml/min, or at least about 10 ml/min, or at least about 12.5 ml/min, or at least about 15 ml/min, or at least about 17.5 ml/min, or at least about 20 ml/min, or at least about 22.5 ml/min, or at least about 25 ml/min, or at least about 27.5 ml/min, or at least about 30 ml/min.

[00196] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic-mixing platform, wherein the microfluidic mixing platform mixes a miscible solvent phase and an aqueous phase at a ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:l, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, solventaqueous, v:v. piggyBac ITR sequences

[00197] In some aspects, a nucleic acid can comprise a piggBac ITR sequence. In some aspects, a nucleic acid can comprise a first piggyBac ITR sequence and a second piggBac ITR sequence.

[00198] In some aspects, a piggyBac ITR sequence can comprise any piggyBac ITR sequence known in the art.

[00199] In some aspects of the methods of the present disclosure, a piggyBac ITR sequence, such as a first piggyBac ITR sequence and/or a second piggyBac ITR sequence in an AAV piggyBac transposon can comprise, consist essentially of, or consist of a Sleeping Beauty transposon ITR, a Helraiser transposon ITR, a Tol2 transposon ITR, a TcBuster transposon ITR or any combination thereof.

Promoter Sequences

[00200] In some aspects, a nucleic acid can comprise a promoter sequence. In some aspects, a promoter sequence can comprise any promoter sequence known in the art. In some aspects, a promoter sequence can comprise any liver-specific promoter sequence known in the art.

[00201] In some aspects, a promoter sequence can comprise a hybrid liver promoter (HLP).

In some aspects, a promoter sequence can comprise an LP1 promoter. In some aspects, a promoter sequence can comprise a leukocyte-specific expression of the pp52 (LSP1) long promoter. In some aspects, a promoter sequence can comprise a thyroxine binding globulin (TBG) promoter.

[00202] In some aspects, a promoter sequence can comprise a wTBG promoter. In some aspects, a promoter sequence can comprise a hepatic combinatorial bundle (HCB) promoter. In some aspects, a promoter sequence can comprise a 2xApoE-hAAT promoter. In some aspects, a promoter sequence can comprise a leukocyte-specific expression of the pp52 (LSP1) plus chimeric intron promoter. In some aspects, a promoter sequence can comprise a cytomegalovirus (CMV) promoter.

Transgene sequences

[00203] In some aspects, a transgene sequence can comprise a nucleic acid sequence that encodes for a methylmalonyl-CoA mutase (MUT1) polypeptide. The MUT1 polypeptide can be any MUT1 polypeptide known in the art. [00204] In some aspects, a transgene sequence can comprise a nucleic acid sequence that encodes for an ornithine transcarbamylase (OTC) polypeptide. The OTC polypeptide can be any OTC polypeptide known in the art.

[00205] In some aspects, a transgene sequence can comprise a nucleic acid sequence that encodes for a Factor VIII (FVIII) polypeptide. The FVin polypeptide can be any FVHI polypeptide known in the art.

[00206] In some aspects, a transgene sequence can comprise a nucleic acid sequence that encodes for an iCAS9 polypeptide.

[00207] In some aspects, a transgene sequence can be codon optimized according to methods known in the art.

[00208] In some aspects, an at least one transgene sequence can be operatively linked to at least one promoter sequence present in the same polynucleotide. poly A sequences

[00209] In some aspects, a nucleic acid can comprise a poly A sequence. In some aspects, a poly A sequence can comprise any poly A sequence known in the art.

Self-cleaving peptide sequence

[00210] In some aspects, a nucleic acid can comprise a self-cleaving peptide sequence. In some aspects, a self-cleaving peptide sequence can comprise any self-cleaving peptide sequence known in the art. In some aspects, a self-cleaving peptide sequence can comprise an 2A selfcleaving peptide sequence known in the art. Non-limiting examples of self-cleaving peptides include a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.

[00211] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a T2A peptide.

[00212] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a GSG-T2A peptide.

[00213] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for an E2A peptide. [00214] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a GSG-E2A peptide.

[00215] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a F2A peptide.

[00216] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a GSG-F2A peptide.

[00217] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a P2A peptide.

[00218] In some aspects, a self-cleaving peptide sequence can comprise a nucleic acid sequence that encodes for a GSG-P2A peptide.

Chimeric Antigen Receptor (CAR)

[00219] A transgene sequence can comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises an ectodomain comprising at least one antigen recognition region; a transmembrane domain, and an endodomain comprising at least one costimulatory domain. The CAR can further comprise a hinge region between the antigen recognition domain and the transmembrane domain.

[00220] The antigen recognition region can comprise at least one single chain variable fragment (scFv), Centyrin, single domain antibody, or a combination thereof. In an aspect, the at least one single domain antibody is a VHH. In an aspect, the at least one single domain antibody is a VH. scFv

[00221] In some aspects, the antigen recognition region of the CAR can comprise one or more scFv compositions to recognize and bind to a specific target protein/antigen. The antigen recognition region can comprise at least two scFvs. The antigen recognition region can comprise at least three scFvs. In an aspect, a CAR of the disclosure is a bi-specific CAR comprising at least two scFvs that specifically bind two distinct antigens.

[00222] The scFv compositions can comprise a heavy chain variable region and a light chain variable region of an antibody. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, and the VH and VL domains are connected with a short peptide linker. An scFv can retain the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

Centyrin

[00223] In some aspects, the antigen recognition region of the CAR can comprise one or more Centyrin compositions to recognize and bind to a specific target protein/antigen. Centyrins that specifically bind an antigen may be used to direct the specificity of a cell, (e.g., a cytotoxic immune cell) towards the specific antigen. A CAR comprising a Centyrin is referred to herein as a CARTyrin.

[00224] Centyrins of the disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding an antigen. Centyrins of the disclosure may comprise a protein scaffold comprising a consensus sequence of at least one fibronectin type III (FN3) domain, wherein the scaffold is capable of specifically binding an antigen. The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be Tenascin-C.

[00225] The consensus sequence can be modified at one or more positions within (a) a A-B loop at positions 13-16 of the consensus sequence; (b) a B-C loop at positions 22-28 of the consensus sequence; (c) a C-D at positions 38-43 of the consensus sequence; (d) a D-E loop at positions 51-54 of the consensus sequence; (e) a E-F loop at positions 60-64 of the consensus sequence; (f) a F-G loop at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). Centyrins of the disclosure may comprise a consensus sequence of at least 5 fibronectin type III (FN3) domains, at least 10 fibronectin type in (FN3) domains or at least 15 fibronectin type III (FN3) domains.

[00226] The term “antibody mimetic” is intended to describe an organic compound that specifically binds a target sequence and has a structure distinct from a naturally-occurring antibody. Antibody mimetics may comprise a protein, a nucleic acid, or a small molecule. The target sequence to which an antibody mimetic of the disclosure specifically binds may be an antigen. Antibody mimetics may provide superior properties over antibodies including, but not limited to, superior solubility, tissue penetration, stability towards heat and enzymes (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimetics include, but are not limited to, an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, and avimer (also known as avidity multimer), a DARPin (Designed Ankyrin Repeat Protein), a Fynomer, a Kunitz domain peptide, and a monobody.

[00227] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one affibody molecule. Affibody molecules of the disclosure comprise a protein scaffold comprising or consisting of one or more alpha helix without any disulfide bridges. Preferably, affibody molecules of the disclosure comprise or consist of three alpha helices. For example, an affibody molecule of the disclosure may comprise an immunoglobulin binding domain. An affibody molecule of the disclosure may comprise the Z domain of protein A.

[00228] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one affilin molecule. In Affilin molecules of the disclosure comprise a protein scaffold produced by modification of exposed amino acids of, for example, either gamma-B crystallin or ubiquitin. Affilin molecules functionally mimic an antibody’s affinity to antigen, but do not structurally mimic an antibody. In any protein scaffold used to make an affilin, those amino acids that are accessible to solvent or possible binding partners in a properly-folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.

[00229] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one affimer molecule. Affimer molecules of the disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide a high affinity binding site for a specific target sequence. Exemplary affimer molecules of the disclosure comprise a protein scaffold based upon a cystatin protein or tertiary structure thereof. Exemplary affimer molecules of the disclosure may share a common tertiary structure of comprising an alphahelix lying on top of an anti-parallel beta-sheet.

[00230] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one affitin molecule. Affitin molecules of the disclosure comprise an artificial protein scaffold, the structure of which may be derived, for example, from a DNA binding protein (e.g., the DNA binding protein Sac7d). Affitins of the disclosure selectively bind a target sequence, which may be the entirety or part of an antigen. Exemplary affitins of the disclosure are manufactured by randomizing one or more amino acid sequences on the binding surface of a DNA binding protein and subjecting the resultant protein to ribosome display and selection. Target sequences of affitins of the disclosure may be found, for example, in the genome or on the surface of a peptide, protein, virus, or bacteria. In some aspects, an affitin molecule may be used as a specific inhibitor of an enzyme. Affitin molecules of the disclosure may include heat-resistant proteins or derivatives thereof.

[00231] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one Alphabody molecule. Alphabody molecules of the disclosure may also be referred to as Cell-Penetrating Alphabodies (CPAB). Alphabody molecules of the disclosure comprise small proteins (typically of less than 10 kDa) that bind to a variety of target sequences (including antigens). Alphabody molecules are capable of reaching and binding to intracellular target sequences. Structurally, alphabody molecules of the disclosure comprise an artificial sequence forming single chain alpha helix (similar to naturally occurring coiled-coil structures). Alphabody molecules of the disclosure may comprise a protein scaffold comprising one or more amino acids that are modified to specifically bind target proteins. Regardless of the binding specificity of the molecule, alphabody molecules of the disclosure maintain correct folding and thermostability.

[00232] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one Anticalin molecule. Anticalin molecules of the disclosure comprise artificial proteins that bind to target sequences or sites in either proteins or small molecules. Anticalin molecules of the disclosure may comprise an artificial protein derived from a human lipocalin. Anticalin molecules of the disclosure may be used in place of, for example, monoclonal antibodies or fragments thereof. Anticalin molecules may demonstrate superior tissue penetration and thermostability than monoclonal antibodies or fragments thereof. Exemplary anticalin molecules of the disclosure may comprise about 180 amino acids, having a mass of approximately 20 kDa. Structurally, anticalin molecules of the disclosure comprise a barrel structure comprising antiparallel beta-strands pairwise connected by loops and an attached alpha helix. In some aspects, anticalin molecules of the disclosure comprise a barrel structure comprising eight antiparallel betastrands pairwise connected by loops and an attached alpha helix.

[00233] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one Avimer molecule. Avimer molecules of the disclosure comprise an artificial protein that specifically binds to a target sequence (which may also be an antigen). Avimers of the disclosure may recognize multiple binding sites within the same target or within distinct targets. When an avimer of the disclosure recognize more than one target, the avimer mimics function of a bi-specific antibody. The artificial protein avimer may comprise two or more peptide sequences of approximately 30-35 amino acids each. These peptides may be connected via one or more linker peptides. Amino acid sequences of one or more of the peptides of the avimer may be derived from an A domain of a membrane receptor. Avimers have a rigid structure that may optionally comprise disulfide bonds and/or calcium. Avimers of the disclosure may demonstrate greater heat stability compared to an antibody.

[00234] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one DARPin. DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprise genetically-engineered, recombinant, or chimeric proteins having high specificity and high affinity for a target sequence. In some aspects, DARPins of the disclosure are derived from ankyrin proteins and, optionally, comprise at least three repeat motifs (also referred to as repetitive structural units) of the ankyrin protein. Ankyrin proteins mediate high-affinity protein-protein interactions. DARPins of the disclosure comprise a large target interaction surface.

[00235] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one Fynomer. Fynomers of the disclosure comprise small binding proteins (about 7 kDa) derived from the human Fyn 8113 domain and engineered to bind to target sequences and molecules with equal affinity and equal specificity as an antibody.

[00236] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one Kunitz domain peptide. Kunitz domain peptides of the disclosure comprise a protein scaffold comprising a Kunitz domain. Kunitz domains comprise an active site for inhibiting protease activity. Structurally, Kunitz domains of the disclosure comprise a disulfide- rich alpha+beta fold. This structure is exemplified by the bovine pancreatic trypsin inhibitor. Kunitz domain peptides recognize specific protein structures and serve as competitive protease inhibitors. Kunitz domains of the disclosure may comprise Ecallantide (derived from a human lipoprotein-associated coagulation inhibitor (LACI)).

[00237] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding at least one monobody. Monobodies of the disclosure are small proteins (comprising about 94 amino acids and having a mass of about 10 kDa) comparable in size to a single chain antibody. These genetically engineered proteins specifically bind target sequences including antigens. Monobodies of the disclosure may specifically target one or more distinct proteins or target sequences. In some aspects, monobodies of the disclosure comprise a protein scaffold mimicking the structure of human fibronectin, and more preferably, mimicking the structure of the tenth extracellular type in domain of fibronectin. The tenth extracellular type in domain of fibronectin, as well as a monobody mimetic thereof, contains seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three complementarity determining regions (CDR3) of an antibody. In contrast to the structure of the variable domain of an antibody, a monobody lacks any binding site for metal ions as well as a central disulfide bond. Multispecific monobodies may be optimized by modifying the loops BC and FG. Monobodies of the disclosure may comprise an adnectin.

VHH

[00238] In some aspects, the antigen recognition region of the CAR can comprise at least one single domain antibodies (SdAb) to recognize and bind to a specific target protein/antigen. In an aspect, the single domain antibody is a VHH. A VHH is a heavy chain antibody found in camelids. A VHH that specifically binds an antigen may be used to direct the specificity of a cell, (e.g., a cytotoxic immune cell) towards the specific antigen. The antigen recognition region can comprise at least two VHHs. The antigen recognition region can comprise at least three VHHs. In an aspect, a CAR of the disclosure is a bi-specific CAR comprising at least two VHHs that specifically bind two distinct antigens. A CAR comprising a VHH is referred to herein as a VCAR.

[00239] At least one VHH protein or VCAR of the disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., , Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001). [00240] Amino acids from a VHH protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.

[00241] Optionally, VHH proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the VHH proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., Immunofilter program of Xencor, Inc. of Monrovia, Calif). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate VHH protein to bind its antigen. In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved. Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used. Screening VHH for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, for example, in vitro display. Competitive assays can be performed with the VHH or VCAR of the disclosure in order to determine what proteins, antibodies, and other antagonists compete for binding to a target protein with the VHH or VCAR of the present disclosure and/or share the epitope region. These assays as readily known to those of ordinary skill in the art evaluate competition between antagonists or ligands for a limited number of binding sites on a protein

VH

[00242] In some aspects, the antigen recognition region of the CAR can comprise at least one single domain antibodies (SdAb) to recognize and bind to a specific target protein/antigen. In an aspect, the single domain antibody is a VH. A VH is a single domain binder derived from common IgG. A VH that specifically binds an antigen may be used to direct the specificity of a cell, (e.g., a cytotoxic immune cell) towards the specific antigen. The antigen recognition region can comprise at least two VHs. The antigen recognition region can comprise at least three VHs. In an aspect, a CAR of the disclosure is a bi-specific CAR comprising at least two VHs that specifically bind two distinct antigens.

[00243] The VH can be isolated or derived from a human sequence. The VH can comprise a human CDR sequence and/or a human framework sequence and a non-human or humanized sequence (e.g., a rat Fc domain). In some aspects, the VH is a fully humanized VH. In some aspects, the VH is neither a naturally occurring antibody nor a fragment of a naturally occurring antibody. In some aspects, the VH is not a fragment of a monoclonal antibody. In some aspects, the VH is a UniDab antibody (TeneoBio). In some aspects, the VH is be modified to remove an Fc domain or a portion thereof. In some aspects, a framework sequence of the VH is modified to, for example, improve expression, decrease immunogenicity or to improve function.

[00244] The VH can be fully engineered using the UniRat (TeneoBio) system and “NGS- based Discovery” to produce the VH. Using this method, the specific VH are not naturally- occurring and are generated using fully engineered systems. The VH are not derived from naturally-occurring monoclonal antibodies (mAbs) that were either isolated directly from the host (for example, a mouse, rat or human) or directly from a single clone of cells or cell line (hybridoma). These VHs were not subsequently cloned from said cell lines. Instead, VH sequences are fully-engineered using the UniRat system as transgenes that comprise human variable regions (VH domains) with a rat Fc domain, and are thus human/rat chimeras without a light chain and are unlike the standard mAb format. The native rat genes are knocked out and the only antibodies expressed in the rat are from transgenes with VH domains linked to a Rat Fc (UniAbs). These are the exclusive Abs expressed in the UniRat. Next generation sequencing (NGS) and bioinformatics are used to identify the full antigen-specific repertoire of the heavychain antibodies generated by UniRat after immunization. Then, a unique gene assembly method is used to convert the antibody repertoire sequence information into large collections of fully- human heavy-chain antibodies that can be screened in vitro for a variety of functions. In some aspects, fully humanized VH are generated by fusing the human VH domains with human Fes in vitro (to generate a non-naturally occurring recombinant VH antibody). In some aspects, the VH are fully humanized, but they are expressed in vivo as human/rat chimera (human VH, rat Fc) without a light chain. Fully humanized VHs are expressed in vivo as human/rat chimera (human VH, rat Fc) without a light chain are about 80kDa (vs 150 kDa). [00245] A CAR of the present disclosure may bind human antigen with at least one affinity selected from a KD of less than or equal to 10 ^-9 M, less than or equal to 10 ^{- 10} M, less than or equal to 10 ^-11 M, less than or equal to 10 ^{- 12} M, less than or equal to 10 ^{- 13} M, less than or equal to 10 ^{- 14} M, and less than or equal to 10 ^{- 15} M. The KD may be determined by any means, including, but not limited to, surface plasmon resonance.

[00246] In an aspect, the antigen recognition region of the disclosed CAR comprises at least one anti-BCMA Centyrin. A CAR comprising the anti-BCMA Centyrin is referred to as a BCMA CARTyrin herein.

[00247] In some aspects, a nanoparticle of the present disclosure can comprise a nucleic acid sequence encoding a BCMA CARTyrin.

[00248] In an aspect, the antigen recognition region of the disclosed CAR comprises at least one anti-PSMA Centyrin. A CAR comprising the anti-PSMA Centyrin is referred to as a PSMA CARTyrin herein.

[00249] In some aspects, a nanoparticle of the present disclosure can comprise a nucleic acid sequence encoding a PSMA CARTyrin.

[00250] In an aspect, the antigen recognition region of the disclosed CAR comprises at least one anti-BCMA VH. A CAR comprising the anti-BCMA VH is referred to as a BCMA VCAR herein.

[00251] In some aspects, a nanoparticle of the present disclosure can comprise a nucleic acid sequence encoding a BCMA VCAR.

[00252] The ectodomain can comprise a signal peptide. The signal peptide can comprise a sequence encoding a human CD2, CD35, CD3ε, CD3y, CD3ζ CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In a preferred aspect, the signal peptide comprises, consists essentially of, or consists of: a human CDS alpha ( CD8α) signal peptide (SP) or a portion thereof.

[00253] The hinge domain or hinge region can comprise a human CD8α, IgG4, CD4 sequence, or a combination thereof In a preferred aspect, the hinge can comprise, consist essentially of, or consist of a human CD8 alpha (CD8a) hinge or a portion thereof.

[00254] The transmembrane domain can comprise, consist essentially of, or consist of a sequence encoding a human CD2, CD35, CD3ε, CD3y, CD3ζ CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. Preferably, the transmembrane domain can comprise, consist essentially of, or consist of a human CD8 alpha (CD8a) transmembrane domain, or a portion thereof.

[00255] The at least one costimulatory domain can comprise, consist essentially of, or consist of a human 4-1BB, CD28, CD3 zeta (CD3ζ, CD40, ICOS, MyD88, OX-40 intracellular domain, or any combination thereof. Preferably, the at least one costimulatory domain comprises a CD3ζ, a 4-1BB costimulatory domain, or a combination thereof.

Transposition systems

[00256] In some aspects, a nucleic acid can comprise a transposon or a nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon.

[00257] In some aspects, a nucleic acid can comprise a transposon or a nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon, and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR, wherein the length of the inter-ITR sequence is equal to or less than 700 nucleotides.

[00258] The transposon or nanotransposon of the disclosure comprises a protein scaffold (e.g., a CAR comprising at least one scFv, single domain antibody or Centyrin). The transposon or nanotransposon can be a plasmid DNA transposon comprising a sequence encoding a protein scaffold (e.g., a CAR comprising at least one scFv, single domain antibody or Centyrin) flanked by two cis-regulatory insulator elements. The transposon or nanotransposon can further comprises a plasmid comprising a sequence encoding a transposase. The sequence encoding the transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence. [00259] The transposon or nanotransposon of the present disclosure can be a piggyBac™ (PB) transposon. In some aspects when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase a piggyBac-like (PBL) transposase or a Super piggyBac™ (SPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[00260] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and PCT Publication No. WO 2010/099296.

[00261] The PB, PBL and SPB transposases recognize transposon-specific inverted terminal repeat sequences (ITR3) on the ends of the transposon, and inserts the contents between the ITR3 at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence) or at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence). The target sequence of the PB or PBL transposon can comprise or consist of 5’-CTAA-3’, 5’-TTAG-3’, 5’- ATAA-3’, 5’-TCAA-3’, 5’AGTT-3’, 5’-ATTA-3’, 5’-GTTA-3’, 5’-TTGA-3’, 5’-TTTA-3’, 5’- TTAC-3’, 5’-ACTA-3’, 5’-AGGG-3’, 5’-CTAG-3’, 5’-TGAA-3’, 5’-AGGT-3’, 5’-ATCA-3’, 5’- CTCC-3’, 5’-TAAA-3’, 5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5’-AATC-3’, 5’-ACAA-3’, 5’- ACAT-3’, 5’-ACTC-3’, 5’-AGTG-3’, 5’-ATAG-3’, 5’-CAAA-3’, 5’-CACA-3’, 5’-CATA-3’, 5’- CCAG-3’, 5’-CCCA-3’, 5’-CGTA-3’, 5’-GTCC-3’, 5’-TAAG-3’, 5’-TCTA-3’, 5’-TGAG-3’, 5’- TGTT-3’, 5’-TTCA-3’5’-TTCT-3’ and 5’-TTTT-3’. The PB or PBL transposon system has no payload limit for the genes of interest that can be included between the ITR3.

[00262] Exemplary amino add sequence for one or more PB, PBL and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810 and U.S. Patent No. 8,399,643. In a preferred aspect, the PB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1.

[00263] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino add substitution at two or more, at three or more or at each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 1. The transposase can be a SPB transposase that comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 1 wherein the amino acid substitution at position 30 can be a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 can be a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 can be a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 can be a substitution of a lysine (K) for an asparagine (N).

[00264] In certain aspects wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the PB, PBL and SPB transposases can further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 are described in more detail in PCT Publication No. WO 2019/173636 and PCT/US2019/049816.

[00265] The PB, PBL or SPB transposases can be isolated or derived from an insect, vertebrate, crustacean or urochordate as described in more detail in PCT Publication No. WO 2019/173636 and PCT/US2019/049816. In preferred aspects, the PB, PBL or SPB transposases is be isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[00266] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred aspect, a hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019/173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[00267] In some aspects, the PB or PBL transposase is integration deficient. An integration deficient PB or PBL transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase. Examples of integration deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019/173636. A list of integration deficient amino acid substitutions is disclosed in US patent No. 10,041,077.

[00268] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019/173636. [00269] A transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U.S. Patent No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase.

[00270] A transposon or nanotransposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon includes Helibatl. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (for example, as disclosed in WO 2019/173636).

[00271] A transposon or nanotransposon of the present disclosure can be a Tol2 transposon.

In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO 2019/173636).

[00272] A transposon or nanotransposon of the present disclosure can be a TcBuster transposon. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (for example, as disclosed in WO 2019/173636). The TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. The polynucleotide encoding a TcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[00273] In some aspects, a mutant TcBuster transposase comprises one or more sequence variations when compared to a wild type TcBuster transposase as described in more detail in PCT Publication No. WO 2019/173636 and PCT/US2019/049816.

[00274] The cell delivery compositions (e.g., transposons) disclosed herein can comprise a nucleic acid molecule encoding a therapeutic protein or therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publication No. WO 2019/173636 and PCT/US2019/049816.

Cells and Modified Cells of the Disclosure

[00275] Cells and modified cells of the disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. Cells and modified cells of the disclosure can be immune cells. The immune cells of the disclosure can comprise lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T-cell), stem memory T cells (TSCM cells), central memory T cells (TCM), stem cell-like T cells, B lymphocytes (B-cells), antigen presenting cells (APCs), cytokine induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.

[00276] The immune precursor cells can comprise any cells which can differentiate into one or more types of immune cells. The immune precursor cells can comprise multipotent stem cells that can self-renew and develop into immune cells. The immune precursor cells can comprise hematopoietic stem cells (HSCs) or descendants thereof. The immune precursor cells can comprise precursor cells that can develop into immune cells. The immune precursor cells can comprise hematopoietic progenitor cells (HPCs).

[00277] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.

[00278] HSCs can be isolated or derived from a primary or cultured stem cell. HSCs can be isolated or derived from an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

[00279] Immune precursor cells can comprise an HSC or an HSC descendent cell. Nonlimiting examples of HSC descendent cells include multipotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocyte cells (T-cells), B lymphocyte cells (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes and macrophages.

[00280] HSCs produced by the disclosed methods can retain features of “primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells. For example, the “primitive” HSCs produced by the disclosed methods retain their “sternness” following division and do not differentiate. Consequently, as an adoptive cell therapy, the “primitive” HSCs produced by the disclosed methods not only replenish their numbers, but expand in vivo. “Primitive” HSCs produced by disclosed the methods can be therapeutically-effective when administered as a single dose. [00281] Primitive HSCs can be CD34+. Primitive HSCs can be CD34+ and CD38

Primitive HSCs can be CD34+, CD38- and CD90+. Primitive HSCs can be CD34+, CD38-, CD90+ and CD45RA-. Primitive HSCs can be CD34+, CD38-, CD9O4-, CD45RA-, and CD49f+. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[00282] Primitive HSCs, HSCs, and/or HSC descendent cells can be modified according to the disclosed methods to express an exogenous sequence (e.g., a chimeric antigen receptor or therapeutic protein). Modified primitive HSCs, modified HSCs, and/or modified HSC descendent cells can be forward differentiated to produce a modified immune cell including, but not limited to, a modified T cell, a modified natural killer cell and/or a modified B-cell.

[00283] The modified immune or immune precursor cells can be NK cells. The NK cells can be cytotoxic lymphocytes that differentiate from lymphoid progenitor cells. Modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some aspects, non-activatedNK cells are derived from CD3-depleted leukapheresis (containing CD14/CD19/CD56+ cells).

[00284] The modified immune or immune precursor cells can be B cells. B cells are a type of lymphocyte that express B cell receptors on the cell surface. B cell receptors bind to specific antigens. Modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[00285] Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike traditional biologies and chemotherapeutics, the disclosed modified-T cells the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, in some aspects, modified-T cells not only drive an initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapses. Alternatively, in some aspects, when it is not desired, the modified-T cells do not persist in the patient.

[00286] Intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory T cells, especially stem cell memory (TSCM) or stem cell-like T cells. Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM) T cells or TCM like cells, effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term modified-T cell engraftment. A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > TSCM > TCM > TEM > TE > T _TE , whereby TN is the parent precursor cell that directly gives rise to TSCM, which then, in turn, directly gives rise to TCM, etc. Compositions of T cells of the disclosure can comprise one or more of each parental T cell subset with TSCM cells being the most abundant (e.g., TSCM > TCM > TEM > TE > T _TE ).

[00287] The immune cell precursor can be differentiated into or is capable of differentiating into an early memory T cell, a stem cell like T-cell, a Naive T cells (TN), a TSCM, a TCM, a TEM, a TE, or a T _TE . The immune cell precursor can be a primitive HSC, an HSC, or a HSC descendent cell of the disclosure. The immune cell can be an early memory T cell, a stem cell like T-cell, a Naive T cells (TN), a TSCM, a TCM, a TEM, a TE, or a T _TE .

[00288] The methods of the disclosure can modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of a plurality of modified T cells in the population expresses one or more cell-surface markers) of an early memory T cell. The population of modified early memory T cells comprises a plurality of modified stem cell-like T cells. The population of modified early memory T cells comprises a plurality of modified TSCM cells. The population of modified early memory T cells comprises a plurality of modified TCM cells.

[00289] The methods of the disclosure can modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface markers) of a stem cell-like T cell. The population of modified stem cell-like T cells comprises a plurality of modified TSCM cells. The population of modified stem cell-like T cells comprises a plurality of modified TCM cells.

[00290] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface markers) of a stem memory T cell (TSCM) or a TscM-like cell; and wherein the one or more cell-surface markers) comprise CD45RA and CD62L. The cell-surface markers can comprise one or more of CD62L, CD45RA, CD28, CCR7, CD 127, CD45RO, CD95, CD95 and IL-2Rβ. The cell-surface markers can comprise one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[00291] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the plurality of modified T cells in the population expresses one or more cell-surface markers) of a central memory T cell (TCM) or a TcM-like cell; and wherein the one or more cell-surface markers) comprise CD45RO and CD62L. The cell-surface markers can comprise one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[00292] The methods of the disclosure can modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface markers) of a naive T cell (TN). The cell-surface markers can comprise one or more of CD45RA, CCR7 and CD62L.

[00293] The methods of the disclosure can modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface markers) of an effector T-cell (modified TEFF). The cell-surface markers can comprise one or more of CD45RA, CD95, and IL- 2Rβ.

[00294] The methods of the disclosure can modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells of the population expresses one or more cell-surface markers) of a stem cell-like T cell, a stem memory T cell (TSCM) or a central memory T cell (TCM). [00295] A plurality of modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express one or more cell-surface markerfs) comprising CD34 or wherein at least about 70% to about 99%, about 75% to about 95% or about 85% to about 95% of the population of modified cells express one or more cell-surface markerfs) comprising CD34 (e.g., comprise the cell-surface marker phenotype CD34+).

[00296] A plurality of modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express one or more cell-surface markerfs) comprising CD34 and do not express one or more cell-surface markerfs) comprising CD38, or wherein at least about 45% to about 90%, about 50% to about 80% or about 65% to about 75% of the population of modified cells express one or more cell-surface markerfs) comprising CD34 and do not express one or more cell-surface markerfs) comprising CD38 (e.g., canprise the cell-surface marker phenotype CD34+ and CD38-).

[00297] A plurality of modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express one or more cell-surface markers) comprising CD34 and CD90 and do not express one or more cell-surface markers) comprising CD38, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified cells express one or more cell-surface markers) comprising CD34 and CD90 and do not express one or more cell-surface markers) comprising CD38 (e.g., comprise the cell-surface marker phenotype CD34+, CD38- and CD90+).

[00298] A plurality of modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express one or more cell-surface markers) comprising CD34 and CD90 and do not express one or more cell-surface markers) comprising CD38 and CD45RA, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified cells express one or more cell-surface markers) comprising CD34 and CD90 and do not express one or more cell-surface markers) comprising CD38 and CD45RA (e.g., comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA-).

[00299] A plurality of modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express one or more cell-surface markers) comprising CD34, CD90 and CD49f and do not express one or more cell-surface markers) comprising CD38 and CD45RA, or wherein at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2% or about 0.04% to about 1% of the population of modified cells express one or more cell-surface markers) comprising CD34, CD90 and CD49f and do not express one or more cell-surface markers) comprising CD38 and CD45RA (e.g., comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+).

[00300] A plurality of modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express one or more cell-surface markers) comprising CD34 and CD90 and do not express one or more cell-surface markers) comprising CD45RA, or wherein at least about 0.2% to about 5%, about 0.2% to about 3% or about 0.4% to about 3% of the population of modified cells express one or more cell-surface markers) comprising CD34 and CD90 and do not express one or more cell-surface markers) comprising CD45RA (e.g., comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA-).

[00301] Compositions and methods of producing and/or expanding the immune cells or immune precursor cells (e.g., the disclosed modified T-cells) and buffers for maintaining or enhancing a level of cell viability and/or a stem-like phenotype of the immune cells or immune precursor cells (e.g., the disclosed modified T-cells) are disclosed elsewhere herein and are disclosed in more detail in U.S. Patent No. 10,329,543 and PCT Publication No. WO 2019/173636.

[00302] Cells and modified cells of the disclosure can be somatic cells. Cells and modified cells of the disclosure can be differentiated cells. Cells and modified cells of the disclosure can be autologous cells or allogenic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment following administration to a subject. Allogeneic cells may be any type of cell. Allogenic cells can be stem cells or can be derived from stem cells. Allogeneic cells can be differentiated somatic cells.

Methods of Expressing a Chimeric Antigen Receptor

[00303] The disclosure provides methods of expressing a CAR on the surface of a cell. The method comprises (a) obtaining a cell population; (b) contacting the cell population to a composition of the present disclosure comprising a CAR or a sequence encoding the CAR, under conditions sufficient to transfer the CAR across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the CAR; and (d) expanding and/or selecting at least one cell from the modified cell population that express the CAR on the cell surface.

[00304] In some aspects, the cell population can comprise leukocytes and/or CD4+ and CD8+ leukocytes. The cell population can comprise CD4+ and CD8+ leukocytes in an optimized ratio. The optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo. The cell population can comprise a tumor cell.

[00305] In some aspects, the conditions sufficient to transfer the CAR or the sequence encoding the CAR, transposon, or vector across a cell membrane of at least one cell in the cell population comprises at least one of an application of one or more pulses of electricity at a specified voltage, a buffer, and one or more supplemental factor(s). In some aspects, the conditions suitable for integration of the sequence encoding the CAR comprise at least one of a buffer and one or more supplemental factors).

[00306] The buffer can comprise PBS, HBSS, OptiMEM, BTXpress, AmaxaNucleofector, Human T cell nucleofection buffer or any combination thereof. The one or more supplemental factors) can comprise (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and (e) a reagent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, the chemokine, the interleukin or any combination thereof can comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, ILS, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha/IL-lFl, IL-1 beta/IL-lF2, IL-12 p70, IL-12/IL-35 p35, IL-13, IL-17/IL-17A, IL-17A/F Heterodimer, IL-17F, IL-18/IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha/TNF-beta, TGF-beta, TNF-alpha, TRANCE/TNFSF11/RANK L or any combination thereof. The salt, the mineral, the metabolite or any combination thereof can comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS/FCS, Human serum, serum-substitute, antibiotics, pH adjusters, Earle’s Salts, 2 -Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgCb, Na2HPO4, NAH2PO4, Sodium lactobionate, Mannitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell medium can comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X- VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof. The inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF 6, TRAF3, IRF-7, NF -KB, Type 1 Interferons, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol in, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-30 (GSK-3 β) (e.g. TWS119), or any combination thereof. Examples of such inhibitors can include Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof. The reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without a NLS sequence, a TREX1 enzyme or any combination thereof.

[00307] The expansion and selection steps can occur concurrently or sequentially. The expansion can occur prior to selection. The expansion can occur following selection, and, optionally, a further (i.e. second) selection can occur following expansion. Concurrent expansion and selection can be simultaneous. The expansion and/or selection steps can proceed for a period of 10 to 14 days, inclusive of the endpoints.

[00308] The expansion can comprise contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell through the CAR, thereby generating an expanded cell population. The antigen can be presented on the surface of a substrate. The substrate can have any form, including, but not limited to a surface, a well, a bead or a plurality thereof, and a matrix. The substrate can further comprise a paramagetic or magnetic component. The antigen can be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet can be used to remove or separate the magnetic beads from the modified and expanded cell population. The antigen can be presented on the surface of a cell or an artificial antigen presenting cell. Artificial antigen presenting cells can include, but are not limited to, tumor cells and stem cells.

[00309] In some aspects wherein the transposon or vector comprises a selection gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying a cell expressing the selection gene as surviving the selection and identifying a cell failing to express the selection gene as failing to survive the selection step.

[00310] The disclosure provides a composition comprising the modified, expanded and selected cell population of the methods described herein. [00311] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publication No. WO 2019/049816 and PCT/US2019/049816.

[00312] The present disclosure provides a cell or a population of cells wherein the cell comprises a composition comprising (a) an inducible transgene construct, comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct, comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein, upon integration of the construct of (a) and the construct of (b) into a genomic sequence of a cell, the exogenous receptor is expressed, and wherein the exogenous receptor, upon binding a ligand or antigen, transduces an intracellular signal that targets directly or indirectly the inducible promoter regulating expression of the inducible transgene (a) to modify gene expression.

[00313] The composition can modify gene expression by decreasing gene expression. The composition can modify gene expression by transiently modifying gene expression (e.g., for the duration of binding of the ligand to the exogenous receptor). The composition can modify gene expression acutely (e.g., the ligand reversibly binds to the exogenous receptor). The composition can modify gene expression chronically (e.g., the ligand irreversibly binds to the exogenous receptor).

[00314] In some aspects, a nucleic acid can comprise a transgene comprising a nucleic acid molecule encoding at least one exogenous receptor. The exogenous receptor can comprise an endogenous receptor with respect to the genomic sequence of the cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell-surface receptors, transmembrane receptors, ligand-gated ion channels, and G-protein coupled receptors.

[00315] The exogenous receptor can comprise a non-naturally occurring receptor. The non- naturally occurring receptor can be a synthetic, modified, recombinant, mutant or chimeric receptor. The non-naturally occurring receptor can comprise one or more sequences isolated or derived from a T-cell receptor (TCR). The non-naturally occurring receptor can comprise one or more sequences isolated or derived from a scaffold protein. In some aspects, including those wherein the non-naturally occurring receptor does not comprise a transmembrane domain, the non- naturally occurring receptor interacts with a second transmembrane, membrane-bound and/or an intracellular receptor that, following contact with the non-naturally occurring receptor, transduces an intracellular signal. The non-naturally occurring receptor can comprise a transmembrane domain. The non-naturally occurring receptor can interact with an intracellular receptor that transduces an intracellular signal. The non-naturally occurring receptor can comprise an intracellular signaling domain. The non-naturally occurring receptor can be a chimeric ligand receptor (CLR). The CLR can be a chimeric antigen receptor (CAR).

[00316] The sequence encoding the inducible promoter of comprises a sequence encoding an NFxB promoter, a sequence encoding an interferon (IFN) promoter or a sequence encoding an interleukin-2 promoter. In some aspects, the IFN promoter is an IFNy promoter. The inducible promoter can be isolated or derived from the promoter of a cytokine or a chemokine. The cytokine or chemokine can comprise IL2, IL3, IL4, IL5, IL6, IL10, IL 12, IL13, IL17A/F, IL21, IL22, IL23, transforming growth factor beta (TGFβ), colony stimulating factor 2 (GM-CSF), interferon gamma (IFNy), Tumor necrosis factor alpha (TNFa), LTa, perforin, Granzyme C (Gzmc), Granzyme B (Gzmb), C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 4 (Ccl4), C-C motif chemokine ligand 3 (Ccl3), X-C motif chemokine ligand 1 (Xcll) or LIF interleukin 6 family cytokine (Lif).

[00317] The inducible promoter can be isolated or derived from the promoter of a gene comprising a surface protein involved in cell differentiation, activation, exhaustion and function. In some aspects, the gene comprises CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL or 4-1BB.

[00318] The inducible promoter can be isolated or derived from the promoter of a gene involved in CD metabolism and differentiation. The inducible promoter can be isolated or derived from the promoter of Nr4al, Nr4a3, Tnfrsf? (4-1BB), Sema7a, Zfp3612, Gadd45b, Dusp5, Dusp6 and Neto2.

[00319] In some aspects, the inducible transgene construct comprises or drives expression of a signaling component downstream of an inhibitory checkpoint signal, a transcription factor, a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand, a metabolic sensing molecule, a protein conferring sensitivity to a cancer therapy, and an oncogene or a tumor suppressor gene. Non-limiting examples of which are disclosed in PCT Publication No. WO 2019/173636 and PCT Application No. PCT/US2019/049816.

Armored Cells [00320] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to enhance their therapeutic potential. Alternatively, or in addition, the modified cells may be further modified to render them less sensitive to immunologic and/or metabolic checkpoints. Modifications of this type “armor” the cells, which, following the modification, may be referred to here as “armored” cells (e.g., armored T-cells). Armored cells may be produced by, for example, blocking and/or diluting specific checkpoint signals delivered to the cells (e.g., checkpoint inhibition) naturally, within the tumor immunosuppressive microenvironment.

[00321] An armored cell of the disclosure can be derived from any cell, for example, a T cell, a NK cell, a hematopoietic progenitor cell, a peripheral blood (PB) derived T cell (including a T cell isolated or derived from G-CSF-mobilized peripheral blood), or an umbilical cord blood (UCB) derived T cell. An armored cell (e.g., armored T-cell) can comprise one or more of a chimeric ligand receptor (CLR comprising a protein scaffold, an antibody, an ScFv, or an antibody mimetic)/chimeric antigen receptor (CAR comprising a protein scaffold, an antibody, an ScFv, or an antibody mimetic), a CARTyrin (a CAR comprising a Centyrin), and/or a VCAR (a CAR comprising a camelid VHH or a single domain VH). An armored cell (e.g., armored T-cell) can comprise an inducible proapoptotic polypeptide as disclosed herein. An armored cell (e.g., armored T-cell) can comprise an exogenous sequence. The exogenous sequence can comprise a sequence encoding a therapeutic protein. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, cell-surface bound, or secreted proteins. Exemplary therapeutic proteins expressed by the armored cell (e.g., armored T-cell) may modify an activity of the armored cell or may modify an activity of a second cell. An armored cell (e.g., armored T- cell) can comprise a selection gene or a selection marker. An armored cell (e.g., armored T-cell) can comprise a synthetic gene expression cassette (also referred to herein as an inducible transgene construct).

[00322] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to silence or reduce expression one or more gene(s) encoding receptors) of inhibitory checkpoint signals to produce an armored cell (e.g., armored CAR T-cell). Receptors of inhibitory checkpoint signals are expressed on the cell surface or within the cytoplasm of a cell. Silencing or reducing expressing of the gene encoding the receptor of the inhibitory checkpoint signal results a loss of protein expression of the inhibitory checkpoint receptors on the surface or within the cytoplasm of an armored cell. Thus, armored cells having silenced or reduced expression of one or more genes encoding an inhibitory checkpoint receptor is resistant, non-receptive or insensitive to checkpoint signals. The resistance or decreased sensitivity of the armored cell to inhibitory checkpoint signals enhances the therapeutic potential of the armored cell in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immunosuppression) are disclosed in PCT Publication No. WO 2019/173636. Preferred examples of inhibitory checkpoint signals that may be silenced include, but are not limited to, PD-1 and TGFβRII.

[00323] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to silence or reduce expression of one or more gene(s) encoding intracellular proteins involved in checkpoint signaling to produce an armored cell (e.g., armored CAR T-cell). The activity of the modified cells may be enhanced by targeting any intracellular signaling protein involved in a checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference to one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in PCT Publication No. WO 2019/173636.

[00324] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to silence or reduce expression of one or more gene(s) encoding a transcription factor that hinders the efficacy of a therapy to produce an armored cell (e.g., armored CAR T-cell). The activity of modified cells may be enhanced or modulated by silencing or reducing expression (or repressing a function) of a transcription factor that hinders the efficacy of a therapy. Non-limiting examples of transcription factors that may be modified to silence or reduce expression or to repress a function thereof include, but are not limited to, the exemplary transcription factors are disclosed in PCT Publication No. WO 2019/173636.

[00325] The modified cells of disclosure (e.g, CAR T-cells) can be further modified to silence or reduce expression of one or more gene(s) encoding a cell death or cell apoptosis receptor to produce an armored cell (e.g., armored CAR T-cell). Interaction of a death receptor and its endogenous ligand results in the initiation of apoptosis. Disruption of an expression, an activity, or an interaction of a cell death and/or cell apoptosis receptor and/or ligand render a modified cell less receptive to death signals, consequently, making the armored cell more efficacious in a tumor environment. Non-limiting examples of cell death and/or cell apoptosis receptors and ligands are disclosed in PCT Publication No. WO 2019/173636. A preferred example of cell death receptor which may be modified is Fas (CD95).

[00326] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to silence or reduce expression of one or more gene(s) encoding a metabolic sensing protein to produce an armored cell (e.g., armored CAR T-cell). Disruption to the metabolic sensing of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose and other molecules) by a modified cell leads to extended retention of T-cell function and, consequently, more tumor cells killed per cell. Non-limiting examples of metabolic sensing genes and proteins are disclosed in PCT Publication No. WO 2019/173636. A preferred example, HIFla and VHL play a role in T-cell function while in a hypoxic environment. An armored T-cell may have silenced or reduced expression of one or more genes encoding HIFla or VHL.

[00327] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to silence or reduce expression of one or more gene(s) encoding proteins that that confer sensitivity to a cancer therapy, including a monoclonal antibody, to produce an armored cell (e.g., armored CAR T-cell). Thus, an armored cell can function and may demonstrate superior function or efficacy whilst in the presence of a cancer therapy (e.g., a chemotherapy, a monoclonal antibody therapy, or another anti-tumor treatment). Non-limiting examples of proteins involved in conferring sensitivity to a cancer therapy are disclosed in PCT Publication No. WO 2019/173636.

[00328] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to silence or reduce expression of one or more gene(s) encoding a growth advantage factor to produce an armored cell (e.g., armored CAR T-cell). Silencing or reducing expression of an oncogene can confer a growth advantage for the cell. For example, silencing or reducing expression (e.g., disrupting expression) of a TET2 gene during a CAR T-cell manufacturing process results in the generation of an armored CAR T-cell with a significant capacity for expansion and subsequent eradication of a tumor when compared to a non-armored CAR T-cell lacking this capacity for expansion. This strategy may be coupled to a safety switch (e.g., an iC9 safety switch described herein), which permits the targeted disruption of an armored CAR T-cell in the event of an adverse reaction from a subject or uncontrolled growth of the armored CAR T-cell. Non-limiting examples of growth advantage factors are disclosed in PCT Publication No. WO 2019/173636. [00329] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to express a modified/chimeric checkpoint receptor to produce an armored T-cell of the disclosure.

[00330] The modified/chimeric checkpoint receptor can comprise a null receptor, decoy receptor or dominant negative receptor. A null receptor, decoy receptor or dominant negative receptor can be modified/chimeric receptor/protein. A null receptor, decoy receptor or dominant negative receptor can be truncated for expression of the intracellular signaling domain. Alternatively, or in addition, a null receptor, decoy receptor or dominant negative receptor can be mutated within an intracellular signaling domain at one or more amino acid positions that are determinative or required for effective signaling. Truncation or mutation of null receptor, decoy receptor or dominant negative receptor can result in loss of the receptor’s capacity to convey or transduce a checkpoint signal to the cell or within the cell.

[00331] For example, a dilution or a blockage of an immunosuppressive checkpoint signal from a PD-L1 receptor expressed on the surface of a tumor cell may be achieved by expressing a modified/chimeric PD-1 null receptor on the surface of an armored cell (e.g., armored CAR T- cell), which effectively competes with the endogenous (non-modified) PD-1 receptors also expressed on the surface of the armored cell to reduce or inhibit the transduction of the immunosuppressive checkpoint signal through endogenous PD-1 receptors of the armored cell. In this non-limiting example, competition between the two different receptors for binding to PD-L1 expressed on the tumor cell reduces or diminishes a level of effective checkpoint signaling, thereby enhancing a therapeutic potential of the armored cell expressing the PD-1 null receptor.

[00332] The modified/chimeric checkpoint receptor can comprise a null receptor, decoy receptor or dominant negative receptor that is a transmembrane receptor, a membrane-associated or membrane-linked receptor/protein or an intracellular receptor/protein. Exemplary null, decoy, or dominant negative intracellular receptors/proteins include, but are not limited to, signaling components downstream of an inhibitory checkpoint signal, a transcription factor, a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand, a metabolic sensing molecule, a protein conferring sensitivity to a cancer therapy, and an oncogene or a tumor suppressor gene. Non-limiting examples of cytokines, cytokine receptors, chemokines and chemokine receptors are disclosed in PCT Publication No. WO 2019/173636. [00333] The modified/chimeric checkpoint receptor can comprise a switch receptor. Exemplary switch receptors comprise a modified/chimeric receptor/protein wherein a native or wild type intracellular signaling domain is switched or replaced with a different intracellular signaling domain that is either non-native to the protein and/or not a wild-type domain. For example, replacement of an inhibitory signaling domain with a stimulatory signaling domain would switch an immunosuppressive signal into an immunostimulatory signal. Alteratively, replacement of an inhibitory signaling domain with a different inhibitory domain can reduce or enhance the level of inhibitory signaling. Expression or overexpression, of a switch receptor can result in the dilution and/or blockage of a cognate checkpoint signal via competition with an endogenous wild-type checkpoint receptor (not a switch receptor) for binding to the cognate checkpoint receptor expressed within the immunosuppressive tumor microenvironment. Armored cells (e.g., armored CAR T-cells) can comprise a sequence encoding a switch receptor, leading to the expression of one or more switch receptors, and consequently, altering an activity of an armored cell. Armored cells (e.g., armored CAR T-cells) can express a switch receptor that targets an intracellularly expressed protein downstream of a checkpoint receptor, a transcription factor, a cytokine receptor, a death receptor, a metabolic sensing molecule, a cancer therapy, an oncogene, and/or a tumor suppressor protein or gene.

[00334] Exemplary switch receptors can comprise or can be derived from a protein including, but are not limited to, the signaling components downstream of an inhibitory checkpoint signal, a transcription factor, a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand, a metabolic sensing molecule, a protein conferring sensitivity to a cancer therapy, and an oncogene or a tumor suppressor gene.

[00335] The modified cells of disclosure (e.g., CAR T-cells) can be further modified to express a CLR/CAR that mediates conditional gene expression to produce an armored T-cell. The combination of the CLR/CAR and the condition gene expression system in the nucleus of the armored T-cell constitutes a synthetic gene expression system that is conditionally activated upon binding of cognate ligand(s) with CLR or cognate antigen(s) with CAR. This system may help to ‘armor’ or enhance therapeutic potential of modified T-cells by reducing or limiting synthetic gene expression at the site of ligand or antigen binding, at or within the tumor environment for example. [00336] The present disclosure provides a gene editing composition and/or a cell comprising the gene editing composition. The gene editing composition can comprise a nanoparticle comprising a nucleic add, wherein the nucleic acid comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or the nuclease domain thereof can comprise one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease.

[00337] The nuclease or the nuclease domain thereof can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a CloOSl nuclease or a nuclease domain thereof. The gene editing composition can comprise a fusion protein. The fusion protein can comprise a nuclease-inactivated Cas9 (dCas9) protein and a CloOSl nuclease or a CloOSl nuclease domain. The gene editing canposition can further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[00338] A transgene can comprise a nucleic sequence encoding a small, Cas9 (Cas9) operatively-linked to an effector. The disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, Cas9 (Cas9). A small Cas9 construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[00339] A transgene can comprise a nucleic sequence encoding an inactivated, small, Cas9 (dSaCas9) operatively-linked to an effector. A transgene can comprise a nucleic sequence encoding a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, inactivated Cas9 (dSaCas9). A small, inactivated Cas9 (dSaCas9) construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[00340] A transgene can comprise a nucleic sequence encoding an inactivated Cas9 (dCas9) operatively-linked to an effector. A transgene can comprise a nucleic sequence encoding a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). An inactivated Cas9 (dCas9) construct of the disclosure can comprise an effector comprising a type US endonuclease.

[00341] The dCas9 can be isolated or derived from Streptoccocus pyogenes. The dCas9 can comprise a dCas9 with substitutions at amino add positions 10 and 840, which inactivate the catalytic site. In some aspects, these substitutions are DI 0A and H840A.

[00342] A cell comprising the gene editing composition can express the gene editing composition stably or transiently. Preferably, the gene editing composition is expressed transiently. The guide RNA can comprise a sequence complementary to a target sequence within a genomic DNA sequence. The target sequence within a genomic DNA sequence can be a target sequence within a safe harbor site of a genomic DNA sequence.

[00343] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in U.S. Patent Publication Nos. 2017/0107541, 2017/0114149, 2018/0187185 and U.S. Patent No. 10,415,024.

Chimeric Stimulator Receptors and Recombinant HLA-E Polypeptides

[00344] Adoptive cell compositions that are “universally” safe for administration to any patient requires a significant reduction or elimination of alloreactivity. Towards this end, cells of the disclosure (e.g., allogenic cells) can be modified to interrupt expression or function of a T-cell Receptor (TCR) and/or a class of Major Histocompatibility Complex (MHC). The TCR mediates graft vs host (GvH) reactions whereas the MHC mediates host vs graft (HvG) reactions. In preferred aspects, any expression and/or function of the TCR is eliminated to prevent T-cell mediated GvH that could cause death to the subject. Thus, in a preferred aspect, the disclosure provides a pure TCR-negative allogeneic T-cell composition (e.g., each cell of the composition expresses at a level so low as to either be undetectable or non-existent).

[00345] Expression and/or function of MHC class I (MHC-I, specifically, HLA-A, HLA-B, and HLA-C) is reduced or eliminated to prevent HvG and, consequently, to improve engraftment of cells in a subject. Improved engraftment results in longer persistence of the cells, and, therefore, a larger therapeutic window for the subject. Specifically, expression and/or function of a structural element of MHC-I, Beta-2-Microglobulin (B2M), is reduced or eliminated. [00346] The above strategies induce further challenges. T Cell Receptor (TCR) knockout (KO) in T cells results in loss of expression of CD3-zeta (CD3z or CD3ζ, which is part of the TCR complex. The loss of CD3£ in TCR-KO T-cells dramatically reduces the ability of optimally activating and expanding these cells using standard stimulation/activation reagents, including, but not limited to, agonist anti-CD3 mAb. When the expression or function of any one component of the TCR complex is interrupted, all components of the complex are lost, including TCR-alpha (TCRa), TCR-beta (TCR0), CD3-gamma (CD3y), CD3-epsilon (CD3ε), CD3-delta (CD35), and CD3-zeta (CD3ζ. Both CD3ε and CD3£ are required for T cell activation and expansion. Agonist anti-CD3 mAbs typically recognize CD3ε and possibly another protein within the complex which, in turn, signals to CD3£. CD3ζ provides the primary stimulus for T cell activation (along with a secondary co-stimulatory signal) for optimal activation and expansion. Under normal conditions, full T-cell activation depends on the engagement of the TCR in conjunction with a second signal mediated by one or more co-stimulatory receptors (e.g., CD28, CD2, 4-1BBL) that boost the immune response. However, when the TCR is not present, T cell expansion is severely reduced when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb. In fact, T cell expansion is reduced to only 20-40% of the normal level of expansion when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb.

[00347] Thus, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[00348] In some aspects, a transgene sequence can comprise a nucleic acid sequence encoding a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical. [00349] The activation component can comprise a portion of one or more of a component of a T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activation component binds. The activation component can comprise a CD2 extracellular domain or a portion thereof to which an agonist binds.

[00350] The signal transduction domain can comprise one or more of a component of a human signal transduction domain, T-cell Receptor (TCR), a component of a TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. The signal transduction domain can comprise a CD3 protein or a portion thereof. The CD3 protein can comprise a CD3( protein or a portion thereof.

[00351] The endodomain can further comprise a cytoplasmic domain. The cytoplasmic domain can be isolated or derived from a third protein. The first protein and the third protein can be identical. The ectodomain can further comprise a signal peptide. The signal peptide can be derived from a fourth protein. The first protein and the fourth protein can be identical. The transmembrane domain can be isolated or derived from a fifth protein. The first protein and the fifth protein can be identical.

[00352] In some aspects, the activation component does not bind a naturally-occurring molecule. In some aspects, the activation component binds a naturally-occurring molecule but the CSR does not transduce a signal upon binding of the activation component to a naturally-occurring molecule. In some aspects, the activation component binds to a non-naturally occurring molecule. In some aspects, the activation component does not bind a naturally-occurring molecule but binds a non-naturally occurring molecule. The CSR can selectively transduces a signal upon binding of the activation component to a non-naturally occurring molecule.

[00353] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof.

[00354] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) wherein the ectodomain comprises a modification. The modification can comprise a mutation or a truncation of the amino acid sequence of the activation component or the first protein when compared to a wild type sequence of the activation component or the first protein. The mutation or a truncation of the amino acid sequence of the activation component can comprise a mutation or truncation of a CD2 extracellular domain or a portion thereof to which an agonist binds. The mutation or truncation of the CD2 extracellular domain can reduce or eliminate binding with naturally occurring CD58.

[00355] In a preferred aspect, the present disclosure provides non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds and wherein the CD2 extracellular domain or a portion thereof to which an agonist binds comprises a mutation or truncation; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof.

[00356] The present disclosure provides a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a transposon or a vector comprising a nucleic acid sequence encoding any CSR disclosed herein.

[00357] The present disclosure provides a cell comprising any CSR disclosed herein. The present disclosure provides a cell comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein.

[00358] A modified cell disclosed herein can be an allogeneic cell or an autologous cell. In some preferred aspects, the modified cell is an allogeneic cell. In some aspects, the modified cell is an autologous T-cell or a modified autologous CAR T-cell. In some preferred aspects, the modified cell is an allogeneic T-cell or a modified allogeneic CAR T-cell.

[00359] The present disclosure provides a composition comprising any CSR disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein or a composition comprising a plurality of modified cells disclosed herein.

[00360] The present disclosure provides a modified T lymphocyte (T-cell), comprising: (a) a modification of an endogenous sequence encoding a T-cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR; and (b) a chimeric stimulatory receptor (CSR) comprising: (i) an ectodomain comprising an activation component, wherein the activation component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.

[00361] The modified T-cell can further comprise an inducible proapoptotic polypeptide. The modified T-cell can further comprise a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

[00362] The modified T-cell can further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising a HLA-E polypeptide can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising a HLA-E polypeptide can further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E can further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding the HLA-E.

[00363] The modified T-cell can further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non- naturally occurring antigen receptor can comprise a chimeric antigen receptor (CAR).

[00364] The CSR can be transiently expressed in the modified T-cell. The CSR can be stably expressed in the modified T-cell. The polypeptide comprising the HLA-E polypeptide can be transiently expressed in the modified T-cell. The polypeptide comprising the HLA-E polypeptide can be stably expressed in the modified T-cell. The inducible proapoptotic polypeptide can be transiently expressed in the modified T-cell. The inducible proapoptotic polypeptide can be stably expressed in the modified T-cell. The non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be transiently expressed in the modified T-cell. The non- naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be stably expressed in the modified T-cell.

[00365] Gene editing compositions, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, as described in detail herein, can be used to target and decrease or eliminate expression of an endogenous T-cell receptor. In preferred aspects, the gene editing compositions target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding an endogenous T-cell receptor. Non-limiting examples of primers (including a T7 promoter, genome target sequence, and gRNA scaffold) for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-a), targeting and deleting TCR-beta (TCR-p), and targeting and deleting beta-2-microglobulin (02M) are disclosed in PCT Application No. PCT/US2019/049816.

[00366] Gene editing compositions, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, can be used to target and decrease or eliminate expression of an endogenous MHCI, MHCII, or MHC activator. In preferred aspects, the gene editing compositions target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding one or more components of an endogenous MHCI, MHCII, or MHC activator. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in PCT Application No. PCT/US2019/049816.

[00367] A detailed description of non-naturally occurring chimeric stimulatory receptors, genetic modifications of endogenous sequences encoding TCR-alpha (TCR-a), TCR-beta (TCR- P), and/or Beta-2-Microglobulin (02M), and non-naturally occurring polypeptides comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide is disclosed in PCT Application No. PCT/US2019/049816.

Formulations. Dosages and Modes of Administration

[00368] The present disclosure provides formulations, dosages and methods for administration of the compositions described herein.

[00369] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant a- the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and in the “Physician's Desk Reference”, 52nd ed., Medical Economics (Montvale, N.J.) 1998. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the composition as well known in the art or as described herein.

[00370] For example, the disclosed LNP compositions of the present invention can further comprise a diluent. In some compositions, the diluent can be phosphate buffered saline (“PBS”).

[00371] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/protein components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic add, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[00372] The compositions can also include a buffer or a pH-adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric add, ascorbic acid, gluconic add, carbonic add, tartaric acid, succinic acid, acetic acid, or phthalic add; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrate.

[00373] Many known and developed modes can be used for administering therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed herein. Nonlimiting examples of modes of administration include bolus, buccal, infusion, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or vaginal means.

[00374] A composition of the disclosure can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms, such as, but not limited to, creams and suppositories; for buccal, or sublingual administration, such as, but not limited to, in the form of tablets or capsules; or intranasally, such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally, such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al. In “Drug Permeation Enhancement;” Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994,), or applications of electric fields to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents being entirely incorporated herein by reference).

[00375] For parenteral administration, any composition disclosed herein can be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. Agents for injection can be a non-toxic, non-orally administrable diluting agent, such as aqueous solution, a sterile injectable solution or suspension in a solvent. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an ordinary solvent or suspending solvent, sterile involatile oil can be used. For these purposes, any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri-glycerides. Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446.

[00376] For pulmonary administration, preferably, a composition or pharmaceutical composition described herein is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. The composition or pharmaceutical composition can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers (e.g., jet nebulizer, ultrasonic nebulizer), dry powder generators, sprayers, and the like. All such devices can use formulations suitable for the administration for the dispensing of a composition or pharmaceutical composition described herein in an aerosol. Such aerosols can be comprised of either solutions (both aqueous and non-aqueous) or solid particles. Additionally, a spray including a composition or pharmaceutical composition described herein can be produced by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered dose inhaler (MDI), a propellant, a composition or pharmaceutical composition described herein, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations and related devices is disclosed in PCT Publication No. WO 2019/049816.

[00377] For absorption through mucosal surfaces, compositions include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application of the emulsions of the disclosure can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, e.g., suppositories, can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019/049816.

[00378] For transdermal administration, a composition or pharmaceutical composition disclosed herein is encapsulated in a delivery device, such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated). A number of suitable devices are known, including microparticles made of synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. No. 5,814,599). A more detailed description of transdermal administration, formulations and suitable devices is disclosed in PCT Publication No. WO 2019/049816. [00379] It can be desirable to deliver the disclosed compounds to the subj ect over prolonged periods of time, for example, for periods of one week to one year from a single administration. Various slow release, depot or implant dosage forms can be utilized.

[00380] Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J. Preferred doses can optionally include about 0.1-99 and/or 100-500 mg/kg/administration, or any range, value or fraction thereof, or to achieve a serum concentration of about 0.1-5000 pg/ml serum concentration per single or multiple administration, or any range, value or fraction thereof. A preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is from about 1 mg/kg, up to about 3, about 6 or about 12 mg/kg of body weight of the subject.

[00381] Alternatively, the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.

[00382] As a non-limiting example, treatment of humans or animals can be provided as a one-time or periodic dosage of the compositions or pharmaceutical compositions disclosed herein about 0.1 to 100 mg/kg or any range, value or fraction thereof per day, on at least one of day 1-40, or, alternatively or additionally, at least one of week 1-52, or, alternatively or additionally, at least one of 1-20 years, or any combination thereof, using single, infusion or repeated doses.

[00383] In aspects where the compositions to be administered to a subject in need thereof are modified cells as disclosed herein, the cells can be administered between about 1x10 ³ and 1x10 ¹⁵ cells; 1x10 ³ and 1x10 ¹⁵ cells, about 1x10 ⁴ and 1x10 ¹² cells; about 1x10 ⁵ and 1x10 ¹⁰ cells; about 1x10 ⁶ and 1x10 ⁹ cells; about 1x10 ⁶ and 1x10 ⁸ cells; about 1x10 ⁶ and 1x10 ⁷ cells; or about 1x10 ⁶ and 25x10 ⁶ cells. In an aspect the cells are administered between about 5x10 ⁶ and 25x10 ⁶ cells. [00384] A more detailed description of pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019/04981.

[00385] The disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of the composition or pharmaceutical composition. In an aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[00386] The disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal or subject. Preferably, the malignant disease is cancer. Non-limiting examples of a malignant disease or disorder include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MBS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.

[00387] In preferred aspects, the treatment of a malignant disease or disorder comprises adoptive cell therapy. For example, in an aspect, the disclosure provides modified cells that express at least one disclosed protein scaffold and/or CAR comprising a protein scaffold (e.g., scFv, single domain antibody, Centyrin, delivered to the cell with a composition of the disclosure) that have been selected and/or expanded for administration to a subject in need thereof. Modified cells can be formulated for storage at any temperature including room temperature and body temperature. Modified cells can be formulated for cryopreservation and subsequent thawing. Modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. Modified cells can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and/or CAR expression level to ensure a minimal level of cell function and CAR expression. Modified cells can be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further expansion and/or prevent cell death.

[00388] Any can comprise administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal or subject in need of such modulation, treatment or therapy. Such a method can optionally further comprise coadministration or combination therapy for treating such diseases or disorders, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and/or after, at least one chemotherapeutic agent (e.g., an alkylating agent, an a mitotic inhibitor, a radiopharmaceutical).

[00389] In some aspects, the subject does not develop graft vs. host (GvH) and/or host vs. graft (HvG) following administration. In an aspect, the administration is systemic. Systemic administration can be any means known in the art and described in detail herein. Preferably, systemic administration is by an intravenous injection or an intravenous infusion. In an aspect, the administration is local. Local administration can be any means known in the art and described in detail herein. Preferably, local administration is by intra-tumoral injection or infusion, intraspinal injection or infusion, intracerebroventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.

[00390] In some aspects, the therapeutically effective dose is a single dose. In some aspects, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses in between that are manufactured simultaneously. In some aspects, where the composition is autologous cells or allogeneic cells, the dose is an amount sufficient for the cells to engraft and/or persist for a sufficient time to treat the disease or disorder.

[00391] In one example, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising a protein scaffold or a CAR comprising a protein scaffold (e.g., e.g., scFv, single domain antibody, Centyrin) the antibody or CAR specifically binds to an antigen on a tumor cell. In aspects where the composition comprises a modified cell or cell population, the cell or cell population may be autologous or allogeneic.

[00392] In some aspects of the methods of treatment described herein, the treatment can be modified or terminated. Specifically, in aspects where the composition used for treatment comprises an inducible proapoptotic polypeptide, apoptosis may be selectively induced in the cell by contacting the cell with an induction agent. A treatment may be modified or terminated in response to, for example, a sign of recovery or a sign of decreasing disease severity/progression, a sign of disease remission/cessation, and/or the occurrence of an adverse event. In some aspects, the method comprises the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and/or efficacy of the cell therapy (for example, when a sign or symptom of the disease reappear or increase in severity and/or an adverse event is resolved).

Protein Scaffold Production. Screening and Purification

[00393] At least one protein scaffold (e.g., monoclonal antibody, a chimeric antibody, a single domain antibody, a VHH, a VH, a single chain variable fragment (scFv), a Centyrin, an antigen-binding fragment (Fab) or a Fab fragment) of the disclosure can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

[00394] Amino acids from a protein scaffold can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.

[00395] Optionally, a protein scaffold can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the scaffold proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., Immunofilter program of Xencor, Inc. of Monrovia, Calif). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate protein scaffold to bind its antigen. In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved. Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used.

[00396] Screening of a protein scaffold for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, for example, in vitro display. This method involves the screening of large collections of peptides for individual members having the desired function or structure. The displayed nucleotide or peptide sequences can be from 3 to 5000 or more nucleotides or amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT Patent Publication Nos. WO 91/17271, WO 91/18980, WO 91/19818, and WO 93/08278.

[00397] Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent Publication Nos. WO 92/05258, WO 92/14843, and WO 96/19256. See also, U.S. Pat. Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, Calif), and Cambridge Antibody Technologies (Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, 5856456, assigned to Enzon; 5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax, 5,427,908, 5,580,717, assigned to Affymax; 5,885,793, assigned to Cambridge Antibody Technologies; 5,750,373, assigned to Genentech, 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra.

[00398] A protein scaffold of the disclosure can bind human or other mammalian proteins with a wide range of affinities (KD). In a preferred aspect, at least one protein scaffold of the present disclosure can optionally bind to a target protein with high affinity, for example, with a KD equal to or less than about IO ^-7 M, such as but not limited to, 0.1-9.9 (or any range or value therein) X IO" ⁸ , IO" ⁹ , 10 ^-10 , 10 ^-11 , IO" ¹² , 10 ^{- 13} , 10 ^-14 , 10 ^{- 15} or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.

[00399] The affinity or avidity of a protein scaffold for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular protein scaffold-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) are preferably made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

[00400] Competitive assays can be performed with a protein scaffold in order to determine what proteins, antibodies, and other antagonists compete for binding to a target protein with the protein scaffold and/or share the epitope region. These assays as readily known to those of ordinary skill in the art evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and/or antibody is immobilized or insolubilized before or after the competition and the sample bound to the target protein is separated from the unbound sample, for example, by decanting (where the protein/antibody was pre-insolubilized) or by centrifuging (where the protein/antibody was precipitated after the competitive reaction). Also, the competitive binding may be determined by whether function is altered by the binding or lack of binding of the protein scaffold to the target protein, e.g., whether the protein scaffold inhibits or potentiates the enzymatic activity of, for example, a label. ELISA and other functional assays may be used, as well known in the art.

Nucleic Acid Molecules

[00401] Nucleic acid molecules of the disclosure encoding a protein scaffold can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.

[00402] Isolated nucleic acid molecules of the disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally, with one or more introns, e.g, but not limited to, at least one specified portion of at least one protein scaffold; nucleic acid molecules comprising the coding sequence for a protein scaffold or loop region that binds to the target protein; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the protein scaffold as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for a specific protein scaffold of the present disclosure. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present disclosure.

[00403] As indicated herein, nucleic acid molecules of the disclosure which comprise a nucleic acid molecule encoding a protein scaffold can include, but are not limited to, those encoding the amino acid sequence of a protein scaffold fragment, by itself; the coding sequence for the entire protein scaffold or a portion thereof; the coding sequence for a protein scaffold, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a protein scaffold can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold comprising a protein scaffold fragment or portion.

Polynucleotides Selectively Hybridizing to a Polynucleotide as Described Herein

[00404] The disclosure provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein. Thus, the polynucleotides can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides. For example, polynucleotides of the present disclosure can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. The polynucleotides can be genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.

[00405] Preferably, the cDNA library comprises at least 80% full-length sequences, preferably, at least 85% or 90% full-length sequences, and, more preferably, at least 95% full- length sequences. The cDNA libraries can be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.

[00406] Optionally, polynucleotides will encode at least a portion of a protein scaffold encoded by the polynucleotides described herein. The polynucleotides embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding a protein scaffold of the present disclosure. See, e.g., Ausubel, supra, Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids [00407] The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as well-known in the art.

[00408] The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present disclosure. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the disclosure.

[00409] Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).

Recombinant Methods for Constructing Nucleic Acids

[00410] The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some aspects, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).

Nucleic Acid Screening and Isolation Methods

[00411] A cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the disclosure. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent, such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.

[00412] Methods of amplification of RNA or DNA are well known in the art and can be used according to the disclosure without undue experimentation, based on the teaching and guidance presented herein.

[00413] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 to Innis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with the tradename NASB A), the entire contents of which references are incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra?)

[00414] For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the disclosure and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

[00415] The isolated nucleic adds of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supray Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

[00416] The disclosure further provides recombinant expression cassettes comprising a nucleic acid of the disclosure. A nucleic acid sequence of the disclosure, for example, a cDNA or a genomic sequence encoding a protein scaffold of the disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the disclosure operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the disclosure.

[00417] In some aspects, isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulate expression of a polynucleotide of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.

Expression Vectors and Host Cells [00418] The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.

[00419] The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[00420] The DNA insert should be operatively linked to an appropriate promoter. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.

[00421] Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenidllin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the abovedescribed host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[00422] Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure. Such cell surface markers include, e.g., but are not limited to, “cluster of designation” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8): 1277-87).

[00423] Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[00424] At least one protein scaffold of the disclosure can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino adds, particularly charged amino acids, can be added to the N-terminus of a protein scaffold to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a protein scaffold of the disclosure to facilitate purification. Such regions can be removed prior to final preparation of a protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

[00425] Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid molecule encoding a protein of the disclosure. Alternatively, nucleic adds of the disclosure can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a protein scaffold of the disclosure. Such methods are well known in the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.

[00426] Illustrative of cell cultures useful for the production of the protein scaffolds, specified portions or variants thereof, are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL- 10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851). In a preferred aspect, the recombinant cell is aP3X63Ab8.653 or an SP2/0-Agl4 cell.

[00427] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells usefill for production of nucleic acids or proteins of the present disclosure are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[00428] When eukaryotic host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.

Protein Scaffold Purification

[00429] A protein scaffold can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N. Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.

[00430] A protein scaffold of the disclosure include purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, E. coli, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the protein scaffold of the disclosure can be glycosylated or can be non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Cdligan, Protein Science, supra, Chapters 12-14, all entirely incorporated herein by reference.

Amino Acid Codes

[00431] The amino acids that make up protein scaffolds of the disclosure are often abbreviated. The amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). A protein scaffold of the disclosure can include one or more amino acid substitutions, deletions or additions, from spontaneous or mutations and/or human manipulation, as specified herein. Amino acids in a protein scaffold of the disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites that are critical for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[00432] As those of skill will appreciate, the disclosure includes at least one biologically active protein scaffold of the disclosure. Biologically active protein scaffolds have a specific activity at least 20%, 30%, or 40%, and, preferably, at least 50%, 60%, or 70%, and, most preferably, at least 80%, 90%, or 95%-99% or more of the specific activity of the native (nonsynthetic), endogenous or related and known protein scaffold. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.

[00433] In another aspect, the disclosure relates to protein scaffolds and fragments, as described herein, which are modified by the covalent attachment of an organic moiety. Such modification can produce a protein scaffold fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group. In particular aspect, the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.

[00434] The modified protein scaffolds and fragments of the disclosure can comprise one or more organic moiedes that are covalently bonded, directly or indirectly, to the antibody. Each organic moiety that is bonded to a protein scaffold or fragment of the disclosure can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term “fatty acid” encompasses mono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymeric group,” as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, a protein scaffold modified by the covalent attachment of polylysine is encompassed by the disclosure. Hydrophilic polymers suitable for modifying protein scaffolds of the disclosure can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxypolyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies the protein scaffold of the disclosure has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG5000 and PEG20,000, wherein the subscript is the average molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

[00435] Fatty acids and fatty add esters suitable for modifying protein scaffolds of the disclosure can be saturated or can contain one or more units of unsaturation. Fatty acids that are suitable for modifying protein scaffolds of the disclosure include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-A9-octadecanoate (C18, oleate), all cis-A5,8,ll,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably, one to about six, carbon atoms.

[00436] The modified protein scaffolds and fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A “modifying agenf ’ as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An “activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a bivalent phosphorous group to form phosphoramidate or phosphorimide linkages. Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example, a divalent C1-C12 group wherein one or more carbon atoms can be replaced by a heteroatom, such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, — (CH2)3 — , — NH— (CH2)6— NH, — (CH2)2— NH— and — CH2— 0— CH2— CH2— 0— CH2— CH2— 0— CH— NH— . Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc- alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate, as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid. (See, for example, Thompson, et al., WO 92/16221, the entire teachings of which are incorporated herein by reference.)

[00437] The modified protein scaffolds of the disclosure can be produced by reacting a protein scaffold or fragment with a modifying agent. For example, the organic moieties can be bonded to the protein scaffold in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG. Modified protein scaffolds and fragments comprising an organic moiety that is bonded to specific sites of a protein scaffold of the disclosure can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

Definitions [00438] In the chemical formulas shown herein, the marking indicates the position where a functional group bonds to another portion of a molecule, Definitions of specific functional groups and chemical terms are described in more detail below.

[00439] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[00440] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

[00441] If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

[00442] The "enantiomeric excess" of a substance is a measure of how pure a desired enantiomer is relative to the undesired enantiomer. Enantiomeric excess is defined as the absolute difference between the mole fraction of each enantiomer which is most often expressed as a percent enantiomeric excess. For mixtures of diastereomers, there are analogous definitions and uses for "diastereomeric excess" and percent diastereomeric excess.

[00443] For example, a sample with 70% of R isomer and 30% of S will have an enantiomeric excess of 40%. This can also be thought of as a mixture of 40% pure R with 60% of a racemic mixture (which contributes 30% R and 30% S to the overall composition).

[00444] One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group," as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In certain embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.

[00445] It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. 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 every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment of diseases or disorders. The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.

[00446] The term "aliphatic," as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl," "alkynyl," and the like. Furthermore, as used herein, the terms "alkyl," "alkenyl," "alkynyl," and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.

[00447] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CIh-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tertbutyl, cyclobutyl, - CH ₂ -cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, - CH ₂ -cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CHi-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. [00448] The term "alkyl" as used herein refers to saturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n- heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[00449] The term "alkenyl" denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.

[00450] The term "alkynyl" as used herein refers to a monovalent group derived form a hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[00451] The term "alkoxy," or "thioalkyl" as used herein refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-4 aliphatic carbon atoms. Examples of alkoxy, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[00452] The term "alkylamino" refers to a group having the structure -NHR', wherein R' is aliphatic, as defined herein. In certain embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-15 aliphatic carbon atoms. In certain other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic group employed in the invention contain 1- 8 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n- propylamino, iso-propylamino, cyclopropylamino, n-butylamino, tert-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like.

[00453] The term "carboxylic acid" as used herein refers to a group of formula -CO2H.

[00454] The term "dialkylamino" refers to a group having the structure --NRR', wherein R and R' are each an aliphatic group, as defined herein. R and R' may be the same or different in a dialkyamino moiety. In certain embodiments, the aliphatic groups contain 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-15 aliphatic carbon atoms. In certain other embodiments, the aliphatic groups contain 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic groups contain 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups contain 1-4 aliphatic carbon atoms. Examples of dialkylamino groups include, but are not limited to, dimethylamino, methyl ethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino, di(n- pentyl)amino, di(hexyl)amino, di(cyclohexyl)amino, and the like. In certain embodiments, R and R' are linked to form a cyclic structure. The resulting cyclic structure may be aromatic or nonaromatic. Examples of cyclic diaminoalkyl groups include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

[00455] Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

[00456] In general, the terms "aryl" and "heteroaryl," as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl," as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, 0, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

[00457] It will be appreciated that aryl and heteroaryl groups can be unsubstituted or substituted, wherein substitution includes replacement of one, two, three, or more of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

[00458] The term "cycloalkyl," as used herein, refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic, or heterocyclic moieties, may optionally be substituted with substituents including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

[00459] The term "heteroaliphatic," as used herein, refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

[00460] The term "haloalkyl" denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

[00461] The term "heterocycloalkyl" or "heterocycle," as used herein, refers to a nonaromatic 5-, 6-, or 7-membered ring or a polycyclic group, including, but not limited to a bi- or tricyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quatemized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a "substituted heterocycloalkyl or heterocycle" group is utilized and as used herein, refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein. [00462] The term "carbocycle," as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is a carbon atom.

[00463] The term "independently selected" is used herein to indicate that the R groups can be identical or different.

[00464] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[00465] The term "heterocyclic," as used herein, refers to a non-aromatic partially unsaturated or fully saturated 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size and bi- and tri-cyclic ring systems which may include aromatic six-membered aryl or aromatic heterocyclic groups fused to a non-aromatic ring. These heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.

[00466] The term "heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

[00467] The term "substituted," whether preceded by the term "optionally" or not, and "substituent," as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained. 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 every position. The substituents may also be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted with fluorine at one or more positions). [00468] "Effective amount": In general, the "effective amount" of an active agent or composition refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, etc. For example, the effective amount of microparticles containing an antigen to be delivered to immunize an individual is the amount that results in an immune response sufficient to prevent infection with an organism having the administered antigen.

[00469] As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[00470] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more standard deviations. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about ’ meaning within an acceptable error range for the particular value should be assumed.

[00471] The disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various aspects, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[00472] The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term "fragment" refers to a portion of the DNA sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the disclosure.

[00473] Nucleic acids or proteins of the disclosure can be constructed by a modular approach including preassembling monomer units and/or repeat units in target vectors that can subsequently be assembled into a final destination vector. Polypeptides of the disclosure may comprise repeat monomers of the disclosure and can be constructed by a modular approach by preassembling repeat units in target vectors that can subsequently be assembled into a final destination vector. The disclosure provides polypeptide produced by this method as well nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced this modular approach.

[00474] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope hereof to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the antibodies hereof as defined herein. Thus, according to an aspect hereof, the term “antibody hereof* in its broadest sense also covers such analogs. Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the antibodies hereof as defined herein.

[00475] "Antibody fragment", and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'- SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDR3 of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDR3 of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g., CHI in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s). The term further includes single domain antibodies (“sdAB”) which generally refers to an antibody fragment having a single monomeric variable antibody domain, (for example, from camelids). Such antibody fragment types will be readily understood by a person having ordinary skill in the art.

[00476] “Binding” refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. [00477] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Aspects defined by each of these transition terms are within the scope of this disclosure.

[00478] The term “epitope” refers to an antigenic determinant of a polypeptide. An epitope could comprise three amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, consists of at least 8, 9, or 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

[00479] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[00480] “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

[00481] “Modulation” or “regulation” of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. [00482] The term “operatively linked” or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.

[00483] Non-covalently linked components and methods of making and using non- covalently linked components, are disclosed. The various components may take a variety of different forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate enables a functional association only or primarily under circumstances where such association is needed for the desired activity. The linkage may be of duration sufficient to allow the desired effect.

[00484] A method for directing proteins to a specific locus in a genome of an organism is disclosed. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.

[00485] The term "scFv" refers to a single-chain variable fragment. scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide. The linker peptide may be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino adds in length. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., Protein G) used to purify antibodies. The term further includes a scFv that is an intrabody, an antibody that is stable in the cytoplasm of the cell, and which may bind to an intracellular protein.

[00486] The term “single domain antibody” means an antibody fragment having a single monomeric variable antibody domain which is able to bind selectively to a specific antigen. A single-domain antibody generally is a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG, which generally have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high concentrations of urea. Examples are those derived from camelid or fish antibodies. Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains.

[00487] The terms “specifically bind” and “specific binding” as used herein refer to the ability of an antibody, an antibody fragment or a nanobody to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens. In some aspects, a specific binding interaction will discriminate between desirable and undesirable antigens in a sample. In some aspects, more than about ten- to 100-fold or more (e.g., more than about 1000- or 10,000- fold). “Specificity” refers to the ability of an immunoglobulin or an immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigenic target versus a different antigenic target and does not necessarily imply high affinity.

[00488] A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[00489] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide" refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of a depicted single strand. A nucleic acid of the disclosure also encompasses substantially identical nucleic acids and complements thereof that retain the same structure or encode for the same protein.

[00490] Probes of the disclosure may comprise a single stranded nucleic acid that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids of the disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[00491] Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides. Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic adds of the disclosure may be obtained by chemical synthesis methods or by recombinant methods.

[00492] Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring. Nucleic adds of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire nucleic add sequence non-naturally occurring. Nucleic adds of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally- occur, rendering the entire nucldc acid sequence non-naturally occurring.

[00493] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.

[00494] As used throughout the disclosure, the term "operably linked" refers to the expression of a gene that is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.

[00495] As used throughout the disclosure, the term "promoter" refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

[00496] As used throughout the disclosure, the term “substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[00497] As used throughout the disclosure, the term "substantially identical" refers to a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

[00498] As used throughout the disclosure, the term "variant" when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[00499] As used throughout the disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[00500] As used throughout the disclosure, the term "variant" when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.

[00501] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference.

[00502] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[00503] As used herein, “conservative” amino add substitutions may be defined as set out in Tables A, B, or C below. In some aspects, fusion polypeptides and/or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1.

Table 1 - Conservative Substitutions I

[00504] Alternately, conservative amino acids can be grouped as described in Lehninger,

(Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in

Table 2.

Table 2 - Conservative Substitutions II

[00505] Alternately, exemplary conservative substitutions are set out in Table 3.

Table 3 - Conservative Substitutions in

[00506] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.

[00507] As used throughout the disclosure, the term “more than one” of the aforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the recited amino acid substitutions. The term “more than one” may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

[00508] Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally-occur, rendering the entire amino acid sequence non- naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally-occur, rendering the entire amino acid sequence non- naturally occurring.

[00509] As used throughout the disclosure, “sequence identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms "identical" or "identity" when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[00510] As used throughout the disclosure, the term "endogenous" refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

[00511] As used throughout the disclosure, the term "exogenous" refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non-naturally occurring genome location.

[00512] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "introducing" is intended presenting to the cell the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell. The methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. Examples

Synthesis of C12 episulfide

[00513] C12 epoxide (1.0 g, 5 mmol) was reacted with KSCN-Silica (5.0 g) in toluene (20 mL) at 90°C for 24 hours in a round bottomed flask equipped with a reflux condenser. The reaction mixture was filtered through celite to give C12 episulfide (1.1 g, quantitative yield).

[00514]

[00515] To a solution of 2-mercaptoethanol (1.0 g, 0.9 mL, 12.8 mmol, 1.0 equiv) in dichloromethane (50 mL), was added 2,2’ -dipyridyl disulfide (4.23 g, 19.2 mmol, 1.5 equiv), and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure to give the crude, which was subsequently purified on a Combi-flash column (elute: hexane/Ethyl Acetate = 3/1) to afford 2-(pyridin-2-yldisulfaneyl)ethan-l-ol as pale-yellow oil (1.56 g, 65% yield).

[00516] Example 1: Synthesis of Compound 3

Compound 3

[00517] Step 1:

[00518] To a solution of amine Al (1.16 g, 8 mmol) and episulfide (9.4 g, 47 mmol) in MeCN (50 mL) was added K2CO3 (14.15 g, 102 mmol), tetra-n-butylammonium iodide, TBAI, (190 mg, 0.5 mmol) and methyl methanethiosulfonate, MMTS, (12.9 g, 102 mmol). The mixture was heated at 80°C for 2 days before quenching with ice cold water (100 mL). The mixture was extracted with DCM (150 mL x 3) and washed with brine (100 mL x 2). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was purified by column chromatography (DCM:EA = 10:1, v/v) to afford Compound 1 as yellow oil (998 mg) in a yield of 11%.

[00519] ¹ HNMR (500 MHz, CDCh) 5 3.66 (t, J = 8.2 Hz, 1H), 3.23 (dd, J = 17.4, 9.6 Hz, 3H), 2.82-2.45 (m, 16H), 2.41 (s, 6H), 2.40 (s, 3H), 2.04 (s, 3H), 1.71 (s, 2H), 1.68-1.46 (m, 15H), 1.28 (d, J = 14.6 Hz, 62H), 0.88 (t, J = 6.8 Hz, 12H). HRMS (ESI): calcd. for C59H123N3S8 [M+H] ⁺ 1130.7, found 1130.5. [00520] Step 2:

[00521] To a solution of Compound 1 (998 mg, 0.88 mmol) in THE (10 mL), H2O (1 mL) and TFA (1 mL) was added PBu3 (6 eq, 5.28 mmol, 1.2 mL) and the mixture was stirred at room temperature under N2 for 2 h. The sample was directly purified on the reverse phase Biotage Selekt with C18 column (70% mobile phase B to 100% mobile phase B; mobile phase A: 0.1 % TEA in H2O and mobile phase B: 0.1 % TEA in MeOH) to afford protonated Compound 2 (480 mg) in a yield of 45%.

[00522] MS (ESI): calcd. for C55H115N3S4 [M+H] ⁺ 946.8, found 946.4.

[00523] Step 3:

[00524] To a solution of Compound 2 (480 mg, 0.396 mmol) in dry DCM (5 mL) was added 2-(2-(Pyridin-2-yl)disulfanyl)ethanol (355 mg, 1.9 mmol) and the mixture was stirred under N2 at room temperature for 1 h. The pH of the crude product was adjusted by adding 20 uL tri ethylamine (TEA) and the sample was directly purified by column chromatography (DCM:MeOH = 20: 1, v/v) to afford desired product Compound 3 as a colorless oil (233 mg) in a yield of 48%.

[00525] ¹ H NMR (500 MHz, CDCh) 5 3.87 (td, J = 5.9, 3.6 Hz, TH), 2.84 (q, J = 5.5 Hz, 8H), 2.77 (s, 4H), 2.68 (ddd, J = 13.2, 6.7, 2.2 Hz, 5H), 2.57-2.44 (m, 9H), 1.86-1.43 (m, 25H), 1.27 (d, J = 6.1 Hz, 61H), 0.88 (t, J = 6.9 Hz, 12H). MS (ESI): calcd. for C63H131N3S4O8 [M+H] ⁺ 1250.8, found 1250.6.

Example 2: Synthesis of Compound 6 Compound 6

[00526] Step 1:

[00527] To a solution of amine A0 (1.9 g, 16 mmol) and episulfide (19 g, 96 mmol) in MeCN (80 mL) was added K2CO3 (29 g, 204 mmol), tetra-n-butylammonium iodide, TBAI, (370 mg, 1 mmol) and methyl methanethiosulfonate, MMTS, (26 g, 205 mmol). The mixture was heated at 80°C for 3 days before quenching with ice cold water (150 mL). The mixture was extracted with DCM (250 mL x 3) and washed with brine (200 mL x 2). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was purified by column chromatography (DCM:EA = 10: 1, v/v) to afford Compound 4 as yellow oil (2.5 g) in a yield of 15%.

[00528] ¹ H NMR (500 MHz, CDCh) 52.77-2.70 (m, 6H), 2.62-2.53 (m, 4H), 2.39 (d, J = 2.6 Hz, 8H), 1.81 (ddd, J = 13.8, 6.7, 4.3 Hz, 3H), 1.65 (dd, J = 10.1, 5.2 Hz, 1H), 1.58-1.42 (m, 8H), 1.27 (d, J = 9.7 Hz, 61H), 0.88 (t, J = 6.8 Hz, 12H). MS (ESI): calcd. for C57H119N3S8 [M+H] ⁺ 1102.7, found 1102.5.

[00529] Step 2:

[00530] To a solution of Compound 4 (1.82 g, 1.65 mmol) in THF (15 mL), H2O (1.5 mL) and TFA (1.5 mL) was added PB113 (6 eq, 9.92 mmol, 2.3 mL) and the mixture was stirred at room temperature under N2 for 2 h. The sample was directly purified on the reverse phase Biotage Selekt with C18 column (70% mobile phase B to 100% mobile phase B; mobile phase A: 0.1 % TFA in H2O and mobile phase B: 0.1 % TFA in MeOH to afford protonated Compound 5 (1.06 g) in a yield of 52%.

[00531] MS (ESI): calcd. for C53H111N3S4 [M+H] ⁺ 918.8, found 918.5.

[00532] Step 3:

[00533] To a solution of Compound 5 (1060 mg, 0.84 mmol) in dry DCM (5 mL) was added 2-(2-(Pyridin-2-yl)disulfanyl)ethanol (750 mg, 4 mmol) and the mixture was stirred under N2 at room temperature for 1 h. The pH of the crude product was adjusted by adding 20 uL TEA and the sample was directly purified by column chromatography (DCM:MeOH = 20:1, v/v) to afford desired product Compound 6 as colorless oil (375 mg) in a yield of 37%.

[00534] ¹ H NMR (500 MHz, CDCh) 5 3.87 (dt, J = 9.8, 5.3 Hz, 8H), 3.16-2.92 (m, 8H), 2.86 (dt, J= 15.5, 7.6 Hz, 14H), 2.73 (ddd, J = 20.6, 15.7, 10.2 Hz, 4H), 2.55 (dd, J= 13.8, 6.1 Hz, 4H), 1.80-1.62 (m, 8H), 1.48 (s, 10H), 1.42-1.34 (m, 4H), 1.27 (d, J = 5.8 Hz, 58H), 0.88 (t, J = 6.9 Hz, 12H). MS (ESI): calcd. for C61H127N3S4O8 [M+H] ⁺ 1222.8, found 1222.5.

Example 3: Synthesis of Compound 9

Compound 9

[00535] Step 1:

[00536] To a solution of amine A96 (950 mg, 10.5 mmol) and episulfide (10.3 g, 45 mmol) in MeCN (25 mL) was added K2CO3 (8.4 g, 60 mmol), tetra-n-butylammonium iodide, TBAI, (185 mg, 0.5 mmol) and methyl methanethiosulfonate, MMTS, (7.6 g, 60 mmol). The mixture was heated at 80°C for 2 days before quenching with ice cold water (100 mL). The mixture was extracted with DCM (200 mL x 3) and washed with brine (100 mL x 2). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was purified by column chromatography (DCM:EA = 10:1, v/v) to afford Compound 7 as yellow oil (1.6 g) in a yield of 18%.

[00537] ¹ HNMR (500 MHz, CDCh) δ 2.92 (s, 2H), 2.89-2.81 (m, 1H), 2.78-2.61 (m, 3H), 2.42-2.37 (m, 6H), 2.31-2.23 (m, 2H), 2.19-2.09 (m, 1H), 1.93-1.72 (m, 2H), 1.64-1.43 (m, 14H), 1.26 (s, 62H), 0.88 (t, J = 6.9 Hz, 10H). MS (ESI): calcd. for C49H102N2S6 [M+H] ⁺ 911.6, found 911.5. [00538] Step 2:

[00539] To a solution of Compound 7 (1.5 g, 1.65 mmol) in THF (15 mL), H2O (1.5 mL) and TFA (1.5 mL) was added PBu3 (4.5 eq, 7.5 mmol, 1.7 mL) and the mixture was stirred at room temperature under N2 for 2 h. The sample was directly purified on the reverse phase Biotage Selekt with C18 column (70% mobile phase B to 100% mobile phase B; mobile phase A: 0.1 % TFA in H2O and mobile phase B: 0.1 % TFA in MeOH) to afford protonated Compound 8 (825 mg) in a yield of 50%.

[00540]

[00541] Step 3:

[00542] To a solution of Compound 8 (411 mg, 0.44 mmol) in dry DCM (5 mL) was added 2-(2-(Pyridin-2-yl)disulfanyl)ethanol (340 mg, 1.8 mmol) and the mixture was stirred under N2 at room temperature for 1 h. The pH of the crude product was adjusted by adding 10 uL TEA and the sample was directly purified by column chromatography (DCM:MeOH = 20:1, v/v) to afford desired product Compound 9 as colorless oil (200 mg) in a yield of 46%.

[00543] ¹ H NMR (500 MHz, CDCh) 53.89 (td, J = 6.0, 2.8 Hz, 6H), 3.48 (s, 2H), 3.40 (s, 1H), 3.24 (s, 3H), 2.89 (tq, J = 18.4, 9.1 Hz, 12H), 2.72 (s, 3H), 2.02 (s, 2H), 1.75 (d, J = 4.3 Hz, 2H), 1.66 (s, 1H), 1.64-1.55 (m, 2H), 1.54-1.43 (m, 3H), 1.43-1.16 (m, 58H), 0.88 (t, J = 6.8 Hz, 9H). MS (ESI): calcd. for C52H108N2O3S6 [M+H] ⁺ 1001.7, found 1001.6.

Example 4: Synthesis of Compound 12 Compound 12

[00544] Step 1:

[00545] To a solution of amine A200 (1.92 g, 8.9 mmol) and episulfide (12.4 g, 62 mmol) in MeCN (60 mL) was added K2CO3 (9.2 g, 67 mmol), tetra-n-butylammonium iodide, TBAI, (300 mg, 0.8 mmol) and methyl methanethiosulfonate, MMTS, (6.4 mL, 66 mmol). The mixture was heated at 80°C for 2 days before quenching with ice cold water (200 mL). The mixture was extracted with DCM (250 mL x 3) and washed with brine (200 mL x 2). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was purified by column chromatography (DCM:EA = 10:1, v/v) to afford Compound 10 as yellow oil (1 g) in a yield of 7.8%.

[00546] ¹ HNMR (500 MHz, CDCh) 5 2.73 (td, J = 8.9, 3.3 Hz, 8H), 2.69-2.42 (m, 19H), 2.41-2.32 (m, 13H), 1.97-1.65 (m, 5H), 1.60-1.42 (m, 9H), 1.42-1.18 (m, 76H), 0.88 (t, J = 6.9 Hz, 15H). MS (ESI): calcd. for C75H155N5S10 [M+H] ⁺ 1446.9, found 1446.8.

[00547] Step 2:

[00548] To a solution of Compound 10 (580 mg, 0.4 mmol) in THF (10 mL), H2O (1 mL) and TFA (1 mL) was added PBu3 (7.5 eq, 3 mmol, 0.8 mL) and the mixture was stirred at room temperature under N2 for 2 h. The sample was directly purified on the reverse phase Biotage Selekt with C18 column (70% mobile phase B to 100% mobile phase B; mobile phase A: 0.1 % TFA in H2O and mobile phase B: 0.1 % TFA in MeOH) to afford protonated Compound 11 (244 mg) in a yield of 52%.

[00549] Step 3:

[00550] To a solution of Compound 11 (183 mg, 0.15 mmol) in dry DCM (5 mL) was added 2-(2-(Pyridin-2-yl)disulfanyl)ethanol (144 mg, 0.8 mmol) and the mixture was stirred under N2 at room temperature for 1 h. The pH of the crude product was adjusted by adding 10 uL TEA and the sample was directly purified by column chromatography (DCM:MeOH = 20:1, v/v) to afford desired product Compound 12 as colorless oil (99 mg) in a yield of 42%.

[00551] ¹ HNMR (500 MHz, CDCh) 53.87 (t, J = 5.9 Hz, 10H), 2.85 (t, J = 5.6 Hz, 12H), 2.73 (td, J = 15.1, 7.0 Hz, 11H), 2.68-2.45 (m, 14H), 1.74 (s, 5H), 1.48 (s, 9H), 1.27 (d, J = 5.8 Hz, 75H), 0.89 (d, J = 6.7 Hz, 15H). MS (ESI): calcd. for , found 1596.8.

Example 5: Synthesis of Compound 13

[00552] Step 1:

[00553] 1-1 (7.6 g, 27 mmol) was treated with ethanol (EtOH) (25 mL) and toluenesulfonic acid (TsOH) (703 mg, 4 mmol, 0.15 equiv) under nitrogen refluxing overnight to yield 1-2 (7.5 g, 90 % yield). Triphenylmethanethiol (TrSH) (5.4 g, 20 mmol, 1.2 equiv) in DMF (40 mL) was reacted with sodium hydride (NaH) (60% dispersion in mineral oil, 717 mg, 18 mmol, 1.1 equiv) at 0 °C for 30 min. A solution of 1.2 (5 g, 16 mmol, 1 equiv) in DMF (5 mL) was added slowly, and the resulting mixture was stirred at room temperature overnight to generate 1.3 (6.1 g, 86.8% yield). 1.3 (5.0 g, 11 mmol, 1 equiv) was reacted with lithium aluminum hydride (LiAlH*) (2.4 M in THF, 5.3 mL, 13 mmol, 1.2 equiv) in tetrahydrofuran (THF) (100 mL) at room temperature to yield 1.4 (1.41 g, 64.2% yield). To a mixture of 1.4 (0.51 g, 2 mmol, 1 equiv) and triethylamine (EtsN) (0.473 g, 5 mmol, 2.0 equiv) in CH2CI2 (8 mL), was added MMTS (649 mg, 5 mmol, 2.2 equiv) under nitrogen protection to afford 1.5 (0.475 g, 77% yield). Swem oxidation at -78 °C was done with 1.5 (475 mg, 1.79 mmol, 1 equiv) in the presence of trifluoroacetic anhydride (TFAA) (566 mg, 2.69 mmol, 1.5 equiv), DMSO (281 mg, 3.59 mmol, 2 equiv) and diisopropylamine (509 mg, 5.03 mmol, 2.8 equiv) in CH2CI2 (35 mL) to get 1.6 (427 mg, 90% yield) after purification. Next, to a mixture of Amine AO (22 mg, 0.18 mmol) and acetic acid (AcOH) (97.8 mg, 1.63 mmol, 8.8 equiv) in THF (1 mL), 2.2 equiv each of 1-6 and sodium triacetoxyborohydride (NaBH(OAc)3) with 0.25 mL THF were added sequentially each hour for 3 hours, for a total of 8.8 equiv (427 mg, 1.63 mmol). The reaction was stirred under nitrogen at room temperature for 16 h and monitored by mass spectrometry. The crude product was purified by flash chromatography using dichloromethane and ethyl acetate to yield Compound 4 (85 mg, 45% yield).

[00554] ’H NMR (300 MHz, CDCh): 5 2.81-2.68 (8H, m), 2.64-2.52 (8H, m), 2.52-2.42 (4H, m), 2.38 (12H, s), 2.23 (3H, s), 1.84-1.78 (4H, m), 1.53-1.43 (6H, m), 1.38-1.19 (62H, m), 0.87 (12H, t, J = 6.60 Hz). MS (APCI): m/z 1101.7 (M+l).

[00555] Step 2:

[00556] To 50 mg of Compound 4 was added tributylphosphine (BusP, 3 equiv), THF (1 mL) and water (0.1 mL). The reaction was stirred for 1 hour at room temperature (20 °C). The solvent was removed, and the crude product was purified by reverse phase chromatography using acetonitrile (0.1% TFA) and water (0.1% TFA) to generate the TFA salt of Compound 5 (33 mg, 79% yield).

[00557] ¹ H NMR (300 MHz, CD3CN): 5 3.36-3.16 (4H, m), 3.12-2.86 (7H, m), 2.82-2.68 (7H, m), 2.64-2.46 (4H, m), 1.78-1.62 (4H, m), 1.58-1.48 (4H, m), 1.42-1.19 (69H, m), 0.87 (12H, m). MS (APCI): m/z 917.8 (M+l).

[00558] Step: 3 [00559] A mixture of Compound 5 (150 mg, 0.16 mmol, 1 equiv) and methyl acrylate (563 mg, 6.54 mmol, 40 equiv), was heated to 55 °C for 7 days. MS showed the formation of desired product. After concentration, the crude was purified by column chromatography to obtain Compound 13 (100 mg, 48%).

[00560] ¹ H NMR (300 MHz, CDCh): δ 3.69 (12H, s), 2.80-2.78 (8H, m), 2.70-2.45 (20H, m), 2.21 (3H, s), 1.81-1.50 (6H, m), 1.50-1.32 (2H, m), 1.32-1.12 (72H, m), 0.87 (12H, t, J 6.6 Hz). MS (APCI): m/z 1263.0 (M+l).

Example 6: Synthesis of Compound 14

Compound 5 was prepared in accordance with Example 5.

[00561] A mixture of Compound 5 and acrylamide was heated to 55 °C for 7 days. MS showed the formation of desired product. After concentration, the crude was purified by column chromatography to obtain Compound 14.

Example 7: Synthesis of Compound 15

[00562] To a solution of 2-1 (5.0 g, 5.22 mL, 33.3 mmol, 1.0 equiv) and p-toluenesulfonyl chloride (1.27 g, 6.66 mmol, 0.2 equiv) in 10 mL dichloromethane, was added triethylamine (0.93 mL, 6.66 mmol, 0.2 equiv), and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured onto ice/water and extracted with dichloromethane (3 x 15 mL). The organic phase was separated and dried over sodium sulfate, and then concentrated. The residue was purified by silica gel column (22% dichloromethane in ethyl acetate) to give product 2-2 as pale-yellow oil (4.1 g, 41%).

[00563] ^T HNMR (300 MHz, CDCh): 57.71 (2H, d, J = 7.95 Hz), 7.28 (2H, d, J = 8.52 Hz), 4.08 (2H, t, J = 4.68 Hz), 3.63-3.48 (10H, m), 2.75 (IH, t, J = 4.83 Hz), 2.37 (3H, s). MS (APCI): m/z 305.1 (M + 1).

[00564] A mixture of 2-2 (4.1 g, 13.55 mmol, 1.0 equiv) and potassium thioacetate (3.09 g,

27.10 mmol, 2.0 equiv) in 40 mL DMF was heated at 90 °C under nitrogen for 16 h. The reaction mixture was cooled to room temperature and diluted with 100 mL dichloromethane. The resulting mixture was washed with water (5 x 100 mL), and the organic layer was dried over sodium sulfate. After concentration, the crude was purified by silica gel column (80% ethyl acetate in hexane) to give product 2-3 as orange-colored oil (2.71 g, 96%).

[00565] ¹ H NMR (300 MHz, CDCh): 53.76-3.58 (12H, m), 3.09 (IH, t, J = 6.30 Hz), 2.33 (3H, s). [00566] A mixture of 2-3 (2.71 g, 13.0 mmol, 1.0 equiv) and potassium carbonate (9.0 g, 65.1 mmol, 5.0 equiv) in 20 mL methanol was stirred at room temperature. After 2 h, a solution of 2,2’-dipyridyl disulfide (4.3 g, 19.5 mmol, 1.5 equiv) in 7 mL methanol was added, the reaction mixture was purged with nitrogen three times and then stirred at room temperature for 2 h. TLC indicated complete reaction. The reaction mixture was diluted with dichloromethane (50 mL), and the mixture was washed with water (3 x 50 mL). After drying over sodium sulfate and concentration, the residue was purified by silica gel column (80% ethyl acetate in hexane) to afford 2-5 as dark orange oil (1.77 g, 49%).

[00567] ¹ H NMR (300 MHz, CDCh): 5 8.45 (1H, m), 7.77-7.64 (2H, m), 7.10-7.08 (1H, m), 3.73 (4H, t, J = 6.30 Hz), 3.65-3.58 (6H, m), 2.99 (2H, t, J = 6.33 Hz), 2.35 (1H, s). MS (APCI): m/z 276.1 (M + 1).

[00568] A mixture of Compound 5 (100 mg, 0.11 mmol, 1.0 equiv) and 2-5 (135 mg, 0.49 mmol, 4.5 equiv) in 3.5 mL chloroform was purged with nitrogen three times, and then the reaction mixture was stirred at room temperature for 1 h. TLC showed complete reaction. After concentration, the crude was purified by silica gel column (10 % methanol in dichloromethane) to afford Compound 15 as pale-yellow oil (74 mg, 43%).

[00569] ^l H NMR (300 MHz, CDCh): 53.74-3.60 (40H, m), 2.88-2.45 (28H, m), 2.22 (3H, s), 1.80 (4H, s), 1.26 (72H, s), 0.87 (12H, t, J = 6.33 Hz). MS (APCI): m/z 1576.0 (M + 1).

Example 8: Synthesis of Compound 16 [00570] A solution of 2-6 (5.47 g, 5.22 ml, 33.3 mmol, 1.0 equiv) and p-toluenesulfonyl chloride (6.5 g, 34.1 mmol, 1.02 equiv) in dichloromethane (35 mL) was cooled to 0 °C, and KOH (7.5 g, 133 mmol, 4.0 equiv) was added slowly. The reaction was stirred at room temperature for 16 h. TLC showed complete reaction. The reaction mixture was poured onto ice/water and extracted with dichloromethane. The organic phase was dried over sodium sulfate concentrated to give product 2-7 as yellow oil (8.5 g, 80% yield), which was used for next step directly without purification. A mixture of 2-7 (8.5 g, 26.73 mmol, 1.0 equiv) and potassium thioacetate (6.09 g, 53.46 mmol, 2.0 equiv) in DMF (85 mL) was heated at 90 °C under nitrogen for 16 h. The reaction mixture was cooled and diluted with dichloromethane, and the resulting solution was washed with water. The organic layer was dried and concentrated to give the crude, which was purified by Combi-flash (eluent: hexane/ethyl acetate = 3/1) to give product 2-8 as orange oil (4.6 g, 78% yield). 2-8 (4.6 g, 20.72 mmol) in a mixture of methanol (30 mL) and aq. HC1 (30 mL) was heated at 100 °C for 2.5 h. After cooled to room temperature, the reaction mixture was diluted with dichloromethane. The mixture was washed with water and saturated sodium bicarbonate, and the organic layer was dried over sodium sulfate. After concentration, the crude was used for the next step without purification. A mixture of 2-9 (533 mg, 2.96 mmol, 1.0 equiv) and 2,2’-dipyridyl disulfide (978 mg, 4.44 mmol, 1.5 equiv) in dichloromethane was purged with nitrogen and then stirred at room temperature overnight. The solvent was removed under reduced pressure to give the crude, which was subsequently purified on Combi-flash column (elute: hexane/Ethyl Acetate = 3/1) to afford compound 2-10 as pale yellow oil (472 mg, 55% yield).

[00571] ^T HNMR (300 MHz, CDCh): 58.44 (1H, d, J = 4.68 Hz), 7.76 (1H, d, J = 9.06 Hz), 7.65 (1H, td, J = 1.92, 0.54 Hz), 7.08 (1H, td, J = 3.74, 1.11 Hz), 3.71 (2H, t, J = 6.3 Hz), 3.65- 3.52 (8H, m), 3.36 (3H, s), 2.98 (2H, t, J = 6.3 Hz). MS (APCI): m/z 289.5 (M ⁺ ).

[00572] A mixture of Compound 5 (129 mg, 0.141 mmol, 1.0 equiv) and 2-10 (183 mg, 0.633 mmol, 4.5 equiv) in chloroform (3 mL) was purged by nitrogen three times and stirred at room for 1 hour. TLC showed that the reaction was completed. The solvent was removed under reduced pressure to give the crude, which was subsequently purified on Combi-flash column (elute: Dichloromethane/MeOH = 95/5) to afford Compound 16 as pale yellow oil (110 mg, 48% yield). [00573] ¹ H NMR (300 MHz, CDCh): δ 3.73-3.53 (40H, m), 3.37 (12H, d, J = 1.65 Hz), 2.83 (8H, t, J = 6.87 Hz), 2.69-2.41 (16H, m), 2.21 (3H, s), 1.81 (4H, m), 1.61-1.25 (72H, m), 0.87 (12H, t, J = 4.92 Hz). MS (APCI): m/z 1629.2 (M ⁺ ).

Example 9: Synthesis of Compound 19

[00574] Step 1: Synthesis of Amine 1

[00575] To a mixture of 1 (10.0 g, 20 mmol, 1 equiv) and pyridine (100 mL), was added a solution of 2 (6.93 g, 20 mmol, 1 equiv) in dimethylformamide (DMF) (20 mL). The reaction mixture was stirred at room temperature overnight to afford 3 (11.15 g, 81% yield). Trifluoroacetic acid (TFA) (50 mL) was slowly added into a solution of 3 (11.15 g, 16.2 mmol, 1 equiv) in dichloromethane (CH2CI2) (50 mL) at 0 °C, and the reaction was stirred at room temperature overnight to generate 4 (10.0 g). Ammonium hydroxide (NH4OH) (20 mL) was slowly added into a solution of 4 (10.0 g, 16.2 mmol, 1 equiv) in methanol (MeOH) (500 mL) over a period of 1 hour, and the reaction mixture was stirred at room temperature overnight to produce 5 (6.6 g, 70% yield), which was then dissolved in dichloromethane (CH2Cl2)/acetic acid (AcOH) (200 mL/200 mL) and treated with 10% Pd/C (3.0 g) under hydrogen (1 atm) to generate the product Amine 1 as a thick oil. The crude was triturated with ethyl acetate (EtOAc) (200 mL) to get pure Amine 1 (2.28 g, 79% yield) as white solid. [00576] ¹ HNMR (300 MHz, CDCh): 54.00 (2H, t, J = 5.22 Hz), 2.82 (2H, t, J = 7.29 Hz), 1.92 (4H, s), 1.88-1.80 (4H, m), 1.64-1.56 (4H, m), 1.50-1.45 (4H, m). MS (APCI): m/z 257.2 (M+l).

[00577] Step 2:

[00578] To a solution of Amine 1 and episulfide in MeCN is added K2CO3, tetra-n- butylammonium iodide, TBAI, and methyl methanethiosulfonate, MMTS. The mixture is heated at 80°C for 2 days before quenching with ice cold water (200 mL). The mixture is extracted with DCM (250 mL x 3) and washed with brine (200 mL x 2). The combined organic layer is dried over Na2SO4 and concentrated under vacuum. The crude is purified by column chromatography (DCM:EA = 10:1, v/v) to afford Compound 17.

[00579] Next, to a solution of Compound 17 in THF (10 mL), H2O (1 mL) and TFA (1 mL) is added PBui and the mixture is stirred at room temperature under N2 for 2 h. The sample is directly purified on the reverse phase Biotage Selekt with Cl 8 column (70% mobile phase B to 100% mobile phase B; mobile phase A: 0.1 % TFA in H2O and mobile phase B: 0.1 % TFA in MeOH) to afford protonated Compound 18. [00580] Then, to a solution of Compound 18 in dry DCM (5 mL) is added 2-(2-(Pyridin-2- yl)disulfanyl)ethanol and the mixture is stirred under N2 at room temperature for 1 h. The pH of the crude product is adjusted by adding 10 uL TEA and the sample is directly purified by column chromatography (DCM:MeOH = 20:1, v/v) to afford desired product Compound 19.

Example 10: Synthesis of Compound 21

[00581] Step 1:

[00582] A solution of amine core A96 (0.48 g, 5.4 mmol) and epoxide (3.4 g, 16.2 mmol) in THF (0.5 mL) was heated at 80°C for 3 days. The mixture was directly purified by column chromatography (DCM:MeOH = 10:1, v/V) to afford Compound 20 as yellow oil (2.8 g) in a yield of 78%.

[00583] Step 2:

[00584] To a solution of Compound 20 (236 mg, 0.324 mmol) in dry DCM (5 mL) was added mesyl chloride (MsCl) (0.97 mmol, 115 mg) and TEA (500 mg). The mixture was stirred at 0°C under N2 for 3 h before adding 2-mercaptoethanol (1.5 mmol). The mixture was further left at room temperature overnight. The mixture was extracted with DCM (150 mL x 2) and washed with brine (100 mL x 2). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was purified by column chromatography (DCM:MeOH = 10:1, v/v) to afford desired product Compound 21 as a colorless oil (130 mg) in a yield of 45%.

[00585] ^T HNMR (500 MHz, CDCh) 5 3.72 (dt, J = 7.5, 5.8 Hz, 5H), 2.72 (tt, J = 6.0, 3.3 Hz, TH), 2.66-2.53 (m, 4H), 2.52-2.34 (m, 4H), 2.19 (s, 2H), 1.48 (d, J = 40.4 Hz, 9H), 1.26 (s, 52H), 0.88 (t, J = 6.9 Hz, 9H). MS (ESI): calcd. for C52H103N2O3S3 [M+H] ⁺ 905.7, found 905.7.

Example 11 : Synthesis of Compound 22

[00586] To a solution of C12-A200 (285 mg, 0.25 mmol) in dry DCM (4 mL) was added

MsCl (1.25 mmol, 152 mg) and TEA (660 mg). The mixture was stirred at 0°C under N2 for 5 h before adding 2-mercaptoethanol (1.5 mmol). The mixture was further left at room temperature overnight. The mixture was extracted with DCM (150 mL x 2) and washed with brine (100 mL x 2). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude was purified by column chromatography (DCM:MeOH = 10:1, v/v) to afford desired product Compound 22 as a colorless oil (112 mg) in a yield of 32%.

[00587] ¹ H NMR (500 MHz, CDCh) 5 3.75 (t, J = 6.6 Hz, 9H), 2.87-2.67 (m, 17H), 2.48 (s, 15H), 1.40 (t, J = 7.4 Hz, 14H), 1.27 (d, J = 6.7 Hz, 77H), 0.88 (t, J = 6.8 Hz, 15H). MS (ESI): calcd. for

Example 12- Preparation of LNPs of Present Disclosure Comprising mRNA and In Vivo Screening

[00588] A. Preparation

[00589] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of Formulas (I)-(IV) and mRNA.

[00590] To formulate the LNPs, various percentages of one of COMPOUNDS 3, 6, or 12, the phospholipid (DOPE or DOPC), the structural lipid cholesterol (Choi) and 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (DMG-PEG2000; Avanti Polar Lipids, Alabaster, Alabama, USA) were combined to prepare LNP compositions.

[00591] Individual 25 mg/ml stock solutions were prepared by solubilizing the lipids in 200- proof HPLC-grade ethanol and stock solutions were stored at -80° C until formulated. At the time of formulation, the lipid stock solutions were briefly allowed to equilibrate to room temp and then placed on a hot plate maintained at a temperature range of 50-55°C. Subsequently, the hot lipid stock solutions were combined to yield desired final mole percentages. A subset of the LNP compositions is shown in Table 4.

Table 4

[00592] A 1 mg/ml solution of the 5’-CleanCap-fLudferase mRNA (TriLink Biotech) to be incorporated into the LNPs was added to 150 mM sodium acetate buffer (pH 5.2) to form a stock solution and kept on ice. The lipid phase was mixed with the aqueous mRNA phase inside a microfluidic chip using a NanoAssemblr® instrument (Precision Nanosystems, Vancouver, BC,

Canada) according to the manufacturer’s instructions to form LNP compositions comprising encapsulated mRNAs. Nanoassemblr process parameters for mRNA encapsulation are shown in the Table 5.

Table 5

[00593] The resultant mRNA LNP compositions were then transferred to a Repligen Float- A-Lyzer dialysis device- having a molecular weight cut off (MWCO) of 8-10kDa (Spectrum Chemical Mfg. Corp, CA, USA) and processed by dialysis against phosphate buffered saline (PBS) (dialysate : dialysis buffer volume at least 1:200 v/v), pH 7.4 overnight at 4°C (or alternatively room temperature for at least 4hours), to remove the 25% ethanol and achieve a complete buffer exchange. In some experiments the LNPs were further concentrated in an Amicon® Ultra-4 centrifugal filter unit, MWCO-30kDa (Millipore Sigma, USA) spun at -4100 x g in an ultracentrifuge. The mRNA LNPs were then stored at 4°C until further use.

[00594] The average particle size diameter of the LNPs was approximately 68-137 nm.

[00595] B. In Vivo Screening

[00596] Adult female BALB/C mice (n=2/group) were intravenously administered 0.5 mg/kg of 5’-CleanCap-fLuciferase mRNA (TriLink Biotech) formulated with the LNP compositions shown in Table 4. One group of mice was treated with vehicle (PBS, Thermo Fisher Scientific, USA) as a negative control.

[00597] The location and extent of luciferase expression in treated and control mice were determined at 4 hr by bioluminescent imaging (BLI) of anesthetized mice using an IVIS Lumina in vivo imaging system (Perkin Elmer) according to the manufacturer’s instructions. Briefly, mice were anesthetized using isoflurane in oxygen, and placed supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 6.

Table 6

[00598] As shown in Table 6, LNP compositions of the present disclosure were capable of delivering mRNA in vivo, predominantly to cells in the liver, and subsequent expression of the encoded protein.

Example 13- Preparation of LNPs of Present Disclosure Comprising DNA and In Vivo Screening

[00599] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of Formulas (I)-(IV) and DNA.

[00600] LNP compositions of the present disclosure comprising Compound No. 6 and DNA encoding firefly luciferase (Nature Technology Corporation) were prepared as described in

Example 12. The LNP compositions are shown in Table 7.

Table ?

[00601] Adult BALB/C mice (n=3) were administered 0.5 mg/kg of total DNA of an LNP composition listed in Table 7. The location and extent of luciferase expression in treated and control mice were determined at 48 hr by bioluminescent imaging (BLI) of anesthetized mice using an IVIS Lumina in vivo imaging system (Perkin Elmer) according to the manufacturer’s instructions. Briefly, mice were anesthetized using isoflurane in oxygen, and placed supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 8.

Table 8

[00602] As shown in Table 8, LNP compositions of the present disclosure were capable of delivering DNA to liver cells and express the encoded transgene in liver cells.

[00603] In addition, administration of LNP compositions of Table 8 were well tolerated in the mice. Most of the mice lost some weight 48 hours after administration but then recovered.

Example 14- Preparation of LNPs of Present Disclosure Comprising DNA and In Vivo Screening

[00604] The following is a nonlimiting example that provides exemplary methods for formulating a plurality of multi-component LNP compositions comprising exemplary compounds of Formulas (I)-(IV) and DNA.

[00605] LNP compositions of the present disclosure comprising Compound No. 6 and DNA encoding firefly luciferase (Nature Technology Corporation) were prepared as described in Example 12 The LNP compositions are shown in Table 9.

Table 9

[00606] Adult BALB/C mice (n=3) were administered 0.5 mg/kg of total DNA of an LNP composition listed in Table 9. The location and extent of luciferase expression in treated and control mice were determined at 48 hr by bioluminescent imaging (BLI) of anesthetized mice using an IVIS Lumina in vivo imaging system (Perkin Elmer) according to the manufacturer’s instructions. Briefly, mice were anesthetized using isoflurane in oxygen, and placed supine on a heated stage. Mice were then administered D-luciferin (Perkin-Elmer #122799) IP, and BLI was performed. The results are shown in Table 10.

Table 10

[00607] As shown in Table 10, LNP compositions of the present disclosure were capable of delivering DNA to liver cells and express the encoded transgene in liver cells.

[00608] In addition, administration of LNP compositions of Table 10 were well tolerated in the mice. Most of the mice lost some weight 48 hours after administration but then recovered.

Example 15- pKa Values for LNPs of Present Disclosure

[00609] pKa values for LNP compositions of the present disclosure were determined using the 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) assay. TNS is an anionic molecule that is non-fluorescent in aqueous solutions but upon binding to cationic lipids exhibits strong fluorescence. LNPs were standardized by diluting to approximately 1 ,7nmol of ionizable lipid per well and were titrated using pH buffered solutions from pH 2 to 12 in a 96-well plate. The TNS reagent (Sigma-Aldrich #T9792) was prepared at 0.05mg/mL in water, 5uL of reagent was dispensed to each well, and fluorescence was measured on a TECAN Infinite M200PRO microplate reader (Excitation: 322nm and Emission: 431nm, gain 75). Fluorescence values for each LNP were normalized to the maximum TNS signal. GraphPad Prism was used to plot the pH and normalized fluorescence values using the XV graphing function. This data was analyzed using nonlinear regression and log(inhibitor) vs normalized response functions to yield the pKa values for each sample, shown in Table 11.

Table 11

[00610] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.