Sulfur-containing compounds and compositions thereof for delivery of nucleic acids

Abstract

Lipid nanoparticle compositions (LNPs), methods for preparing LNPs, methods of using same, including but not limited to, for the treatment of certain diseases and disorders, including but not limited to, liver damage, kits for in vivo, ex vivo, and in vitro nucleic acid delivery to various cell types, including T cells and hepatocytes.

Description

[Technical Field]

[0001] There is a long-standing, unmet need in the art for compositions and methods for delivering nucleic acids to cells in vivo, ex vivo, and in vitro, and for genetically modifying cells. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors such as AAV, can cause acute toxicity and adverse side effects in patients. The present disclosure provides improved compositions, methods, and kits for delivering 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 for using the same. These lipid nanoparticle compositions and methods enable the delivery of nucleic acids to cells with high efficiency and low toxicity. Thus, the compositions and methods of the present disclosure have broad applicability in a variety of fields, including gene therapy and the production of cellular therapeutics. Summary of the Invention

[0002] In some embodiments, novel lipid nanoparticles ("LNPs") are provided that comprise novel compounds. In one embodiment, the novel compounds are compounds of formulas (I)-(IV).

[0003] In some aspects, a pharmaceutical composition is provided comprising a composition of the present disclosure and at least one pharmaceutically acceptable excipient or diluent.

[0004] In some embodiments, methods are provided for 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.

[0005] In some embodiments, methods of genetically modifying at least one cell are provided, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0006] In some aspects, methods are provided for 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.

[0007] In some embodiments, methods are provided for 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.

[0008] In some aspects, cells modified according to the methods of the present disclosure are provided.

[0009] Any aspect and / or embodiment described herein may 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. As used herein, the singular includes the plural unless the context clearly dictates otherwise. By way of example, 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 elements. Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" are understood to mean 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 may 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 this 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. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will become apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure provides novel compounds, novel lipid nanoparticle compositions (LNPs) comprising the novel compounds, methods for preparing LNPs, and methods for using the same. In a non-limiting example, the compositions and methods of the present disclosure can be used for gene delivery and cell therapy. In a non-limiting example, the compositions and methods of the present disclosure can be widely used for delivering nucleic acids to hepatocytes in vivo, ex vivo, or in vitro for the treatment of certain diseases and disorders, including but not limited to, liver damage. In a non-limiting example, the compositions and methods of the present disclosure can be widely used for delivering nucleic acids 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 widely used for delivering nucleic acids to primary resting 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 widely used for delivering nucleic acids for vaccination purposes. In a non-limiting example, the compositions and methods of the present disclosure can be used broadly to deliver nucleic acids to induce expression of secreted therapeutic proteins.

[0013] Compositions of the present disclosure 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 embodiments, the lipid nanoparticle can further comprise at least one structural lipid. In some embodiments, the lipid nanoparticle can further comprise at least one phospholipid. In some embodiments, the 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.

[0014] compound In some embodiments, the present disclosure provides a compound of formula (I):

[0015] [ka] A is a substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, each R1 is independently hydrogen, substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, and at least one occurrence of R1 is hydrogen; each of B, C, and D is independently hydrogen, a substituted or unsubstituted branched or unbranched C1-15 aliphatic, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, or (CHR6)CH(SXLY)R6; X is S or CH2; Y is -OH,

[0016] [ka] where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic; compounds wherein each R6 is independently hydrogen, substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, and at least one occurrence of R6 is hydrogen; or a pharmaceutically acceptable salt thereof.

[0017] 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.

[0018] 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 -(CHR6)CH(SXLY)R6, C and D are different.

[0019] 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.

[0020] In some embodiments, X is S and Y is —OH.

[0021] In some embodiments, X is S. In some embodiments, X is CH. In certain of these embodiments, Y is —OH.

[0022] In some embodiments, L is unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0023] In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXLY)R6, each of the two occurrences of R1 and R6 that are not hydrogen are the same. In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXLY)R6, each of the two occurrences of R1 and R6 that are not hydrogen are different.

[0024] In certain embodiments, when two of B, C, and D are -(CHR6)CH(SXLY)R6, each of the three occurrences of R1 and R6 that are not hydrogen are the same. In certain embodiments, when two of B, C, and D are -(CHR6)CH(SXLY)R6, two of the three occurrences of R1 and R6 that are not hydrogen are the same. In certain embodiments, when two of B, C, and D are -(CHR6)CH(SXLY)R6, each of the three occurrences of R1 and R6 that are not hydrogen are different.

[0025] In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-15 alkyl. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-10 alkyl. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

[0026] In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic.

[0027] In some embodiments, R1 is

[0028] [ka] is.

[0029] In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-15 alkenyl.

[0030] [ka] is.

[0031] In some embodiments, R1 is

[0032] [ka]

[0033] [ka] is.

[0034] In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-15 alkyl. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-10 alkyl. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

[0035] In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic.

[0036] In some embodiments, R6 is

[0037] [ka] is.

[0038] In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-15 alkenyl.

[0039] [ka] is.

[0040] In some embodiments, R6 is

[0041] [ka]

[0042] [ka] is.

[0043] In some embodiments,

[0044] [ka] teeth,

[0045] [ka] is.

[0046] In some embodiments,

[0047] [ka] In some embodiments,

[0048] [ka] is.

[0049] In some embodiments,

[0050] [ka] is a substituted or unsubstituted branched or unbranched aliphatic. In some embodiments,

[0051] [ka] is a substituted or unsubstituted branched or unbranched alkyl. In some embodiments,

[0052] [ka] is a substituted or unsubstituted branched or unbranched heteroaliphatic.

[0053] 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 is C 12 H 25 In certain of these embodiments, R6 is C 12 H 25 In certain of these embodiments, L is unsubstituted C alkyl. In certain of these embodiments,

[0054] [ka] is a C3 alkyl.

[0055] In some embodiments, the disclosure provides compounds of the formula:

[0056] [ka]

[0057] In some embodiments, the present disclosure provides a compound of formula (II):

[0058] [ka] a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group, 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 R1 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; Y is -OH,

[0059] [ka] where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic compound; or a pharmaceutically acceptable salt thereof. In some embodiments, X is S and Y is —OH.

[0060] In some embodiments, X is S. In some embodiments, X is CH. In certain of these embodiments, Y is —OH.

[0061] In some embodiments, L is unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0062] In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-15 alkyl. In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-10 alkyl. In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-5 alkyl.

[0063] In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-15 aliphatic. In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-10 aliphatic. In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-5 aliphatic.

[0064] In some embodiments, each of R1, R2, R3, and R4 is independently:

[0065] [ka] is.

[0066] In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-15 alkenyl. In some embodiments, each of R1, R2, R3, and R4 is independently

[0067] [ka] is.

[0068] In some embodiments, each of R1, R2, R3, and R4 is independently:

[0069] [ka]

[0070] [ka] is.

[0071] In some embodiments, each of R1, R2, R3, and R4 is independently unsubstituted branched or unbranched C1-15 heteroaliphatic.

[0072] In some embodiments, each of R1, R2, R3, and R4 is independently C 10 H 21 is.

[0073] 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.

[0074] In some embodiments,

[0075] [ka] teeth,

[0076] [ka] is.

[0077] In some embodiments,

[0078] [ka] In some embodiments,

[0079] [ka] is.

[0080] In some embodiments,

[0081] [ka] is a substituted or unsubstituted branched or unbranched aliphatic. In some embodiments,

[0082] [ka] is a substituted or unsubstituted branched or unbranched alkyl. In some embodiments,

[0083] [ka] is a substituted or unsubstituted branched or unbranched heteroaliphatic.

[0084] In some embodiments, the disclosure provides compounds of the formula:

[0085] [ka]

[0086] In some embodiments, the present disclosure provides a compound of formula (III):

[0087] [ka] During the ceremony, Each of Ra and Rb is independently

[0088] [ka] or

[0089] [ka] and X is S or CH2; Y is -OH,

[0090] [ka] where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic; R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group, x is an integer between 1 and 10 inclusive; y is an integer between 1 and 10 inclusive; Each Ry and Rz is independently

[0091] [ka] a compound, or a pharmaceutically acceptable salt thereof.

[0092] In some embodiments, Ra is

[0093] [ka] and Rb is

[0094] [ka] is.

[0095] In some embodiments, Ra is

[0096] [ka] In some embodiments, Ra is

[0097] [ka] In some embodiments, Ra is

[0098] [ka] is.

[0099] In some embodiments, Rb is

[0100] [ka] In some embodiments, Rb is

[0101] [ka] In some embodiments, Rb is

[0102] [ka] is.

[0103] In some embodiments, x is 1 and y is 2.

[0104] In some embodiments, X is S and Y is —OH.

[0105] In some embodiments, X is S. In some embodiments, X is CH. In certain of these embodiments, Y is —OH.

[0106] In some embodiments, L is unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0107] In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-15 alkyl. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-10 alkyl. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

[0108] In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic.

[0109] In some embodiments, R7 is C 10 H 21 is.

[0110] In some embodiments, Ra is

[0111] [ka] is.

[0112] In some embodiments, Rb is

[0113] [ka] is.

[0114] In some embodiments, the disclosure provides compounds of the formula:

[0115] [ka]

[0116] In some embodiments, the present disclosure provides a compound of formula (IV):

[0117] [ka] During the ceremony, Each of Re and Rf independently represents

[0118] [ka] and x is an integer between 1 and 10 inclusive; Each Ry and Rz is independently

[0119] [ka] and X is S or CH2; Y is -OH,

[0120] [ka] where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic; R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments, Re is

[0122] [ka] is.

[0123] In some embodiments, Rf is

[0124] [ka] is.

[0125] In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3.

[0126] In some embodiments, X is S and Y is —OH.

[0127] In some embodiments, X is S. In some embodiments, X is CH. In certain of these embodiments, Y is —OH.

[0128] In some embodiments, L is unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is unsubstituted C2 alkyl.

[0129] In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-15 alkyl. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-10 alkyl. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

[0130] In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic.

[0131] It will be understood that the compounds of any one of the formulas disclosed herein, and any pharmaceutically acceptable salts thereof, include stereoisomers, mixtures of stereoisomers, and polymorphs of all isomeric forms of the compounds.

[0132] It will be understood that the compounds disclosed herein may be presented without a specific configuration (e.g., without a specific stereochemistry). Such presentation is intended to encompass all possible isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, presentation of a compound herein without a specific configuration is intended to refer to each of the possible isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixture thereof.

[0133] It should be understood that the compounds of any formula described herein include the compounds themselves, as well as, if applicable, their salts and solvates. Salts can be formed, for example, 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).

[0134] General Methods for the Preparation of Compounds of Formulae (I)-(IV) of the Present Disclosure The compounds of the present invention can be prepared by any method known in the art. The compounds can be prepared from commercially available starting materials such as amines and thioepoxide compounds. The compounds can also be prepared by total synthesis from commercially available starting materials.

[0135] Generally, the first step in the preparation of compounds of Formulas (I)-(IV) of the present disclosure is the selection of an appropriate amine for preparing the "A" amine core precursor. Non-limiting examples of amines that can be selected to prepare the "A" amine core precursor include:

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] In some embodiments, the next step in the preparation of compounds of Formulas (I)-(IV) is to react an amine with a terminal thioepoxide to form a precursor compound. Below is an exemplary reaction scheme between a primary amine and a thioepoxide to form a precursor compound by ring-opening of the thioepoxide and attachment to an amine:

[0140] [ka] wherein R is an aliphatic group as defined herein.

[0141] In some embodiments, one equivalent of amine is reacted with one equivalent of thioepoxide compound. In other embodiments, one equivalent of amine is reacted with 1, 2, 3, 4, 5, or more equivalents of thioepoxide compound. The amount of thioepoxide compound can be limited to prevent functionalization of all amino groups. The resulting compounds may contain secondary and / or primary amino groups and may be further functionalized, for example, with different terminal thioepoxides or different electrophiles. Further functionalization of such amines can result in compounds with different thioepoxide-derived tails.

[0142] In some embodiments, the amine and thioepoxide react at an unsubstituted carbon of the thioepoxide to form the compound:

[0143] [ka] This results in:

[0144] In some embodiments, the amine and thioepoxide react at a substituted carbon of the thioepoxide to form the compound:

[0145] [ka] This results in:

[0146] Thioepoxide compounds useful in the present invention include any thioepoxide compound, its racemate or stereoisomers, all varying in chain length and degree of saturation of functional groups. 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 compound has the formula:

[0147] [ka] It is of the type.

[0148] In some embodiments, reaction of an amine with a terminal thioepoxide produces a compound having a thiol moiety attached to the carbon beta to the amino group (the "β carbon").

[0149] In certain embodiments, the preparation of compounds of Formulas (I)-(IV) involves the reaction of an amine with a terminal thioepoxide under conditions to form an "A" amine precursor compound having a disulfide bonded to the β-carbon of the amino group. For example, an amine can be reacted with a thioepoxide compound in the presence of methanethiosulfonate to form a precursor compound having a disulfide-linked methyl group bonded to the β-carbon of the amino group.

[0150] In some embodiments, the disulfide can then be treated with a reducing agent to form a free thiol.

[0151] In some embodiments, the final step in the preparation of compounds of Formulas (I)-(IV) involves 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 β-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 β-carbon of the amino group.

[0152] In some embodiments, the present disclosure provides a compound of the formula:

[0153] [ka]

[0154] [ka]

[0155] [ka] or more equivalents of one amine of the formula:

[0156] [ka] and a thioepoxide-containing compound of formula (I) to (IV):

[0157] [ka]

[0158] [ka] and reacting under conditions to form a compound selected from the group consisting of: wherein A, B, C, D, X, L, Y, R1, R2, R3, R4, Ra, Rb, Re and Rf are as defined herein.

[0159] In some embodiments, the present disclosure provides a compound of the formula:

[0160] [ka] with one or more equivalents of an amine of the formula:

[0161] [ka] and a thioepoxide-containing compound of the formula:

[0162] [ka] reacting under conditions to form a compound of Formula (b):

[0163] [ka] By reducing the compound formula:

[0164] [ka] and forming a compound of formula (I) (c) Formula:

[0165] [ka] The compound formula:

[0166] [ka] By reacting with the compound formula:

[0167] [ka] and producing a compound of formula (I): wherein A, R1, L, Y, B, C, and D are as described herein.

[0168] In some embodiments, the present disclosure provides a compound of the formula:

[0169] [ka] with one or more equivalents of an amine of the formula:

[0170] [ka] and a thioepoxide-containing compound of the formula:

[0171] [ka] reacting under conditions to form a compound of Formula (b):

[0172] [ka] By reducing the compound formula:

[0173] [ka] and forming a compound of formula (I) (c) Formula:

[0174] [ka] The compound formula:

[0175] [ka] By reacting with the compound formula:

[0176] [ka] and producing a compound of formula (I): wherein R7, L and Y are as described herein.

[0177] In some embodiments, the preparation of compounds of Formulas (I)-(IV) comprises the reaction of an amine with an aldehyde to form an amine precursor compound. Below is an exemplary reaction scheme between an amine and a terminal aldehyde to form the precursor compound by reductive amination:

[0178] [ka] wherein R is an aliphatic group as defined herein and Z is hydrogen or —SR.

[0179] In some embodiments, reaction of an amine with a terminal aldehyde produces a compound with a thiol moiety linked to the beta carbon of the amino group.

[0180] In some embodiments, the preparation of compounds of Formulas (I)-(IV) involves the reaction of an amine with a terminal aldehyde under conditions to form an "A" amine precursor compound having a disulfide attached to the β-carbon of the amino group. For example, an amine can be reacted with an aldehyde compound containing a methyl disulfide group β to the aldehyde to form a precursor compound having a methyl disulfide attached to the β-carbon of the amino group.

[0181] In some embodiments, the disulfide can then be treated with a reducing agent to form a free thiol.

[0182] In some embodiments, the final step in the preparation of compounds of Formulas (I)-(IV) involves 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 β-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 β-carbon of the amino group.

[0183] In some embodiments, the present disclosure provides: (a) Formula:

[0184] [ka] (b) preparing an aldehyde of formula:

[0185] [ka] to an aldehyde of the formula:

[0186] [ka] to condense with an amine of the formula:

[0187] [ka] (c) forming a compound of formula:

[0188] [ka] by reducing a compound of the formula:

[0189] [ka] and (d) forming a compound of formula:

[0190] [ka] Compounds with formula:

[0191] [ka] to form a compound of the formula:

[0192] [ka] wherein A, R1, L, Y, B, C, and D are as described herein.

[0193] In some embodiments, one equivalent of amine is reacted with one equivalent of aldehyde compound. In other embodiments, one equivalent of amine is reacted with 1, 2, 3, 4, 5, or more equivalents of aldehyde compound. The amount of aldehyde compound can be limited to prevent functionalization of all amino groups. The resulting compound may contain secondary and / or primary amino groups and may be further functionalized, for example, with a different aldehyde or a different electrophile. Further functionalization of such amines can result in compounds having tails derived from different aldehyde compounds.

[0194] In some embodiments, the preparation of compounds of Formulas (I)-(IV) involves the selection or formation of an amino alcohol precursor compound. For example, an exemplary amine can be reacted with an epoxide to form the amino alcohol precursor compound by ring-opening of the epoxide. In some embodiments, the next step in the preparation of compounds of Formulas (I)-(IV) involves further functionalization of the amino alcohol moiety. For example, the amine precursor compound can be treated with 2-mercaptoethanol to add a sulfur-linked ethyl alcohol group to the precursor compound at the β-carbon of the amino group.

[0195] LNP component In some embodiments, the LNPs of the disclosure have at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or less The at least one compound of the present disclosure may comprise at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or at least about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one compound of the present disclosure. In some embodiments, the at least one compound is at least one compound of Formulas (I)-(IV) described herein.

[0196] In some embodiments, LNPs of the present disclosure can comprise about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of at least one compound of the present disclosure. In some embodiments, the at least one compound is at least one compound of Formulas (I)-(IV) described herein.

[0197] In some embodiments, the LNPs are at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or less At least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or at least about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one structured lipid.

[0198] In some embodiments, the LNP may further comprise at least about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of at least one structured lipid.

[0199] In some embodiments, the LNPs are at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or less and may further comprise at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or at least about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one phospholipid.

[0200] In some embodiments, the LNP may further comprise at least about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of at least one phospholipid.

[0201] In some embodiments, the LNPs may further comprise at least about 0.25 mol%, or at least about 0.5 mol%, or at least about 0.75 mol%, or at least about 1.0 mol%, or at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol% PEGylated lipids.

[0202] A. Structured lipid In some embodiments, the structured lipid may be a steroid. In some embodiments, the structured lipid may be a sterol. In some embodiments, the structured lipid may comprise cholesterol. In some embodiments, the structured lipid may comprise ergosterol. In some embodiments, the structured lipid may be a phytosterol.

[0203] B. Phospholipids As used herein, the term "phospholipid" is used in its broadest sense to refer to any amphipathic molecule containing a polar (hydrophilic) head group containing phosphate and two hydrophobic fatty acid chains. In some embodiments, the phospholipid may comprise dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the phospholipid may comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the phospholipid may be selected from the group consisting of DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt)), DEPC (1,2-dierucoyl-sn-glycero-3-phosphocholine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-dierucoyl-sn-glycero-3[phopho]choline), and the like. Sulfo-rac-(1-glycerol) (sodium salt)), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPA-NA (1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt)), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DLPG-NA (1,2-dilauroyl 1,2-dilauroyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)), DLPG-NH4 (1,2-dilauroyl-sn-glycero-3-phospho-rac-(1-glycerol) (ammonium salt)), DLPS-NA (1,2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt)), DMPA-NA (1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt)), DMPC(1,2 -dimyristoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DMPG-NH4 (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DMPG-NH4 / NA(1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium / ammonium salt)), DMPS-NA (1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt)), DOPA-NA (1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanol) diolamine), DOPG-NA (1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DOPS-NA (1,2-dioleoyl-sn-glycero-3-phosphoserine(sodium salt)), DPPA-NA (1,2-dipalmitoyl-sn-glycero-3-phosphate(sodium salt)), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPE (1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DPPG-NH4 (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DPPS-NA (1,2-dipalmitoyl-sn-glycero-3-phosphoserine(sodium salt)), DSPA-NA (1,2-distearoyl-sn-glycero-3-phosphate) (sodium salt)), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPG-NA (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DSPG-NH4 (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DSPS-NA (1,2-distearoyl-sn-glycero-3-phosphoserine (sodium salt)), EPC (egg PC), HEPC (hydrogenated egg PC), HSPC (hydrogenated soybean PC), LYSOPC MYRISTIC (1-myristoyl-sn-glycero-3-phosphocholine), LYSOPC PALMITIC (1-palmitoyl-sn-glycero-3-phosphocholine), LYSOPC STEARIC (1-stearoyl-sn-glycero-3-phosphocholine), milk sphingomyelin (MPPC, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC (1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) amine), POPG-NA (1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)](sodium salt)), PSPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SPPC (1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or any combination thereof.

[0204] C.PEGylated lipid As used herein, the term "PEGylated lipid" refers to any lipid modified (e.g., covalently attached) with at least one polyethylene glycol molecule. In some embodiments, the PEGylated lipid may comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (hereinafter referred to as DMG-PEG2000).

[0205] LNP composition In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one compound of the present disclosure, and at least one structured lipid.

[0206] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one compound of the present disclosure, and at least one PEGylated lipid.

[0207] In some embodiments, the at least one structured lipid is a mixture of two structured lipids.

[0208] In some embodiments, the at least one PEGylated lipid is a mixture of two PEGylated lipids.

[0209] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one compound of the present disclosure, at least one structured lipid, at least one PEGylated lipid, or any combination thereof.

[0210] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structured lipid, and at least one PEGylated lipid.

[0211] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one compound of the present disclosure, at least one structured lipid, at least one phospholipid, at least one PEGylated lipid, or any combination thereof.

[0212] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structured lipid, at least one phospholipid, and at least one PEGylated lipid.

[0213] In some embodiments, at least one compound of the present disclosure is a compound of Formulas (I)-(IV).

[0214] In some embodiments, at least one compound of the present disclosure is a mixture of two or more compounds of Formulas (I)-(IV).

[0215] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 54 mol% of at least one compound of Formula (I)-(IV), about 35 mol% of at least one structural lipid, about 10 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44 mol% to about 64 mol% of at least one compound of Formula (I)-(IV), about 25 mol% to about 45 mol% of at least one structural lipid, about 0.1 mol% to about 20 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 49 mol% to about 59 mol% of at least one compound of Formula (I)-(IV), about 30 mol% to about 40 mol% of at least one structured lipid, about 5 mol% to about 15 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid.

[0216] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 43.3 mol% of at least one compound of Formula (I)-(IV), about 43.3 mol% of at least one structural lipid, about 12 mol% of at least one phospholipid, and about 1.5 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 33.3 mol% to about 53.3 mol% of at least one compound of Formula (I)-(IV), about 33.3 mol% to about 53.3 mol% of at least one structural lipid, about 2 mol% to about 22 mol% of at least one phospholipid, and about 0.1 mol% to about 11.5 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 38.3 mol% to about 48.3 mol% of at least one compound of Formula (I)-(IV), about 38.3 mol% to about 48.3 mol% of at least one structured lipid, about 7 mol% to about 17 mol% of at least one phospholipid, and about 0.5 mol% to about 6.5 mol% of at least one PEGylated lipid.

[0217] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 33.5 mol% of at least one compound of Formula (I)-(IV), about 33.5 mol% of at least one structural lipid, about 32 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 23.5 mol% to about 43.5 mol% of at least one compound of Formula (I)-(IV), about 23.5 mol% to about 43.5 mol% of at least one structural lipid, about 22 mol% to about 42 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 28.5 mol% to about 38.5 mol% of at least one compound of Formula (I)-(IV), about 28.5 mol% to about 38.5 mol% of at least one structured lipid, about 27 mol% to about 37 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid.

[0218] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 49.6 mol% of at least one compound of Formula (I)-(IV), about 39.9 mol% of at least one structural lipid, about 9.5 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 39.6 mol% to about 59.6 mol% of at least one compound of Formula (I)-(IV), about 29.9 mol% to about 49.9 mol% of at least one structural lipid, about 0.1 mol% to about 19.5 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44.6 mol% to about 54.6 mol% of at least one compound of Formula (I)-(IV), about 34.9 mol% to about 44.9 mol% of at least one structural lipid, about 4.5 mol% to about 14.5 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid.

[0219] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 52.1 mol% of at least one compound of Formula (I)-(IV), about 45 mol% of at least one structural lipid, about 1.9 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 42.1 mol% to about 62.1 mol% of at least one compound of Formula (I)-(IV), about 35 mol% to about 55 mol% of at least one structural lipid, about 0.1 mol% to about 11.9 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 47.1 mol% to about 57.1 mol% of at least one compound of Formula (I)-(IV), about 40 mol% to about 50 mol% of at least one structured lipid, about 0.5 mol% to about 6.9 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid.

[0220] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 60 mol% of at least one compound of Formula (I)-(IV), about 30 mol% of at least one structural lipid, about 9 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 50 mol% to about 70 mol% of at least one compound of Formula (I)-(IV), about 20 mol% to about 40 mol% of at least one structural lipid, about 0.1 mol% to about 19 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 55 mol% to about 65 mol% of at least one compound of Formula (I)-(IV), about 25 mol% to about 35 mol% of at least one structured lipid, about 4 mol% to about 14 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid.

[0221] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 34 mol% to about 60 mol% of at least one compound of Formula (I)-(IV), about 30 mol% to about 60 mol% of at least one structured lipid, about 5 mol% to about 11.9 mol% of at least one phospholipid, and about 1 mol% to about 2 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 24 mol% to about 70 mol% of at least one compound of Formula (I)-(IV), about 20 mol% to about 70 mol% of at least one structured lipid, about 0.1 mol% to about 21.9 mol% of at least one phospholipid, and about 0.1 mol% to about 12 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 29 mol% to about 65 mol% of at least one compound of Formula (I)-(IV), about 25 mol% to about 65 mol% of at least one structured lipid, about 1 mol% to about 16.9 mol% of at least one phospholipid, and about 0.5 mol% to about 7 mol% of at least one PEGylated lipid.

[0222] In some embodiments, lipid nanoparticles containing at least one nucleic acid may comprise about 49.6 mol% to about 60 mol% of at least one compound of Formula (I)-(IV), about 30 mol% to about 45 mol% of at least one structured lipid, about 0.2 mol% to about 9.5 mol% of at least one phospholipid, and about 1 mol% to about 1.5 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 39.6 mol% to about 70 mol% of at least one compound of Formula (I)-(IV), about 20 mol% to about 55 mol% of at least one structured lipid, about 0.1 mol% to about 19.5 mol% of at least one phospholipid, and about 0.1 mol% to about 11.5 mol% of at least one PEGylated lipid. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44.6 mol% to about 65 mol% of at least one compound of Formula (I)-(IV), about 25 mol% to about 50 mol% of at least one structured lipid, about 0.1 mol% to about 14.5 mol% of at least one phospholipid, and about 0.5 mol% to about 6.5 mol% of at least one PEGylated lipid.

[0223] In some embodiments, the compound of Formulas (I)-(IV) included in the LNP composition is one of compounds 3, 6, 9, 12-16, and 19.

[0224] In some embodiments of the aforementioned LNPs, the structural lipid can be cholesterol. In some embodiments of the aforementioned LNPs, the phospholipid can be DOPE. In some embodiments of the aforementioned LNPs, the phospholipid can be DSPC. In some embodiments of the aforementioned LNPs, the phospholipid can be DOPC. In some embodiments of the aforementioned LNPs, the PEGylated lipid can be DMG-PEG2000.

[0225] In some embodiments of the aforementioned LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPE, and the PEGylated lipid can be DMG-PEG2000.

[0226] In some embodiments of the aforementioned LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPC, and the PEGylated lipid can be DMG-PEG2000.

[0227] In some embodiments of the aforementioned LNPs, at least one nucleic acid molecule is a DNA molecule. In one embodiment, at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, at least one DNA molecule is a DNA nanoplasmid.

[0228] In some embodiments, lipid nanoparticles containing at least one nucleic acid may contain at least one nucleic acid molecule. In some embodiments, lipid nanoparticles may contain multiple nucleic acid molecules. In some embodiments, at least one nucleic acid molecule or multiple nucleic acid molecules may be incorporated into the lipid nanoparticle.

[0229] In some embodiments, the lipid nanoparticles may comprise lipids and nucleic acids in a specific ratio (weight / weight).

[0230] In some embodiments, lipid nanoparticles comprising at least one nucleic acid have a lipid to nucleic acid 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 The lipid:nucleic acid (weight / weight) ratio may be 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.

[0231] In some embodiments, lipid nanoparticles comprising at least one nucleic acid have a lipid to nucleic acid 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. 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 lipid:nucleic acid (weight / weight).

[0232] In some embodiments, lipid nanoparticles comprising at least one nucleic acid may comprise lipid and nucleic acid in a lipid:nucleic acid (weight / weight) ratio of about 10:1, or about 25:1, or about 40:1.

[0233] In some embodiments of the aforementioned LNPs, at least one nucleic acid molecule is an RNA molecule. In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the mRNA molecule further comprises a 5'-CAP.

[0234] Thus, the present disclosure provides lipid nanoparticles comprising about 54 mol% of at least one compound of Formula (I)-(IV), about 35 mol% of at least one structured lipid, about 10 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44 mol% to about 64 mol% of at least one compound of Formula (I)-(IV), about 25 mol% to about 45 mol% of at least one structured lipid, about 0.1 mol% to about 20 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 49 mol% to about 59 mol% of at least one compound of Formula (I) to (IV), about 30 mol% to about 40 mol% of at least one structured lipid, about 5 mol% to about 15 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0235] In some embodiments, lipid nanoparticles are provided comprising about 43.3 mol% of at least one compound of Formula (I)-(IV), about 43.3 mol% of at least one structured lipid, about 12 mol% of at least one phospholipid, and about 1.5 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 33.3 mol% to about 53.3 mol% of at least one compound of Formula (I)-(IV), about 33.3 mol% to about 53.3 mol% of at least one structured lipid, about 2 mol% to about 22 mol% of at least one phospholipid, and about 0.1 mol% to about 11.5 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 38.3 mol% to about 48.3 mol% of at least one compound of Formula (I)-(IV), about 38.3 mol% to about 48.3 mol% of at least one structured lipid, about 7 mol% to about 17 mol% of at least one phospholipid, and about 0.5 mol% to about 6.5 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0236] In some embodiments, lipid nanoparticles are provided comprising about 33.5 mol% of at least one compound of Formula (I)-(IV), about 33.5 mol% of at least one structured lipid, about 32 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 23.5 mol% to about 43.5 mol% of at least one compound of Formula (I)-(IV), about 23.5 mol% to about 43.5 mol% of at least one structured lipid, about 22 mol% to about 42 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., mRNA molecule). In some embodiments, the present disclosure provides lipid nanoparticles comprising about 28.5 mol% to about 38.5 mol% of at least one compound of Formula (I) to (IV), about 28.5 mol% to about 38.5 mol% of at least one structured lipid, about 27 mol% to about 37 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid, wherein the lipid nanoparticles further comprise at least one RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 30:1 to about 50:1 (w / w) or about 35:1 to about 45:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 40:1 (w / w).

[0237] In some embodiments, the nucleic acid molecule is a DNA molecule. Thus, the present disclosure provides lipid nanoparticles comprising about 49.6 mol% of at least one compound of Formula (I)-(IV), about 39.9 mol% of at least one structured lipid, about 9.5 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 39.6 mol% to about 59.6 mol% of at least one compound of Formula (I)-(IV), about 29.9 mol% to about 49.9 mol% of at least one structured lipid, about 0.1 mol% to about 19.5 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44.6 mol% to about 54.6 mol% of at least one compound of Formula (I)-(IV), about 34.9 mol% to about 44.9 mol% of at least one structured lipid, about 4.5 mol% to about 14.5 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA nanoplasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w).

[0238] In some embodiments, lipid particles are provided comprising about 52.1 mol% of at least one compound of Formula (I)-(IV), about 45 mol% of at least one structured lipid, about 1.9 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 42.1 mol% to about 62.1 mol% of at least one compound of Formula (I)-(IV), about 35 mol% to about 55 mol% of at least one structured lipid, about 0.1 mol% to about 11.9 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 47.1 mol% to about 57.1 mol% of at least one compound of Formula (I)-(IV), about 40 mol% to about 50 mol% of at least one structured lipid, about 0.5 mol% to about 6.9 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA nanoplasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 100:1 (w / w).

[0239] In some embodiments, lipid particles are provided comprising about 60 mol% of at least one compound of Formula (I)-(IV), about 30 mol% of at least one structured lipid, about 9 mol% of at least one phospholipid, and about 1 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 50 mol% to about 70 mol% of at least one compound of Formula (I)-(IV), about 20 mol% to about 40 mol% of at least one structured lipid, about 0.1 mol% to about 19 mol% of at least one phospholipid, and about 0.1 mol% to about 11 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 55 mol% to about 65 mol% of at least one compound of Formula (I) to (IV), about 25 mol% to about 35 mol% of at least one structured lipid, about 4 mol% to about 14 mol% of at least one phospholipid, and about 0.5 mol% to about 6 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA nanoplasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 110:1 to about 130:1 (w / w), or about 115:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 120:1 (w / w).

[0240] In some embodiments, lipid particles are provided comprising about 34 mol% to about 60 mol% of at least one compound of Formula (I)-(IV), about 30 mol% to about 60 mol% of at least one structured lipid, about 5 mol% to about 11.9 mol% of at least one phospholipid, and about 1 mol% to about 2 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 24 mol% to about 70 mol% of at least one compound of Formula (I)-(IV), about 20 mol% to about 70 mol% of at least one structured lipid, about 0.1 mol% to about 21.9 mol% of at least one phospholipid, and about 0.1 mol% to about 12 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 29 mol% to about 65 mol% of at least one compound of Formula (I) to (IV), about 25 mol% to about 65 mol% of at least one structured lipid, about 1 mol% to about 16.9 mol% of at least one phospholipid, and about 0.5 mol% to about 7 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA nanoplasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 130:1 (w / w), or about 75:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w) to about 120:1 (w / w).

[0241] In some embodiments, lipid particles are provided comprising about 49.6 mol% to about 60 mol% of at least one compound of Formula (I)-(IV), about 30 mol% to about 45 mol% of at least one structured lipid, about 0.2 mol% to about 9.5 mol% of at least one phospholipid, and about 1 mol% to about 1.5 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 39.6 mol% to about 70 mol% of at least one compound of Formula (I)-(IV), about 20 mol% to about 55 mol% of at least one structured lipid, about 0.1 mol% to about 19.5 mol% of at least one phospholipid, and about 0.1 mol% to about 11.5 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the present disclosure provides lipid nanoparticles comprising about 44.6 mol% to about 65 mol% of at least one compound of Formula (I) to (IV), about 25 mol% to about 50 mol% of at least one structured lipid, about 0.1 mol% to about 14.5 mol% of at least one phospholipid, and about 0.5 mol% to about 6.5 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In one embodiment, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA nanoplasmid. In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 70:1 to about 130:1 (w / w), or about 75:1 to about 125:1 (w / w). In some embodiments, the lipid to nucleic acid ratio in the nanoparticles can be about 80:1 (w / w) to about 120:1 (w / w).

[0242] In some embodiments, the compound of Formulas (I)-(IV) included in the LNP composition is one of compounds 3, 6, 9, 12-16, and 19.

[0243] In some embodiments of the aforementioned LNPs, the structural lipid can be cholesterol. In some embodiments of the aforementioned LNPs, the phospholipid can be DOPE. In some embodiments of the aforementioned LNPs, the phospholipid can be DSPC. In some embodiments of the aforementioned LNPs, the phospholipid can be DOPC. In some embodiments of the aforementioned LNPs, the PEGylated lipid can be DMG-PEG2000.

[0244] In some embodiments of the aforementioned LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPE, and the PEGylated lipid can be DMG-PEG2000.

[0245] In some embodiments of the aforementioned LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPC, and the PEGylated lipid can be DMG-PEG2000.

[0246] Pharmaceutical Compositions of the Present Disclosure In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure. In some embodiments, 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, and the at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon. In some embodiments, the at least one nucleic acid molecule encoding the at least one transposase may be an RNA molecule (e.g., an mRNA molecule), and the at least one nucleic acid molecule encoding the at least one transposon may be a DNA molecule (e.g., a DoggyBone DNA molecule or a DNA nanoplasmid).

[0247] In some embodiments, the present disclosure provides a composition comprising at least one cell contacted by at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a composition comprising at least one cell genetically modified using at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a composition comprising at least one cell genetically modified using any of the methods of the present disclosure.

[0248] In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell contacted by at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using at least one nanoparticle of the present disclosure. In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one cell genetically modified using any of the methods of the present disclosure.

[0249] Methods of the present disclosure The present disclosure provides a method for 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 for 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.

[0250] In all of the methods, compositions, and kits of the present disclosure, at least one cell may be a hepatocyte. Hepatocytes may include, but are not limited to, hepatic parenchymal cells, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells. In all of the methods, compositions, and kits of the present disclosure, at least one cell may be a T cell. T cells may be resting T cells, activated T cells, stem memory T cells (TscM cells), central memory T cells (T CM ), or stem cell-like T cells.

[0251] In some aspects of any of the methods of the present disclosure, the cells 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.

[0252] The present disclosure provides a method for 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 for genetically modifying at least one cell, comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0253] In some embodiments, genetic modification of a cell can include delivering at least one exogenous nucleic acid to a cell, causing the cell to express at least one protein that the cell does not normally express, or causing the cell to express at least one protein at a level higher than the level at which the cell normally expresses at least one protein, or causing the cell to express at least one protein at a level lower than the level at which the cell normally expresses at least one protein. In some embodiments, genetic modification of a cell can include delivering at least one exogenous nucleic acid to a cell, causing the at least one exogenous nucleic acid to be integrated into the genome of the at least one cell.

[0254] In all methods of the present disclosure, T cells can be activated before, simultaneously with, or after contacting the T cells with at least one composition or at least one nanoparticle of the present disclosure. In some embodiments, T cells can be activated using standard techniques known in the art, including, but not limited to, contacting the T cells with a CD3 / CD28 / CD2 activator solution, anti-CD3 antibody beads, anti-CD28 antibody beads, anti-CD2 antibody beads, anti-CD3 and anti-CD28 antibody beads, a tetrameric antibody complex that binds to CD3, CD28, and CD2 cell surface ligands, or any combination thereof.

[0255] In some aspects, the 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 contact with at least one composition or nanoparticle of the present disclosure.

[0256] In some aspects, T cells can be activated after contact with at least one composition or nanoparticle of the present disclosure 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.

[0257] In some embodiments of the aforementioned 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).

[0258] In some embodiments of the aforementioned 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 before step b).

[0259] In some aspects, the methods of the present disclosure can produce 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 of the plurality of cells express at least one protein encoded by at least one nucleic acid delivered to the plurality of cells via a nanoparticle of the present disclosure.

[0260] In some aspects, the methods of the disclosure can produce 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 stem memory T cells.

[0261] In some aspects, the methods of the disclosure can produce 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 stem memory T cells (T SCM ) or express one or more cell surface markers of TscM-like cells, the one or more cell surface markers including CD62L and CD45RA.

[0262] The present disclosure provides methods of treating at least one disease in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0263] 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.

[0264] The present disclosure provides methods 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 with at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.The present disclosure provides methods 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 genetically modified using the compositions and / or methods of the present disclosure.

[0265] In some embodiments, the at least one disease may be a malignant disease, including but not limited to cancer. In some embodiments, the at least one disease may be a metabolic liver disease (MLD). In some embodiments, the at least one disease may be a urea cycle disorder (UCD). The MLD and / or UCD may include, but are not limited to, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), or any combination thereof.

[0266] In some embodiments, the at least one disease may be hemophilia A.

[0267] Thus, the present disclosure provides a method for treating hemophilia A in a subject in need thereof, the method 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.

[0268] The present disclosure provides a method for treating ornithine transcarbamylase (OTC) deficiency in a subject in need thereof, the method 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.

[0269] The present disclosure provides a method for treating methylmalonic acidemia (MMA) in a subject in need thereof, the method 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.

[0270] nucleic acid molecule In some embodiments, the nucleic acid molecule can be an RNA molecule.Therefore, in some embodiments, the lipid nanoparticle can comprise at least one RNA molecule.At least one RNA molecule can be encapsulated in the lipid nanoparticle.In some embodiments, the RNA molecule can be an mRNA molecule.In some embodiments, the lipid nanoparticle can comprise at least one mRNA molecule.The mRNA molecule can be encapsulated in the lipid nanoparticle.

[0271] In some embodiments, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some embodiments, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some embodiments, the non-naturally occurring nucleic acid molecule can contain 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 embodiments, the nucleic acid molecule can be a modified nucleic acid molecule. In some embodiments, the modified nucleic acid molecule can contain at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0272] In some embodiments, mRNA molecules can be capped using any method and / or capping moiety known in the art. mRNA molecules can be capped with an m7G(5')ppp(5')G moiety. The m7G(5')ppp(5')G moiety is also referred to herein as "Cap0." mRNA molecules can be capped with a CleanCap® moiety. The CleanCap® moiety can include an m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. The CleanCap® moiety can include an m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. mRNA molecules can be capped with an anti-reverse cap analog (ARCA®) moiety. The ARCA® moiety can include an m7(3'-O-methyl)G(5')ppp(5')G moiety. The mRNA molecule can be capped with a CleanCap® 3'OMe moiety (CleanCap® + ARCA®).

[0273] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0274] At least one modified nucleic acid may contain 5-methoxyuridine (5moU). In some embodiments, 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 the mRNA molecule are 5-methoxyuridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. Without being 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).

[0275] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0276] At least one modified nucleic acid is N1-methylpseudouridine (me 1Ψ). In some embodiments, 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 the mRNA molecule are N1-methylpseudouridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are N1-methylpseudouridine bases. Without wishing to be bound by theory, N1-methylpseudouridine can improve protein expression (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853).

[0277] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0278] At least one modified nucleic acid can comprise a pseudouridine ('Ψ). In some embodiments, 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 the mRNA are pseudouridine bases. In some embodiments, all of the uridine bases in the mRNA molecule are pseudouridine bases. Without being bound by theory, pseudouridine can improve protein expression and reduce immunogenicity (Li et al., Bioconjugate Chem. 2016, 27, 3, 849-853 and Vaidyanathan et al. Molecular Therapy-Nucleic Acids, 2018, 12, 530-542).

[0279] In some embodiments, the mRNA molecule may comprise at least one modified nucleic acid.

[0280] At least one modified nucleic acid may contain 5-methylcytidine (5-MeC). In some embodiments, 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 the mRNA molecule are 5-MeC bases. In some embodiments, all of the cytidine bases in the mRNA molecule are 5-MeC bases.

[0281] In some embodiments, the nucleic acid molecule may comprise a DNA molecule. Thus, in some embodiments, the lipid nanoparticle may comprise a DNA molecule. In some embodiments, the DNA molecule may be a circular DNA molecule, such as, but not limited to, a DNA plasmid or a DNA nanoplasmid. Thus, in some embodiments, the lipid nanoparticle may comprise a circular DNA molecule. In some embodiments, the lipid nanoparticle may comprise a Doggybone DNA molecule. In some embodiments, the lipid nanoparticle may comprise a DNA plasmid. In some embodiments, the lipid nanoparticle may comprise a DNA nanoplasmid. In some embodiments, the DNA molecule may be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0282] The DNA plasmid or DNA nanoplasmid is 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 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 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 It may be 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.

[0283] In some embodiments, the nucleic acid molecule incorporated into the lipid nanoparticles of the present disclosure may comprise at least one transgene sequence. In some embodiments, the transgene sequence may comprise a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transgene sequence may comprise a nucleotide sequence encoding at least one transposase. In some embodiments, the transgene sequence may comprise a nucleotide sequence encoding at least one transposon. In some embodiments, the transposon may comprise a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transposon may comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, wherein the at least one therapeutic protein is operably linked to at least one promoter sequence.

[0284] In some aspects, the 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.

[0285] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform, hi some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0286] In some embodiments, a microfluidic mixing platform can use a microfluidic device to combine a miscible solvent phase containing the lipid components of the nanoparticle with an aqueous phase containing the lipid nanoparticle cargo (e.g., nucleic acid, DNA, mRNA, etc.) to produce the lipid nanoparticles of the present disclosure. In some embodiments, 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 the microfluidic channel, microscopic features within the channel allow for controlled homogeneous mixing, which can produce the lipid nanoparticles of the present disclosure.

[0287] In some embodiments, the microfluidic mixing platform may include, but is not limited to, NanoAssembler® Spark (Precision NanoSystems), NanoAssembler® Ignite™ (Precision NanoSystems), NanoAssembler® Benchtop (Precision NanoSystems), NanoAssembler® Blaze (Precision NanoSystems), or NanoAssembler® GMP System (Precision NanoSystems).

[0288] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes at a flow 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.

[0289] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes a miscible solvent phase and an aqueous phase in a solvent:aqueous (v:v) 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 3:1, 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.

[0290] piggyBac ITR sequences In some embodiments, the nucleic acid may comprise a piggBac ITR sequence. In some embodiments, the nucleic acid may comprise a first piggyBac ITR sequence and a second piggBac ITR sequence.

[0291] In some embodiments, the piggyBac ITR sequences can include any piggyBac ITR sequences known in the art.

[0292] In some embodiments of the methods of the present disclosure, the piggyBac ITR sequences, such as the first piggyBac ITR sequence and / or the second piggyBac ITR sequence in an AAV piggyBac transposon, can comprise, consist essentially of, or consist of Sleeping Beauty transposon ITRs, Helraiser transposon ITRs, Tol2 transposon ITRs, TcBuster transposon ITRs, or any combination thereof.

[0293] Promoter sequence In some aspects, the nucleic acid may comprise a promoter sequence. In some aspects, the promoter sequence may comprise any promoter sequence known in the art. In some aspects, the promoter sequence may comprise any liver-specific promoter sequence known in the art.

[0294] In some embodiments, the promoter sequence may comprise a hybrid liver promoter (HLP). In some embodiments, the promoter sequence may comprise an LP1 promoter. In some embodiments, the promoter sequence may comprise a pp52 (LSP1) long promoter for leukocyte-specific expression. In some embodiments, the promoter sequence may comprise a thyroxine-binding globulin (TBG) promoter.

[0295] In some embodiments, the promoter sequence may comprise a wTBG promoter. In some embodiments, the promoter sequence may comprise a liver combinatorial bundle (HCB) promoter. In some embodiments, the promoter sequence may comprise a 2xApoE-hAAT promoter. In some embodiments, the promoter sequence may comprise a pp52 (LSP1) + chimeric intron promoter for leukocyte-specific expression. In some embodiments, the promoter sequence may comprise a cytomegalovirus (CMV) promoter.

[0296] Transgene sequence In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide. The MUT1 polypeptide may be any MUT1 polypeptide known in the art.

[0297] In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. The OTC polypeptide may be any OTC polypeptide known in the art.

[0298] In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding a factor VIII (FVIII) polypeptide. The FVIII polypeptide may be any FVIII polypeptide known in the art.

[0299] In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding an iCAS9 polypeptide.

[0300] In some embodiments, the transgene sequence may be codon optimized according to methods known in the art.

[0301] In some embodiments, at least one transgene sequence may be operably linked to at least one promoter sequence present in the same polynucleotide.

[0302] Poly(A) sequence In some embodiments, the nucleic acid can comprise a polyA sequence. In some embodiments, the polyA sequence can comprise any polyA sequence known in the art.

[0303] Self-cleaving peptide sequence In some embodiments, the nucleic acid may comprise a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence may comprise any self-cleaving peptide sequence known in the art. In some embodiments, the self-cleaving peptide sequence may comprise a 2A self-cleaving peptide sequence known in the art. Non-limiting examples of self-cleaving peptides include T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide.

[0304] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a T2A peptide.

[0305] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-T2A peptide.

[0306] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding an E2A peptide.

[0307] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-E2A peptide.

[0308] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding an F2A peptide.

[0309] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-F2A peptide.

[0310] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a P2A peptide.

[0311] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-P2A peptide.

[0312] Chimeric antigen receptor (CAR) The transgene sequence can include a nucleic acid sequence encoding a CAR, the CAR comprising 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.

[0313] The antigen recognition region may comprise at least one single-chain variable fragment (scFv), centrin, single-domain antibody, or a combination thereof. In some embodiments, at least one single-domain antibody is a VHH. In some embodiments, at least one single-domain antibody is a VH.

[0314] scFv In some embodiments, the antigen recognition region of the CAR can comprise one or more scFv compositions that recognize and bind to specific target proteins / antigens. The antigen recognition region can comprise at least two scFvs. The antigen recognition region can comprise at least three scFvs. In some embodiments, the CAR of the present disclosure is a bispecific CAR that comprises at least two scFvs that specifically bind to two different antigens.

[0315] An scFv composition can contain the heavy and light chain variable regions of an antibody. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, with the VH and VL domains linked by a short peptide linker. Despite the removal of the constant region and the introduction of the linker, the scFv can retain the specificity of the original immunoglobulin.

[0316] Sentinel In some embodiments, the antigen recognition region of the CAR may comprise one or more centirin compositions that recognize and bind to a specific target protein / antigen. Centirin, which specifically binds to an antigen, can be used to target the specificity of cells (e.g., cytotoxic immune cells) to a specific antigen. CARs containing centirin are referred to herein as CARTyrin.

[0317] The centilin of the present disclosure may comprise a protein scaffold, wherein the scaffold is capable of specifically binding to an antigen. The centilin of the present 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 to 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.

[0318] The consensus sequence can be modified at one or more positions within (a) the AB loop at positions 13-16 of the consensus sequence, (b) the BC loop at positions 22-28 of the consensus sequence, (c) the CD loop at positions 38-43 of the consensus sequence, (d) the DE loop at positions 51-54 of the consensus sequence, (e) the EF loop at positions 60-64 of the consensus sequence, (f) the FG loop at positions 75-81 of the consensus sequence, or (g) any combination of (a)-(f). The centrin of the present disclosure can comprise a consensus sequence of at least five fibronectin type III (FN3) domains, at least ten fibronectin type III (FN3) domains, or at least 15 fibronectin type III (FN3) domains.

[0319] The term "antibody mimetic" is intended to describe an organic compound that specifically binds to a target sequence and has a structure different from that of naturally occurring antibodies. Antibody mimetics may include proteins, nucleic acids, or small molecules. The target sequence to which the antibody mimetics of the present disclosure specifically bind may be an antigen. Antibody mimetics may offer superior properties over antibodies, including, but not limited to, superior solubility, tissue penetration, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower production costs. Exemplary antibody mimetics include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, and avimers (also known as avidity multimers), DARPins (designed ankyrin repeat proteins), finomers, Kunitz domain peptides, and monobodies.

[0320] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one affibody molecule. The affibody molecules of the present disclosure comprise a protein scaffold that comprises or consists of one or more α-helices that do not contain any disulfide bridges. Preferably, the affibody molecules of the present disclosure comprise or consist of three α-helices. For example, the affibody molecules of the present disclosure may comprise an immunoglobulin-binding domain. The affibody molecules of the present disclosure may comprise the Z domain of Protein A.

[0321] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one affilin molecule. Affilin molecules of the present disclosure include, for example, protein scaffolds produced by modifying exposed amino acids of either gamma-B crystallin or ubiquitin. Affilin molecules functionally mimic the affinity of antibodies for antigens, but do not structurally mimic antibodies. In any protein scaffold used to generate affilins, amino acids accessible for dissolution in a properly folded protein molecule or to potential binding partners 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.

[0322] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one Affimer molecule. Affimer molecules of the present disclosure comprise a protein scaffold comprising a highly stable protein engineered to display peptide loops that provide high-affinity binding sites for specific target sequences. Exemplary Affimer molecules of the present disclosure include a protein scaffold based on a cystatin protein or its tertiary structure. Exemplary Affimer molecules of the present disclosure may share a common tertiary structure comprising an α-helix positioned on top of an antiparallel β-sheet.

[0323] In some embodiments, the transgene sequence can include a nucleic acid sequence encoding at least one affitin molecule. The affitin molecules of the present disclosure include artificial protein scaffolds, the structure of which can be derived, for example, from DNA-binding proteins (e.g., the DNA-binding protein Sac7d). The affitins of the present disclosure selectively bind to a target sequence, which can be all or part of an antigen. Exemplary affitins of the present disclosure are produced by randomizing one or more amino acid sequences on the binding surface of a DNA-binding protein and subjecting the resulting protein to ribosome display and selection. The target sequence for the affitins of the present disclosure can be found, for example, in a genome or on the surface of a peptide, protein, virus, or bacterium. In some embodiments, the affitin molecules can be used as specific inhibitors of enzymes. The affitin molecules of the present disclosure can also include a heat-stable protein or a derivative thereof.

[0324] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one alphabody molecule. The alphabody molecules of the present disclosure are sometimes referred to as cell-permeable alphabodies (CPABs). The alphabody molecules of the present disclosure comprise small proteins (typically less than 10 kDa) that bind to a variety of target sequences (including antigens). The alphabody molecules are capable of reaching and binding to intracellular target sequences. Structurally, the alphabody molecules of the present disclosure comprise an artificial sequence that forms a single-stranded alpha helix (similar to a naturally occurring coiled-coil structure). The alphabody molecules of the present disclosure may comprise a protein scaffold comprising one or more amino acids modified to specifically bind to a target protein. Regardless of the molecule's binding specificity, the alphabody molecules of the present disclosure maintain correct folding and thermal stability.

[0325] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one anticalin molecule. The anticalin molecules of the present disclosure include artificial proteins that bind to target sequences or sites in either proteins or small molecules. The anticalin molecules of the present disclosure may also include artificial proteins derived from human lipocalins. The anticalin molecules of the present disclosure may be used, for example, in place of monoclonal antibodies or fragments thereof. The anticalin molecules may exhibit superior tissue penetration and thermal stability compared to monoclonal antibodies or fragments thereof. An exemplary anticalin molecule of the present disclosure may contain approximately 180 amino acids with a mass of approximately 20 kDa. Structurally, the anticalin molecules of the present disclosure comprise a barrel structure comprising antiparallel beta strands connected in pairs by loops and an attached alpha helix. In some embodiments, the anticalin molecules of the present disclosure comprise a barrel structure comprising eight antiparallel beta strands connected in pairs by loops and an attached alpha helix.

[0326] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one avimer molecule. The avimer molecules of the present disclosure include engineered proteins that specifically bind to a target sequence (which may be an antigen). The avimers of the present disclosure can recognize multiple binding sites within the same target or different targets. When the avimers of the present disclosure recognize two or more targets, they mimic the function of a bispecific antibody. The engineered avimer protein may comprise two or more peptide sequences, each of approximately 30-35 amino acids. These peptides may be linked via one or more linker peptides. The amino acid sequence of one or more peptides of the avimer may be derived from the A domain of a membrane receptor. Avimers have a rigid structure that may optionally include disulfide bonds and / or calcium. The avimers of the present disclosure may exhibit higher thermal stability compared to antibodies.

[0327] In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding at least one DARPin. DARPins (designed ankyrin repeat proteins) of the present disclosure comprise engineered, recombinant, or chimeric proteins with high specificity and high affinity for target sequences. In some embodiments, DARPins of the present disclosure are derived from ankyrin proteins and optionally comprise at least three repeat motifs (also called repeat structural units) of ankyrin proteins. Ankyrin proteins mediate high-affinity protein-protein interactions. DARPins of the present disclosure comprise a large target interaction surface.

[0328] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one Fynomer. The Fynomers of the present disclosure comprise small binding proteins (approximately 7 kDa) derived from the human Fyn SH3 domain and engineered to bind target sequences and molecules with affinity and specificity equivalent to antibodies.

[0329] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one Kunitz domain peptide. The Kunitz domain peptides of the present disclosure include protein scaffolds containing Kunitz domains. The Kunitz domains contain active sites for inhibiting protease activity. Structurally, the Kunitz domains of the present disclosure include a disulfide-rich α+β fold. This structure is exemplified by bovine pancreatic trypsin inhibitor. The Kunitz domain peptides recognize specific protein structures and act as competitive protease inhibitors. The Kunitz domains of the present disclosure may include ecallantide (derived from human lipoprotein-associated coagulation inhibitor (LACI)).

[0330] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding at least one monobody. The monobodies of the present disclosure are small proteins (containing approximately 94 amino acids and having a mass of approximately 10 kDa) comparable in size to single-chain antibodies. These engineered proteins specifically bind to target sequences, including antigens. The monobodies of the present disclosure may specifically target one or more distinct proteins or target sequences. In some embodiments, the monobodies of the present disclosure comprise a protein scaffold that mimics the structure of human fibronectin, more preferably the structure of the 10th extracellular type III domain of fibronectin. The 10th extracellular type III domain of fibronectin, as well as its monobody mimetics, contains seven beta sheets that form a barrel and three exposed loops on each side that correspond to the three complementarity-determining regions (CDRs) of an antibody. In contrast to the structure of an antibody variable domain, monobodies lack any binding sites for metal ions and a central disulfide bond. Multispecific monobodies can be optimized by modifying the BC and FG loops. Monobodies of the present disclosure may include adnectins.

[0331] VHH In some embodiments, the antigen recognition region of the CAR may comprise at least one single domain antibody (sdAb) that recognizes and binds to a specific target protein / antigen. In one embodiment, the single domain antibody is a VHH. VHHs are heavy chain antibodies found in camelids. VHHs that specifically bind to an antigen can be used to direct the specificity of cells (e.g., cytotoxic immune cells) to a specific antigen. The antigen recognition region may comprise at least two VHHs. The antigen recognition region may comprise at least three VHHs. In one embodiment, the CAR of the present disclosure is a bispecific CAR comprising at least two VHHs that specifically bind to two different antigens. A CAR comprising VHHs is referred to herein as a VCAR.

[0332] At least one VHH protein or VCAR of the present disclosure may optionally be produced by a cell line, mixed cell line, immortalized cell, or clonal population of immortalized cells, as known in the art (see, e.g., Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (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, NY (1997-2001).

[0333] Amino acids from a VHH protein can be modified, added and / or deleted to reduce immunogenicity or to decrease, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as is known in the art.

[0334] Optionally, VHH proteins can be engineered to retain high affinity for antigens and other favorable biological properties. To achieve this goal, VHH proteins can be optionally prepared by a process of analysis of parent sequences and various conceptual engineered products using three-dimensional models of the parent and engineered sequences. Three-dimensional models are publicly available and familiar to those skilled in the art. Computer programs are available that can illustrate and display the likely three-dimensional conformations of selected candidate sequences and measure their potential immunogenicity (e.g., the Immunofilter program from Xencor, Inc., Monrovia, Calif.). Inspection of these displays allows for analysis of the expected role of residues in the function of the candidate sequences, i.e., analysis of residues that affect the ability of the candidate VHH protein to bind to its antigen. In this way, residues can be selected and combined from parent and reference sequences to achieve desired characteristics, such as affinity for the target antigen. Alternatively or in addition to the above procedures, other suitable engineering methods can be used. Screening of VHHs for specific binding to similar proteins or fragments can be conveniently accomplished using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. Competition assays can be performed with the VHHs or VCARs of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the VHHs or VCARs of the present disclosure for binding to the target protein and / or share epitope regions. These assays, readily known to those skilled in the art, evaluate competition between antagonists or ligands for a limited number of binding sites on a protein.

[0335] VH In some embodiments, the antigen recognition region of the CAR may comprise at least one single domain antibody (sdAb) that recognizes and binds to a specific target protein / antigen. In one embodiment, the single domain antibody is a VH. The VH is a single domain binder derived from a common IgG. The VH that specifically binds to an antigen can be used to direct the specificity of cells (e.g., cytotoxic immune cells) to a specific antigen. The antigen recognition region may comprise at least two VHs. The antigen recognition region may comprise at least three VHs. In one embodiment, the CAR of the present disclosure is a bispecific CAR that comprises at least two VHs that specifically bind to two different antigens.

[0336] The VH can be isolated or derived from human sequences. The VH can comprise human CDR sequences and / or human framework sequences and non-human or humanized sequences (e.g., a rat Fc domain). In some embodiments, the VH is a fully humanized VH. In some embodiments, the VH is not a naturally occurring antibody or a fragment of a naturally occurring antibody. In some embodiments, the VH is not a fragment of a monoclonal antibody. In some embodiments, the VH is a UniDab antibody (TeneoBio). In some embodiments, the VH is modified to remove the Fc domain or a portion thereof. In some embodiments, the framework sequences of the VH are modified, for example, to improve expression, reduce immunogenicity, or improve function.

[0337] VHs can be fully engineered using the UniRat (TeneoBio) system and "NGS discovery" to produce VHs. Using this method, specific VHs are not naturally occurring and are generated using a fully engineered system. The VHs are not derived from naturally occurring monoclonal antibodies (mAbs), either isolated directly from a host (e.g., mouse, rat, or human) or from a single clone of a cell or cell line (hybridoma). These VHs were not subsequently cloned from the cell line. Instead, the VH sequences are fully engineered using the UniRat system as transgenes containing human variable regions (VH domains) with a rat Fe domain, and are therefore human / rat chimeras without light chains, distinct from the standard mAb format. The native rat gene has been knocked out, and the only antibodies expressed in rats are derived from transgenes with VH domains linked to Rat Fe (UniAbs). These are the exclusive Abs expressed in UniRat. Next-generation sequencing (NGS) and bioinformatics are used to identify the full antigen-specific repertoire of heavy chain antibodies produced by UniRat after immunization. A unique gene assembly method is then used to convert the antibody repertoire sequence information into a large collection of fully human heavy chain antibodies that can be screened in vitro for various functions. In some embodiments, fully humanized VHs are generated by fusing a human VH domain with a human Fc in vitro (generating a non-naturally occurring recombinant VH antibody). In some embodiments, VHs are fully humanized, but are expressed in vivo as human / rat chimeras (human VH, rat Fc) without light chains. Fully humanized VHs are expressed in vivo as human / rat chimeras (human VH, rat Fc) without light chains and are approximately 80 kDa (versus 150 kDa).

[0338] The CAR of this disclosure is K D But, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13M or less, 10 -14 M or less, and 10 -15 M or less. D can be determined by any means, including but not limited to, surface plasmon resonance.

[0339] In some embodiments, the antigen recognition region of the disclosed CARs comprises at least one anti-BCMA centirin. CARs comprising anti-BCMA centirin are referred to herein as BCMA CARTyrin.

[0340] In some aspects, the nanoparticles of the present disclosure may comprise a nucleic acid sequence encoding a BCMA CARTyrin.

[0341] In some embodiments, the antigen recognition region of the disclosed CARs comprises at least one anti-PSMA centirin. CARs comprising anti-PSMA centirin are referred to herein as PSMA CARTyrin.

[0342] In some aspects, the nanoparticles of the present disclosure can include a nucleic acid sequence encoding PSMA CARTyrin.

[0343] In one embodiment, the antigen recognition region of the disclosed CARs comprises at least one anti-BCMA VH. A CAR comprising an anti-BCMA VH is referred to herein as a BCMA VCAR.

[0344] In some embodiments, the nanoparticles of the present disclosure may comprise a nucleic acid sequence encoding a BCMA VCAR.

[0345] The ectodomain can comprise a signal peptide. The signal peptide can comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In a preferred embodiment, the signal peptide comprises, consists essentially of, or consists of human CD8 alpha (CD8α) signal peptide (SP), or a portion thereof.

[0346] The hinge domain or region may comprise a human CD8α, IgG4, CD4 sequence, or a combination thereof. In a preferred embodiment, the hinge may comprise, consist essentially of, or consist of a human CD8 alpha (CD8α) hinge or a portion thereof.

[0347] The transmembrane domain can comprise, consist essentially of, or consist of a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, 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 (CD8α) transmembrane domain or a portion thereof.

[0348] At least one costimulatory domain can comprise, consist essentially of, or consist of human 4-1BB, CD28, CD3 zeta (CD3ζ), CD40, ICOS, MyD88, OX-40 intracellular domain, or any combination thereof. Preferably, at least one costimulatory domain comprises a CD3ζ, 4-1BB costimulatory domain, or a combination thereof.

[0349] Transposition System In some embodiments, the nucleic acid may comprise a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or sequence encoding the first ITR; (b) a second ITR or sequence encoding the second ITR; and (c) an intra-ITR sequence or sequence encoding the intra-ITR, wherein the intra-ITR sequence may comprise the transposon sequence or sequence encoding the transposon.

[0350] In some embodiments, the nucleic acid may comprise a transposon or nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR; (b) a second ITR or a sequence encoding the second ITR; and (c) an intra-ITR sequence or an intra-ITR coding sequence, 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 an inter-ITR coding sequence, wherein the length of the inter-ITR sequence is 700 nucleotides or less.

[0351] The transposon or nanotransposon of the present disclosure comprises a protein scaffold (e.g., a CAR comprising at least one scFv, single domain antibody, or centilin). The transposon or nanotransposon may be a plasmid DNA transposon comprising a sequence encoding the protein scaffold (e.g., a CAR comprising at least one scFv, single domain antibody, or centilin) flanked by two cis-regulatory insulator sequences. The transposon or nanotransposon may further comprise 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.

[0352] The transposon or nanotransposon of the present disclosure may be a piggyBac™ (PB) transposon. In some embodiments, 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.

[0353] Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are described in detail in U.S. Patent Nos. 6,218,182, 6,962,810, 8,399,643, and WO 2010 / 099296.

[0354] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) on the ends of the transposon and insert their contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) or the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of the PB or PBL transposon are 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'-AA The PB or PBL transposon system can comprise or consist of AT-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 is not payload-restricted for the gene of interest that can be contained between the ITRs.

[0355] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, and 8,399,643. In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:1.

[0356] The PB or PBL transposase may comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 1. The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 1, in which the amino acid substitution at position 30 may be an isoleucine (I) to valine (V), the amino acid substitution at position 165 may be a glycine (G) to serine (S), the amino acid substitution at position 282 may be a methionine (M) to valine (V), and the amino acid substitution at position 538 may be an asparagine (N) to lysine (K).

[0357] In certain embodiments, where the transposase comprises the above-described mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases may also comprise mutations at positions 3, 46, 82, 103, 119, 125, 177, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 282, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1 It may further comprise an amino acid substitution at one or more of positions 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.

[0358] As described in more detail in International Publication No. WO 2019 / 173636 and International Application No. PCT / US2019 / 049816, the PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordata. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or the insect Bombyx mori (GenBank Accession No. BAD11135).

[0359] 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 embodiment, the 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 Nos. 6,218,185, 6,962,810, 8,399,643, and WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0360] In some embodiments, 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 integrates the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643 and WO 2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0361] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to nuclear localization signals are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and WO 2019 / 173636.

[0362] The transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Pat. No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0363] The transposon or nanotransposon of the present disclosure may be a Helraiser transposon. Exemplary Helraiser transposons include Helbat1. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO2019 / 173636).

[0364] The transposon or nanotransposon of the present disclosure may be a Tol2 transposon. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO 2019 / 173636).

[0365] The transposon or nanotransposon of the present disclosure may be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in WO 2019 / 173636). The TcBuster transposase may comprise or consist of naturally occurring or non-naturally occurring amino acids. The polynucleotide encoding the TcBuster transposase may comprise or consist of naturally occurring or non-naturally occurring nucleic acid sequences.

[0366] In some embodiments, the mutant TcBuster transposase comprises one or more sequence mutations when compared to a wild-type TcBuster transposase, as described in more detail in WO 2019 / 173636 and PCT / US2019 / 049816.

[0367] The cell delivery compositions (e.g., transposons) disclosed herein can include nucleic acid molecules encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in International Publication No. WO 2019 / 173636 and International Application No. PCT / US2019 / 049816.

[0368] Cells and modified cells of the present disclosure The cells and modified cells of the present disclosure may be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of the present disclosure may be immune cells. The immune cells of the present disclosure include lymphocyte progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (T SCM cells), central memory T cells (T CM ), 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.

[0369] Immune progenitor cells may include any cell that can differentiate into one or more types of immune cells. Immune progenitor cells may include pluripotent stem cells that can self-renew and become immune cells. Immune progenitor cells may include hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells may include progenitor cells that can become immune cells. Immune progenitor cells may include hematopoietic progenitor cells (HPCs).

[0370] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All blood cells differentiated from 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.

[0371] HSCs can be isolated or derived from primary or cultured stem cells. HSCs can be isolated or derived from embryonic stem cells, pluripotent stem cells, pluripotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0372] Immune progenitor cells can include HSCs or HSC progeny, including, but not limited to, pluripotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), B lymphocytes (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0373] HSCs produced by the disclosed methods can be isolated or derived from adult stem cells and, while committed to a single lineage, retain the characteristics of "primitive" stem cells that share the characteristics of embryonic stem cells. For example, "primitive" HSCs produced by the disclosed methods retain their "stemness" after division and do not differentiate. As a result, as adoptive cell therapy, "primitive" HSCs produced by the disclosed methods not only replenish their numbers but also expand in vivo. "Primitive" HSCs produced by the disclosed methods can be therapeutically effective when administered as a single dose.

[0374] 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-, CD90+, CD45RA- and CD49f+. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA- and CD49f+.

[0375] Primitive HSCs, HSCs, and / or HSC progeny can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). The modified primitive HSCs, modified HSCs, and / or modified HSC progeny can be forward differentiated to produce modified immune cells, including, but not limited to, modified T cells, modified natural killer cells, and / or modified B cells.

[0376] The modified immune cells or immune progenitor cells can be NK cells. NK cells can be cytotoxic lymphocytes differentiated from lymphocyte progenitor cells. The modified NK cells can be derived from modified hematopoietic stem progenitor cells (HSPCs) or modified HSCs. In some embodiments, the non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0377] The modified immune cells or immune precursor cells can be B cells. B cells are a type of lymphocyte that express a B cell receptor on the cell surface. The B cell receptor binds to a specific antigen. The modified B cells can be derived from modified hematopoietic stem progenitor cells (HSPCs) or modified HSCs.

[0378] The modified T cells of the present disclosure can be derived from modified hematopoietic stem progenitor cells (HSPCs) or modified HSCs. Unlike traditional biologics and chemotherapeutics, the disclosed modified T cells have the ability to rapidly regenerate upon antigen recognition, thereby potentially avoiding the need for repeated treatments. To accomplish this, in some embodiments, the modified T cells not only drive an initial response but also persist as a stable population of viable memory T cells in the patient, preventing the possibility of relapse. Alternatively, in some embodiments, the modified T cells do not persist in the patient if undesired.

[0379] Antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as early memory T cells, especially stem memory cells (T SCMIntensive efforts have been made to develop engineered T cell products containing stem cell-like T cells. The stem cell-like engineered T cells of the present disclosure have the greatest capacity for self-renewal as well as central memory (T CM ) T cells or T CM -like cells, effector memory (T EM ) and effector T cells (T E ), thereby resulting in better tumor eradication and long-term engraftment of modified T cells. A linear differentiation pathway leads to the differentiation of these cells, i.e., naive T cells (T N )>T SCM >T CM >T EM >T E >T TE In this case, T N is T SCM The parent progenitor cells that directly give rise to T CM The T cell compositions of the present disclosure directly generate one or more of each parental T cell subset, such as the most abundant T SCM may be included with cells (e.g., T SCM >T CM >T EM >T E >T TE ).

[0380] Immune progenitor cells include early memory T cells, stem cell-like T cells, and naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE The immune progenitor cells can be primitive HSCs, HSCs, or HSC progeny cells of the present disclosure. The immune cells can be early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE It could be.

[0381] The disclosed methods 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 therebetween, of a plurality of modified T cells in the population express one or more cell surface markers of early memory T cells. 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 can comprise a plurality of modified T cells. SCM The population of modified early memory T cells comprises multiple modified T CM Contains cells.

[0382] The disclosed methods can modify and / or produce a population of engineered 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 therebetween, of a plurality of engineered T cells in the population express one or more cell surface markers of stem cell-like T cells. A population of engineered stem cell-like T cells can include a plurality of engineered T cells. SCM The population of engineered stem cell-like T cells comprises a plurality of engineered T CM Contains cells.

[0383] In some embodiments, 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 therebetween, of the plurality of engineered T cells in the population are stem memory T cells (T SCM ) or T SCMThe cells express one or more cell surface markers of CD45RA-like cells, and the one or more cell surface markers may include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. The cell surface markers may include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0384] In some embodiments, 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 engineered T cells in the population are central memory T cells (T CM ) or T CM The cells express one or more cell surface markers of CD45RO-like cells, and the one or more cell surface markers include CD45RO and CD62L. The cell surface markers may include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0385] The disclosed methods can modify and / or produce a population of engineered 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 therebetween, of a plurality of engineered T cells in the population are naive T cells (T N ) The cell surface markers may include one or more of CD45RA, CCR7, and CD62L.

[0386] The disclosed methods can modify and / or produce a population of engineered 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 therebetween, of a plurality of engineered T cells in the population are effector T cells (engineered T EFF ) The cell surface markers may include one or more of CD45RA, CD95, and IL-2Rβ.

[0387] The disclosed methods can modify and / or produce a population of engineered 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 therebetween, of a plurality of engineered T cells of the population are stem cell-like T cells, stem memory T cells (T SCM ) or central memory T cells (T CM ) express one or more cell surface markers.

[0388] A plurality of the modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 a transgene or a sequence encoding a transgene, and at least 70%, at least 75%, at least 80%, at least 99.9%, at least 100% of the population of modified cells comprise a transgene or a sequence encoding a transgene (e.g., a CAR). 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 including CD34, or 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 markers including CD34 (e.g., comprising the cell surface marker phenotype CD34+).

[0389] A plurality of the modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 a transgene or a sequence encoding a transgene, and 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 comprise a transgene or a sequence encoding a transgene. 5%, 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% express one or more cell surface markers including CD34 and do not express one or more cell surface markers including CD38, or 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 markers including CD34 and do not express one or more cell surface markers including CD38 (e.g., comprising the cell surface marker phenotypes CD34+ and CD38-).

[0390] A plurality of the modified cells of the population comprise a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 a transgene or a sequence encoding a transgene, and 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% are CD34 and CD or 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 including CD34 and CD90 and do not express one or more cell surface markers including CD38 (e.g., comprising cell surface marker phenotypes CD34+, CD38-, and CD90+).

[0391] A plurality of the modified cells of the population comprise a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 a transgene or a sequence encoding a transgene, and 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% of the population of modified cells. , 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 cells are 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% ... and do not express one or more cell surface markers, including CD38 and CD45RA, or 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, including CD34 and CD90, and do not express one or more cell surface markers, including CD38 and CD45RA (e.g., comprising the cell surface marker phenotypes CD34+, CD38-, CD90+, CD45RA-).

[0392] A plurality of the modified cells of the population comprise a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 a transgene or a sequence encoding a transgene, and 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 100% of the modified cells of the population comprise a transgene or a sequence encoding a transgene (e.g., a CAR). , 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% express CD34, CD90 and CD49f. and do not express one or more cell surface markers including CD34, CD90, and CD49f, or 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 including CD34, CD90, and CD49f, and do not express one or more cell surface markers including CD38 and CD45RA (e.g., comprising cell surface marker phenotypes CD34+, CD38-, CD90+, CD45RA-, and CD49f+).

[0393] A plurality of the modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 a transgene or a sequence encoding a transgene, and at least 90% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene (e.g., a CAR). 1%, 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 1 0%, 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 including CD34 and CD90 and do not express one or more cell surface markers including CD45RA, or 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 including CD34 and CD90 and do not express one or more cell surface markers including CD45RA (e.g., comprising cell surface marker phenotypes CD34+, CD90+, and CD45RA-).

[0394] Compositions and methods for producing and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified T cells), and buffers for maintaining or enhancing cell viability and / or levels of stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells), are disclosed elsewhere herein and in more detail in U.S. Pat. No. 10,329,543 and WO 2019 / 173636.

[0395] The cells and modified cells of the present disclosure can be somatic cells. The cells and modified cells of the present disclosure can be differentiated cells. The cells and modified cells of the present disclosure can be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0396] Methods for expressing chimeric antigen receptors The present disclosure provides a method for expressing a CAR on the surface of a cell, the method comprising: (a) obtaining a cell population; (b) contacting the cell population with a composition of the disclosure comprising a CAR or a sequence encoding a CAR under conditions sufficient to translocate the CAR across the cell membrane of at least one cell in the cell population, thereby producing 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 expresses the CAR on its cell surface.

[0397] In some embodiments, the cell population may comprise leukocytes and / or CD4+ and CD8+ leukocytes. The cell population may comprise an optimized ratio of CD4+ and CD8+ leukocytes. The optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo. The cell population may comprise tumor cells.

[0398] In some embodiments, the conditions sufficient to translocate the CAR or CAR-encoding sequence, transposon, or vector across the cell membrane of at least one cell in the cell population comprise application of one or more electrical pulses at a designated voltage, a buffer, and at least one of one or more cofactors. In some embodiments, the conditions suitable for integration of the CAR-encoding sequence comprise at least one of a buffer and one or more cofactors.

[0399] The buffer may include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more supplemental factors may include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof, (b) salts, minerals, metabolites, or any combination thereof, (c) cell culture media, (d) inhibitors of cellular DNA sensing, metabolic, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof, and (e) one or more nucleic acid modifying or stabilizing reagents. Recombinant human cytokines, chemokines, interleukins or any combination thereof include IL2, IL7, IL12, IL15, IL21, ILI, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, ILI0, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-γ, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 The antibodies may include p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32β, IL-32γ, IL-33, LAP (TGF-β1), lymphotoxin-alpha / TNF-β, TGF-β, TNF-alpha, TRANCE / TNFSFI I / RANK L, or any combination thereof.The salts, minerals, metabolites, or any combination thereof may include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacers, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, crown-5, or any combination thereof. The cell culture medium may include 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. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or combinations thereof, include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, proinflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-ILIB, PBK, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), or any combination thereof. Examples of such inhibitors include bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.Reagents that modify or stabilize one or more nucleic acids include pH adjusters, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

[0400] The expansion and selection steps can occur simultaneously or sequentially. Expansion can occur prior to selection. Expansion can occur following selection, and optionally, expansion can be followed by a further (i.e., second) selection. Simultaneous expansion and selection can be co-timed. The expansion and / or selection steps can proceed over a period of 10-14 days, including the endpoint.

[0401] The expansion may include contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell via the CAR, thereby generating an expanded cell population. The antigen may be presented on the surface of a substrate. The substrate may have any form, including, but not limited to, a surface, a well, a bead or beads, and a matrix. The substrate may further include a paramagnetic or magnetic component. The antigen may be presented on the surface of a substrate, where the substrate is a magnetic bead, and a magnet can be used to remove or separate the magnetic bead from the modified and expanded cell population. The antigen may be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells may include, but are not limited to, tumor cells and stem cells.

[0402] In some embodiments in which 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 cells that express the selection gene as those that survive the selection and cells that do not express the selection gene as those that do not survive the selection step.

[0403] The present disclosure provides compositions comprising the modified, expanded, and selected cell populations of the methods described herein.

[0404] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in International Publication No. WO 2019 / 049816 and International Application No. PCT / US2019 / 049816.

[0405] The present disclosure provides a cell or population of cells, the cells including compositions 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 when the constructs of (a) and (b) are integrated into the genomic sequence of the cell, the exogenous receptor is expressed, and when the exogenous receptor binds to a ligand or antigen, it transduces an intracellular signal that directly or indirectly targets the inducible promoter, which modifies expression of the inducible transgene (a), thereby modifying gene expression.

[0406] The composition may alter gene expression by reducing gene expression. The composition may alter gene expression by transiently altering gene expression (e.g., while a ligand is bound to an exogenous receptor). The composition may alter gene expression acutely (e.g., when a ligand reversibly binds to an exogenous receptor). The composition may alter gene expression chronically (e.g., when a ligand is irreversibly bound to an exogenous receptor).

[0407] In some embodiments, the nucleic acid may comprise a transgene comprising a nucleic acid molecule encoding at least one exogenous receptor. The exogenous receptor may comprise an endogenous receptor in relation 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.

[0408] The exogenous receptor may include a non-naturally occurring receptor. The non-naturally occurring receptor may be a synthetic receptor, a modified receptor, a recombinant receptor, a mutant receptor, or a chimeric receptor. The non-naturally occurring receptor may include one or more sequences isolated or derived from a T cell receptor (TCR). The non-naturally occurring receptor may include one or more sequences isolated or derived from a scaffold protein. In some embodiments, including those in which the non-naturally occurring receptor does not include a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound, and / or intracellular receptor that transduces an intracellular signal following contact with the non-naturally occurring receptor. The non-naturally occurring receptor may include a transmembrane domain. The non-naturally occurring receptor may interact with an intracellular receptor that transduces an intracellular signal. The non-naturally occurring receptor may include an intracellular signaling domain. The non-naturally occurring receptor may be a chimeric ligand receptor (CLR). The CLR may be a chimeric antigen receptor (CAR).

[0409] The sequence encoding the inducible promoter includes a sequence encoding the NFK13 promoter, a sequence encoding an interferon (IFN) promoter, or a sequence encoding an interleukin-2 promoter. In some embodiments, the IFN promoter is an IFNγ promoter. The inducible promoter can be isolated or derived from a cytokine or chemokine promoter. The cytokine or chemokine can include IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor beta (TGFβ), colony-stimulating factor 2 (GM-CSF), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), LTα, perforin, granzyme C (Gzmc), granzyme B (Gzmb), CC-C motif chemokine ligand 5 (CCL5), CC-C motif chemokine ligand 4 (Ccl4), CC-C motif chemokine ligand 3 (Ccl3), XC motif chemokine ligand 1 (Xcl1), or LIF interleukin 6 family cytokine (Lif).

[0410] Inducible promoters can be isolated from or derived from promoters of genes including surface proteins involved in cell differentiation, activation, exhaustion, and function, hi some embodiments, the genes include CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0411] The inducible promoter can be isolated from or derived from the promoters of genes involved in CD metabolism and differentiation, such as Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6, and Neto2.

[0412] In some embodiments, the inducible transgene construct comprises or promotes expression of downstream signaling components of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapy, and oncogenes or tumor suppressor genes, non-limiting examples of which are disclosed in WO 2019 / 173636 and PCT / US2019 / 049816.

[0413] armed cells The modified cells (e.g., CART cells) of the present disclosure can be further modified to enhance their therapeutic potential. Alternatively, or in addition, the modified cells can be further modified to reduce their sensitivity to immune and / or metabolic checkpoints. This type of modification "arms" the cells, and after modification, they can be referred to herein as "armed" cells (e.g., armed T cells). Armed cells can be produced, for example, by blocking and / or attenuating certain checkpoint signals (e.g., checkpoint inhibition) that are naturally delivered to cells within the tumor immunosuppressive microenvironment.

[0414] The armed cells of the present disclosure can be derived from any cell, such as T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells (including T cells isolated or derived from G-CSF-mobilized peripheral blood), or umbilical cord blood (UCB)-derived T cells. Armed cells (e.g., armed T cells) can comprise one or more of chimeric ligand receptors (CLRs comprising a protein scaffold, antibody, scFv, or antibody mimetic) / chimeric antigen receptors (CARs comprising a protein scaffold, antibody, scFv, or antibody mimetic), CARTyrin (CARs comprising centrin), and / or VCARs (CARs comprising camelid VHHs or single-domain VHs). Armed cells (e.g., armed T cells) can comprise an inducible pro-apoptotic polypeptide disclosed herein. Armed cells (e.g., armed T cells) can comprise exogenous sequences. The exogenous sequences can include sequences encoding therapeutic proteins. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, cell surface-associated, or secreted proteins. Exemplary therapeutic proteins expressed by armed cells (e.g., armed T cells) may modify the activity of the armed cell or may modify the activity of a second cell. Armed cells (e.g., armed T cells) may contain a selection gene or selection marker. Armed cells (e.g., armed T cells) may contain a synthetic gene expression cassette (also referred to herein as an inducible transgene construct).

[0415] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce the expression of one or more genes encoding receptors for inhibitory checkpoint signals to produce armed cells (e.g., armed CAR T cells). Receptors for inhibitory checkpoint signals are expressed on the cell surface or in the cytoplasm of the cell. Silencing or reducing the expression of genes encoding receptors for inhibitory checkpoint signals results in loss of protein expression of the inhibitory checkpoint receptor on the surface or in the cytoplasm of the armed cells. Thus, armed cells in which the expression of one or more genes encoding inhibitory checkpoint receptors is silenced or reduced are resistant, non-receptive, or insensitive to checkpoint signals. The reduced resistance or sensitivity of armed cells to inhibitory checkpoint signals enhances the therapeutic potential of the armed cells in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immunosuppression) are disclosed in WO 2019 / 173636. Preferred examples of inhibitory checkpoint signals that can be halted include, but are not limited to, PD-1 and TGFβRII.

[0416] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce expression of one or more genes encoding intracellular proteins involved in checkpoint signaling to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells can be enhanced by targeting any intracellular signaling protein involved in a checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference with one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in WO 2019 / 173636.

[0417] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce the expression of one or more genes encoding transcription factors that interfere with the effectiveness of a therapy to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells can be enhanced or modified by silencing or reducing the expression (or inhibiting the function) of a transcription factor that interferes with the effectiveness of a therapy. Non-limiting examples of transcription factors that can be modified to silence or reduce the expression or inhibit the function include, but are not limited to, the exemplary transcription factors disclosed in WO 2019 / 173636.

[0418] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce the expression of one or more genes encoding cell death or cell apoptosis receptors to produce armed cells (e.g., armed CAR T cells). The interaction of a cell death receptor with its endogenous ligand results in the initiation of apoptosis. Disruption of the expression, activity, or interaction of a cell death and / or cell apoptosis receptor and / or ligand makes the modified cell less susceptible to death signals, thereby making the armed cell more effective in the tumor environment. Non-limiting examples of cell death and / or cell apoptosis receptors and ligands are disclosed in WO 2019 / 173636. A preferred example of a cell death receptor that can be modified is Fas (CD95).

[0419] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce the expression of one or more genes encoding metabolic sensing proteins to produce armed cells (e.g., armed CAR T cells). Disruption of metabolic sensing of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose, and other molecules) by the modified cells results in prolonged retention of T cell function, resulting in the death of more tumor cells per cell. Non-limiting examples of metabolic sensing genes and proteins are disclosed in WO 2019 / 173636. Preferred examples, HIF1a and VHL, play a role in T cell function even in hypoxic environments. Armed T cells may have silenced or reduced expression of one or more genes encoding HIF1a or VHL.

[0420] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce expression of one or more genes encoding proteins that confer sensitivity to a cancer therapy, such as a monoclonal antibody, to produce armed cells (e.g., armed CAR T cells). Armed cells can thus function in the presence of a cancer therapy (e.g., chemotherapy, monoclonal antibody therapy, or another anti-tumor therapy) and may exhibit superior function or efficacy. Non-limiting examples of proteins involved in conferring sensitivity to a cancer therapy are disclosed in WO 2019 / 173636.

[0421] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to silence or reduce expression of one or more genes encoding growth advantage factors to produce armed cells (e.g., armed CART cells). Silencing or reducing expression of an oncogene can confer a growth advantage to the cells. For example, silencing or reducing (e.g., disrupting expression of) the TET2 gene during the CART cell manufacturing process results in the generation of armed CAR T cells with significant growth capacity and subsequent eradication of tumors when compared to disarmed CAR T cells that lack this growth capacity. This strategy can be coupled to a safety switch (e.g., the iC9 safety switch described herein) to allow for targeted destruction of armed CART cells in the event of an adverse reaction from the subject or uncontrolled growth of the armed CART cells. Non-limiting examples of growth advantage factors are disclosed in WO 2019 / 173636.

[0422] The modified cells (e.g., CAR T cells) of the present disclosure can be further modified to express modified / chimeric checkpoint receptors to produce armed T cells of the present disclosure.

[0423] The modified / chimeric checkpoint receptor may include a null receptor, a decoy receptor, or a dominant-negative receptor. The null receptor, decoy receptor, or dominant-negative receptor may be a modified / chimeric receptor / protein. The null receptor, decoy receptor, or dominant-negative receptor may be truncated to express the intracellular signaling domain. Alternatively, or in addition, the null receptor, decoy receptor, or dominant-negative receptor may be mutated at one or more amino acid positions within the intracellular signaling domain that are critical or required for effective signal transduction. Truncation or mutation of the null receptor, decoy receptor, or dominant-negative receptor may result in the loss of the receptor's ability to transmit or transduce a checkpoint signal into or within a cell.

[0424] For example, dilution or blocking of immunosuppressive checkpoint signals derived from PD-L1 receptors expressed on the surface of tumor cells can be achieved by expressing an engineered / chimeric PD-1 null receptor on the surface of armed cells (e.g., armed CAR T cells), which effectively competes with endogenous (unmodified) PD-1 receptors also expressed on the surface of the armed cells, reducing or inhibiting the transmission of immunosuppressive checkpoint signals through the armed cells' endogenous PD-1 receptors. In this non-limiting example, competition between the two different receptors for binding to PD-L1 expressed on tumor cells reduces or diminishes the level of effective checkpoint signaling, thereby enhancing the therapeutic potential of armed cells expressing PD-1 null receptors.

[0425] The engineered / chimeric checkpoint receptor may include a null receptor, a decoy receptor, or a dominant-negative receptor that is a transmembrane receptor, a membrane-bound or membrane-linked receptor / protein, or an intracellular receptor / protein. Exemplary null, decoy, or dominant-negative intracellular receptor / protein include, but are not limited to, downstream signaling components of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapy, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in WO 2019 / 173636.

[0426] The engineered / chimeric checkpoint receptor may include a switch receptor. Exemplary switch receptors include engineered / chimeric receptors / proteins in which a native or wild-type intracellular signaling domain has been switched or replaced with a different intracellular signaling domain that is non-native to the protein and / or is not the wild-type domain. For example, replacing an inhibitory signaling domain with a stimulatory signaling domain switches an immunosuppressive signal to an immunostimulatory signal. Alternatively, replacing 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 may result in dilution and / or blockage of a cognate checkpoint signal through competition with endogenous wild-type checkpoint receptors (not the switch receptor) for binding to cognate checkpoint receptors expressed within the immunosuppressive tumor microenvironment. Armed cells (e.g., armed CAR T cells) can include a sequence encoding a switch receptor, resulting in the expression of one or more switch receptors, thereby altering the activity of the armed cell. Armed cells (e.g., armed CAR T cells) can express switch receptors that target checkpoint receptors, transcription factors, cytokine receptors, death receptors, metabolic sensing molecules, cancer therapeutics, oncogenes, and / or intracellularly expressed proteins downstream of tumor suppressor proteins or genes.

[0427] Exemplary switch receptors may include or be derived from proteins including, but not limited to, signaling components downstream of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapy, and oncogenes or tumor suppressor genes.

[0428] The engineered cells (e.g., CAR T cells) of the present disclosure can be further engineered to express a CLR / CAR that mediates conditional gene expression to produce armed T cells. The combination of the CLR / CAR and conditional gene expression system in the nucleus of the armed T cell constitutes a synthetic gene expression system that is conditionally activated upon binding of the cognate ligand to the CLR or the cognate antigen to the CAR. This system can serve to enhance the "arming" or therapeutic potential of the engineered T cell, for example, by reducing or limiting synthetic gene expression at or within the ligand or antigen binding site, the tumor environment, or within it.

[0429] The present disclosure provides a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition can include nanoparticles comprising a nucleic acid, wherein the nucleic acid includes a sequence encoding a DNA-binding domain and a sequence encoding a nuclease protein or its nuclease domain. The sequence encoding the nuclease protein or its nuclease domain can include a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain can include one or more of a CRISPR / Cas protein, a TAL effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0430] The nuclease or nuclease domain thereof may comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease may comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition may comprise a fusion protein. The fusion protein may comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition may further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0431] The transgene can include a nucleic acid sequence encoding a mini-Cas9 (Cas9) operably linked to an effector. The present disclosure provides fusion proteins comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a mini-Cas9 (Cas9). The mini-Cas9 constructs of the present disclosure can include an effector that comprises a Type IIS endonuclease.

[0432] The transgene can include a nucleic acid sequence encoding an inactivated small Cas9 (dSaCas9) operably linked to an effector. The transgene can include a nucleic acid sequence encoding a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, where the effector comprises a small inactivated Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) construct of the present disclosure can include an effector that comprises a type IIS endonuclease.

[0433] The transgene can include a nucleic acid sequence encoding an inactivated Cas9 (dCas9) operably linked to an effector. The transgene can include a nucleic acid sequence encoding a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, where the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) construct of the present disclosure can include an effector comprising a type IIS endonuclease.

[0434] dCas9 can be isolated or derived from Streptococcus pyogenes. dCas9 can include dCas9 with substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A.

[0435] The cell containing the gene editing composition can stably or transiently express the gene editing composition. Preferably, the gene editing composition is transiently expressed. The guide RNA can include a sequence complementary to a target sequence in the genomic DNA sequence. The target sequence in the genomic DNA sequence can be a target sequence in a safe harbor site of the genomic DNA sequence.

[0436] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in U.S. Patent Application Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and U.S. Patent No. 10,415,024.

[0437] Chimeric stimulatory receptors and recombinant HLA-E polypeptides An adoptive cell composition that is "universally" safe for administration to any patient requires a significant reduction or elimination of alloreactivity. To this end, the cells (e.g., allogeneic cells) of the present disclosure can be modified to disrupt the expression or function of T cell receptors (TCRs) and / or major histocompatibility complex (MHC) classes. TCRs mediate graft-versus-host (GvH) reactions, while MHCs mediate host-versus-graft (HvG) reactions. In preferred embodiments, any expression and / or function of TCRs is eliminated to prevent T cell-mediated GvH, which can cause death in the subject. Thus, in preferred embodiments, the present disclosure provides pure TCR-negative allogeneic I cell compositions (e.g., each cell of the composition expresses TCRs at such low levels that they are either undetectable or absent).

[0438] The 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, thereby improving cell engraftment in a subject. Improved engraftment results in longer cell persistence and therefore a wider therapeutic window in a subject. Specifically, the expression and / or function of beta-2-microglobulin (B2M), a structural component of MHC-I, is reduced or eliminated.

[0439] The above strategies pose additional challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), which is part of the TCR complex. Loss of CD3ζ in TCR-KO T cells dramatically reduces the ability of these cells to be optimally activated and proliferated using standard stimulating / activating reagents, including, but not limited to, agonistic anti-CD3 mAbs. Disruption of the expression or function of any one component of the TCR complex results in the loss of all components of the complex, including TCR-alpha (TCRα), TCR-beta (TCRβ), CD3-gamma (CD3γ), CD3-epsilon (CD3ε), CD3-delta (CD3δ), and CD3-zeta (CD3ζ). Both CD3ε and CD3ζ are required for T cell activation and proliferation. Agonistic anti-CD3 mAbs typically recognize CD3ε and possibly another protein within the complex, which then signal to CD3ζ. CD3ζ provides the primary stimulus (together with a secondary costimulatory signal) for T cell activation for optimal activation and proliferation. Under normal conditions, full T cell activation depends on engagement of the TCR in conjunction with a second signal mediated by one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL) that boosts the immune response. However, in the absence of the TCR, T cell proliferation is significantly reduced when stimulated using standard activation / stimulation reagents containing agonist anti-CD3 mAbs. In fact, T cell proliferation is reduced by only 20–40% of normal proliferation levels when stimulated using standard activation / stimulation reagents containing agonist anti-CD3 mAbs.

[0440] Accordingly, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising an activating moiety, wherein the activating moiety is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[0441] In some aspects, the transgene sequence may comprise a nucleic acid sequence encoding a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising an 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 signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[0442] The activating component may 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 activating component binds. The activating component may comprise a CD2 extracellular domain or a portion thereof to which an agonist binds.

[0443] The signaling domain may comprise one or more of a human signaling domain component, 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. The signaling domain may comprise a CD3 protein or a portion thereof. The CD3 protein may comprise a CD3 zeta protein or a portion thereof.

[0444] The endodomain may further comprise a cytoplasmic domain. The cytoplasmic domain may be isolated or derived from a third protein. The first protein and the third protein may be the same. The ectodomain may further comprise a signal peptide. The signal peptide may be derived from a fourth protein. The first protein and the fourth protein may be the same. The transmembrane domain may be isolated or derived from a fifth protein. The first protein and the fifth protein may be the same.

[0445] In some embodiments, the activating component does not bind to a naturally occurring molecule. In some embodiments, the activating component binds to a naturally occurring molecule, but CSR does not transduce a signal upon binding of the activating component to a naturally occurring molecule. In some embodiments, the activating component binds to a non-naturally occurring molecule. In some embodiments, the activating component does not bind to a naturally occurring molecule, but binds to a non-naturally occurring molecule. CSR can selectively transmit a signal when the activating component binds to a non-naturally occurring molecule.

[0446] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation moiety, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, and the activation moiety 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 signaling domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and the at least one signaling domain comprises a CD3 zeta protein or a portion thereof.

[0447] The present disclosure also provides non-naturally occurring chimeric stimulating receptors (CSRs) whose ectodomains comprise modifications. The modifications can include mutations or truncations in the amino acid sequence of the activating component or first protein compared to the wild-type sequence of the activating component or first protein. The mutations or truncations in the amino acid sequence of the activating component can include mutations or truncations in the CD2 extracellular domain or a portion thereof to which an agonist binds. Mutations or truncations in the CD2 extracellular domain can reduce or eliminate binding to naturally occurring CD58.

[0448] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation moiety, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, and the activation moiety comprises an agonist-binding CD2 extracellular domain or a portion thereof, wherein the agonist-binding CD2 extracellular domain or portion thereof comprises a mutation or truncation; (b) a transmembrane domain, wherein the transmembrane domain comprises the CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signaling domain, wherein the cytoplasmic domain comprises the CD2 cytoplasmic domain or a portion thereof, and the at least one signaling domain comprises a CD3ζ protein or a portion thereof.

[0449] The present disclosure provides a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a transposon or vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0450] The present disclosure provides a cell comprising any of the CSRs disclosed herein.The present disclosure provides a cell comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0451] The modified cells disclosed herein can be allogeneic or autologous cells. In some preferred embodiments, the modified cells are allogeneic cells. In some embodiments, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0452] The present disclosure provides a composition comprising any of the CSRs disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs 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.

[0453] The present disclosure provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), where the modification reduces or eliminates the level of expression or activity of the TCR; and (b) a chimeric stimulating receptor (CSR), where the CSR comprises: (i) an ectodomain comprising an activating component, where the activating component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signaling domain, where the at least one signaling domain is isolated or derived from a second protein, where the first protein and the second protein are not identical.

[0454] The modified T cells can further comprise an inducible pro-apoptotic polypeptide. The modified T cells can further comprise a modification of the endogenous sequence encoding beta-2-microglobulin (B2M), which reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

[0455] The engineered T cell may 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 an HLA-E polypeptide may further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a linker, the linker being positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising HLA-E may 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 HLA-E.

[0456] The engineered T cells may further comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor may comprise a chimeric antigen receptor (CAR).

[0457] The CSR may be transiently expressed in the modified T cells. The CSR may be stably expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be transiently expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide may be stably expressed in the modified T cells. An inducible pro-apoptotic polypeptide may be transiently expressed in the modified T cells. An inducible pro-apoptotic polypeptide may be stably expressed in the modified T cells. A sequence encoding a non-naturally occurring antigen receptor or therapeutic protein may be transiently expressed in the modified T cells. A sequence encoding a non-naturally occurring antigen receptor or therapeutic protein may be stably expressed in the modified T cells.

[0458] As described in detail herein, gene editing compositions, including but not limited to RNA-guided fusion proteins comprising dCas9-Clo051, can be used to target and reduce or eliminate the expression of endogenous T cell receptors. In a preferred embodiment, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (such as a promoter) of a gene encoding an endogenous T cell receptor. Non-limiting examples of primers (including T7 promoters, genomic target sequences, and gRNA scaffolds) for generating guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-α), TCR-beta (TCR-β), and beta-2-microglobulin (β2M) are disclosed in International Application PCT / US2019 / 049816.

[0459] Gene editing compositions, including but not limited to RNA-guided fusion proteins comprising dCas9-Clo051, can be used to target and reduce or eliminate the expression of endogenous MHC1, MHCII, or MHC activators. In a preferred embodiment, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (such as a promoter) of a gene encoding one or more components of endogenous MHC1, MHCII, or MHC activators. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in International Application PCT / US2019 / 049816.

[0460] A detailed description of genetic modifications of endogenous sequences encoding non-naturally occurring chimeric stimulating receptors, TCR-alpha (TCR-α), TCR-beta (TCR-β), and / or beta-2-microglobulin (β2M), and non-naturally occurring polypeptides, including HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptides, is disclosed in International Application PCT / US2019 / 049816.

[0461] Formulation, dosage and mode of administration The present disclosure provides formulations, dosages and methods for administration of the compositions described herein.

[0462] The disclosed compositions and pharmaceutical compositions may further comprise any suitable auxiliary agent, such as, but not limited to, at least one of a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of and methods for preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers can be routinely selected to be appropriate for the mode of administration, solubility, and / or stability of the composition, as is well known in the art or as described herein.

[0463] 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").

[0464] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acid, esterified sugars, and polysaccharides or sugar polymers), which can be present alone or in combination and comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, e.g., human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0465] The composition may also contain a buffer or pH adjuster. Typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts such as citrate.

[0466] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracavity, intracelial, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or intravaginal means.

[0467] The compositions of the present disclosure can be administered parenterally (subcutaneously, intramuscularly, or intravenously) or for any other administration, particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration, particularly in semi-solid form, 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, in the form of powders, nasal drops, or aerosols or specific medications; or transdermally, such as, but not limited to, in the form of gels, ointments, lotions, suspensions, or patch delivery systems containing chemical enhancers such as dimethyl sulfoxide to either modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D.S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994,) or the application of an electric field to create a transient transport pathway, such as by electroporation, or to increase the mobility of charged drugs through the skin, such as by iontophoresis, or the application of ultrasound, such as by sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entireties).

[0468] For parenteral administration, any composition disclosed herein may be formulated with a pharmaceutically acceptable parenteral vehicle, or provided separately, as a solution, suspension, emulsion, particle, powder, or lyophilized powder. Parenteral formulations may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of plant origin, hydrogenated naphthalenes, and the like. Aqueous or oily suspensions for injection can be prepared according to known methods using appropriate emulsifiers or wetting agents and suspending agents. Injectable preparations may be non-toxic parenterally administrable diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Acceptable vehicles or solvents include water, Ringer's solution, isotonic saline, and the like, and sterile fixed oils can be used as common solvents or suspension media. For these purposes, any type of fixed oil and fatty acid can be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids, and natural, synthetic, or semi-synthetic mono-, di-, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas-pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446.

[0469] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in particle sizes effective to reach the lungs or sinuses of the lower respiratory tract. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, capable of depositing aerosolized formulations in a patient's sinus cavities or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, sprayers, and the like. All such devices can employ formulations suitable for administration of the compositions or pharmaceutical compositions described herein for dispensing in an aerosol. Such aerosols can be composed of either solutions (both aqueous and non-aqueous) or solid particles. Furthermore, aerosols containing the compositions or pharmaceutical compositions described herein can be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In metered-dose inhalers (MDIs), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations and associated devices is disclosed in WO 2019 / 049816.

[0470] For absorption via mucosal surfaces, the composition comprises an emulsion comprising a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption via the mucosal surface by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present disclosure may include the corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration may be solid and may contain excipients such as lactose, or may be aqueous or oily solution nasal sprays. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in WO 2019 / 049816.

[0471] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device such as a liposome or polymer nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise specified). Several suitable devices are known, including microparticles made from synthetic polymers such as polyhydroxy acids, e.g., polylactic acid, polyglycolic acid and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in International Publication No. WO 2019 / 049816.

[0472] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, for example, for a period of one week to one year with a single administration. A variety of sustained-release, depot, or implant dosage forms are available.

[0473] Suitable dosage is well known in the art.See, for example, 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, California (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, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value, or fraction thereof, or can achieve a serum concentration of about 0.1-5000 μg / mL serum concentration, or any range, value, or fraction thereof, per single or multiple administrations. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg to about 3, about 6, or about 12 mg / kg of subject body weight.

[0474] Alternatively, the administered dose may vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, type of concurrent treatment, frequency of treatment, and the desired effect.

[0475] By way of non-limiting example, human or animal treatment can be provided as a single or periodic dose of a composition or pharmaceutical composition disclosed herein of about 0.1 to 100 mg / kg per day, or any range, value, or fraction thereof, at least once every 1 to 40 days, or alternatively or additionally, at least once every 1 to 52 weeks, or alternatively or additionally, at least once every 1 to 20 years, or any combination thereof, using a single infusion or repeated doses.

[0476] In embodiments where the composition administered to a subject in need thereof is a modified cell disclosed herein, the cells are administered in a concentration of about 1 x 10 3 ~1×10 15 cells, 1 x 10 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×0 12 cells, approximately 1 x 10 5 ~1×10 10 cells, approximately 1 x 10 6 ~1×10 9 cells, approximately 1 x 10 6 ~1×10 8 cells, approximately 1 x 10 6 ~1×10 7 cells, or approximately 1 x 10 6 ~25×10 6 In one embodiment, the cells are administered in amounts of about 5 x 10 6 ~25×10 6 It is administered in cells.

[0477] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in WO 2019 / 04981.

[0478] The present disclosure provides uses of the disclosed compositions and pharmaceutical compositions to treat a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, by, for example, administering or contacting a cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In some aspects, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0479] The present disclosure provides methods 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 malignant diseases or disorders include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell, or FAB. These include ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, malignant paraneoplastic syndrome / hypercalcemia, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis carcinoma, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, and cancer-related bone pain.

[0480] In preferred embodiments, the treatment of a malignant disease or disorder involves adoptive cell therapy. For example, in certain embodiments, the present disclosure provides modified cells expressing at least one of the disclosed protein scaffolds and / or CARs, including a selected and / or expanded protein scaffold (e.g., an scFv, single domain antibody, centilin delivered to cells with a composition of the present disclosure) for administration to a subject in need thereof. The modified cells can be formulated for storage at any temperature, including room temperature and body temperature. The modified cells can be formulated for cryopreservation and subsequent thawing. The modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from a sterile package. The modified cells can be formulated in a pharmaceutically acceptable carrier with indicators of cell viability and / or CAR expression level to ensure a minimum level of cell function and CAR expression. The modified cells can be formulated in a pharmaceutically acceptable carrier at a predetermined density with one or more reagents to inhibit further proliferation and / or prevent cell death.

[0481] Any of these methods may include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods may optionally further include co-administration or combination therapy for treating such a disease or disorder, and the administration of any of the compositions or pharmaceutical compositions disclosed herein may further include the administration of at least one chemotherapeutic agent (e.g., alkylating agent, antimitotic agent, radiopharmaceutical) prior to, simultaneously with, and / or after the administration of at least one chemotherapeutic agent (e.g., alkylating agent, antimitotic agent, radiopharmaceutical).

[0482] In some embodiments, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG) after administration. In certain embodiments, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In certain embodiments, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0483] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, 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 therebetween produced simultaneously. In some embodiments, when the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0484] In one example, the present 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., an scFv, single domain antibody, centilin), where the antibody or CAR specifically binds to an antigen on a tumor cell. In aspects where the composition comprises an engineered cell or cell population, the cell or cell population can be autologous or allogeneic.

[0485] In some aspects of the therapeutic methods described herein, treatment can be modified or terminated. Specifically, in aspects in which the composition used in treatment comprises an inducible pro-apoptotic polypeptide, apoptosis can be selectively induced in cells by contacting the cells with an inducer. Treatment can be modified or terminated, for example, in response to signs of recovery or signs of decreased disease severity / progression, signs of disease remission / halt, and / or the occurrence of adverse events. In some aspects, the method includes administering an inhibitor of the inducer to inhibit the modification of the cell therapy, thereby restoring the function and / or effectiveness of the cell therapy (e.g., when signs or symptoms of disease recurrence or increased severity and / or adverse events are resolved).

[0486] Protein scaffold production, screening and purification At least one protein scaffold of the present disclosure (e.g., a monoclonal antibody, a chimeric antibody, a single domain antibody, a VHH, a VH, a single chain variable fragment (scFv), a centilin, an antigen-binding fragment (Fab) or a Fab fragment) may optionally be produced by a cell line, a mixed cell line, an immortalized cell or a clonal population of immortalized cells, as known in the art. For example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001), Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989), Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989), Colligan, et al. 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, NY, (1997-2001).

[0487] Amino acids from the protein scaffold can be modified, added and / or deleted to reduce immunogenicity or to decrease, 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.

[0488] Optionally, protein scaffolds can be engineered to retain high affinity for antigens and other favorable biological properties. To achieve this goal, scaffold proteins can optionally be prepared by a process of analysis of parent sequences and various conceptual engineered products using three-dimensional models of the parent and engineered sequences. Three-dimensional models are publicly available and familiar to those skilled in the art. Computer programs are available that can illustrate and display the potential three-dimensional conformations of selected candidate sequences and measure their potential immunogenicity (e.g., the Immunofilter program from Xencor, Inc., Monrovia, Calif.). Inspection of these displays allows for analysis of the expected role of residues in the function of the candidate sequence, i.e., analysis of residues that affect the ability of the candidate protein scaffold to bind to its antigen. In this way, residues can be selected and combined from parent and reference sequences to achieve desired characteristics, such as affinity for the target antigen. Alternatively or in addition to the above procedures, other suitable engineering methods can be used.

[0489] Screening protein scaffolds for specific binding to similar proteins or fragments can be conveniently accomplished using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. This method involves screening large collections of peptides for individual members with the desired function or structure. The displayed nucleotide or peptide sequences can be 3 to 5,000 or more nucleotides or amino acids in length, often 5 to 100 amino acids in length, and often about 8 to 25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves displaying peptide sequences on the surface of bacteriophage or cells. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are described in WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0490] Other systems for generating libraries of peptides include aspects of both in vitro chemical synthesis and recombinant methods. See WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also U.S. Patent Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from suppliers such as Invitrogen (Carlsbad, CA) and Cambridge Antibody Technologies (Cambridgeshire, UK). See, for example, U.S. Patent 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, and 5,856,456, all assigned to Enzon; U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500, all assigned to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717, all assigned to Affymax; and Cambridge Antibody See US Patent No. 5,885,793 assigned to Genentech; US Patent No. 5,750,373 assigned to Genentech; US Patent Nos. 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.

[0491] The protein scaffolds of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In preferred embodiments, at least one protein scaffold of the present disclosure binds to human or other mammalian proteins with high affinity, e.g., about 10 KD, as determined by surface plasmon resonance or Kinexa methods performed by one of skill in the art. -7 M or less, for example, but not limited to, 0.1 to 9.9 (or any range or value therein) x 10-8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 or any range or value therein.

[0492] The affinity or avidity of a protein scaffold for an antigen can be experimentally determined using any suitable method (see, for example, Berzofsky, et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company: New York, NY (1992), and the methods described herein). The measured affinity of a particular protein scaffold-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed using standardized solutions of the protein scaffold and antigen, and standardized buffers such as those described herein.

[0493] Competition assays can be performed using protein scaffolds to determine which proteins, antibodies, and other antagonists compete with the protein scaffold for binding to a target protein and / or share epitope regions. These assays, readily known to those skilled in the art, evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The proteins and / or antibodies are immobilized or insolubilized before or after competition, and the target protein-bound sample is separated from the unbound sample by, for example, decanting (if the protein / antibody was pre-insolubilized) or centrifugation (if the protein / antibody was precipitated after the competition reaction). Competitive binding can also be determined by whether binding or lack of binding of the protein scaffold to the target protein alters function, e.g., whether the protein scaffold inhibits or enhances, for example, the enzymatic activity of a label. ELISAs and other functional assays can be used, as are well known in the art.

[0494] nucleic acid molecule The nucleic acid molecule of the present 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 combination 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 the non-coding strand, also called the antisense strand.

[0495] Isolated nucleic acid molecules of the present disclosure can include, for example, but are not limited to, nucleic acid molecules comprising at least one specified portion of at least one protein scaffold, an open reading frame (ORF), optionally having one or more introns, a nucleic acid molecule comprising a coding sequence for a protein scaffold or loop region that binds to a target protein, and nucleic acid molecules that comprise a substantially different nucleotide sequence than those described above, but that still encode a protein scaffold as described herein and / or as known in the art due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Thus, it is routine for one of skill in the art to generate such degenerate nucleic acid variants that encode a particular protein scaffold of the present disclosure. See, e.g., Ausubel, et al., supra; such nucleic acid variants are included in the present disclosure.

[0496] As provided herein, nucleic acid molecules of the present disclosure, including nucleic acid molecules encoding protein scaffolds, can include, but are not limited to, those that encode the amino acid sequence of a protein scaffold fragment by themselves, coding sequences for the entire protein scaffold or a portion thereof, coding sequences for a protein scaffold, fragment, or portion, and additional sequences, such as coding sequences for at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, and additional non-coding sequences, including, but not limited to, non-coding 5' and 3' sequences, e.g., transcribed and non-translated sequences that play a role in transcription, mRNA processing (e.g., ribosome binding and mRNA stability), such as splicing and polyadenylation signals, additional coding sequences that encode additional amino acids, such as those that provide additional functionality. Thus, the protein scaffold-encoding sequence can be fused to a marker sequence, e.g., a sequence encoding a peptide that facilitates purification of the fusion protein scaffold comprising the protein scaffold fragment or portion.

[0497] Polynucleotides that selectively hybridize to the polynucleotides described herein The present disclosure provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Thus, the polynucleotides can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the 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 complementary to isolated genomic or cDNA sequences, or cDNAs from a human or mammalian nucleic acid library.

[0498] Preferably, the cDNA library contains 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 library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, used with sequences having reduced sequence identity to complementary sequences. Medium and high stringency conditions can optionally be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0499] Optionally, the polynucleotide encodes at least a portion of a protein scaffold encoded by a polynucleotide described herein. The polynucleotide comprises a nucleic acid sequence that can be used for selective hybridization to a polynucleotide encoding a protein scaffold of the present disclosure. See, e.g., Ausubel, supra; Colligan, supra, each of which is incorporated herein by reference in its entirety.

[0500] Nucleic acid constructs Isolated nucleic acids of the present disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are well known in the art.

[0501] A nucleic acid may advantageously contain sequences in addition to the polynucleotide of the present disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotide of the present disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the protein of the present disclosure. A nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of the polynucleotide of the present disclosure.

[0502] 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 introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0503] Recombinant methods for constructing nucleic acids The isolated nucleic acid compositions of the present disclosure, e.g., RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).

[0504] Nucleic Acid Screening and Isolation Methods cDNA or genomic libraries can be screened using probes based on the sequences of the polynucleotides disclosed herein. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will understand that various degrees of hybridization stringency can be used in assays, and that either the hybridization or wash medium can be stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and target must be 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, hybridization stringency can be conveniently varied by changing the polarity of the reactant solution, e.g., by manipulating the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies according to the stringency of the hybridization medium and / or wash medium. The degree of complementarity is optimally 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.

[0505] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance presented herein.

[0506] Known methods of DNA or RNA amplification include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 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, 5,142,033 to Gyllensten et al., and 5,142,033 to Innis). Nos. 4,889,818 to Gelfand et al., 4,994,370 to Silver et al., 4,766,067 to Biswas, and 4,656,134 to Ringold), and RNA-mediated amplification using antisense RNA to a target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al., under the trade name NASBA), the entire contents of which are incorporated herein by reference. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0507] For example, polymerase chain reaction (PCR) techniques can be used to amplify the sequences of the polynucleotides and related genes of the present disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, for generating nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of procedures sufficient to guide one of skill in the art through in vitro amplification methods can be found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as U.S. Pat. No. 4,683,202 to Mullis et al. (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, for example, the Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to improve yields of long PCR products.

[0508] Synthetic methods for constructing nucleic acids The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., supra). 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. Those skilled in the art will recognize that chemical synthesis of DNA may be limited to sequences of about 100 bases or more, while longer sequences can be obtained by ligating shorter sequences.

[0509] Recombinant Expression Cassette The present disclosure further provides a recombinant expression cassette comprising a nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure, for example, a cDNA or genomic sequence encoding a protein scaffold of the present disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically comprises a polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acid of the present disclosure.

[0510] In some aspects, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a suitable location (upstream, downstream, or within an intron) of a non-heterologous form of a polynucleotide of the disclosure to up- or down-regulate expression of the polynucleotide of the disclosure. For example, endogenous promoters can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0511] Expression vectors and host cells The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques well known in the art (see, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each incorporated herein by reference in its entirety).

[0512] Polynucleotide can be optionally linked to a vector that contains a selectable marker for propagation in a host.Generally, plasmid vector is introduced into a precipitate such as calcium phosphate precipitate or in a complex with charged lipid.If vector is virus, it can be packaged in vitro using a suitable packaging cell line, and then transduced into host cell.

[0513] The DNA insert must be operably linked to a suitable promoter. The expression construct further contains sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably includes a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the beginning and the end of the translated mRNA, with UAA and UAG being preferred for mammalian or eukaryotic cell expression.

[0514] Expression vectors preferably, but optionally, include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Shbla 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. Patent Nos. 5,122,464, 5,770,359, 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, as well as genes encoding ampicillin, zeocin (Shbla 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. Patent Nos. 5,122,464, 5,770,359, 5,827,739), blasticidin (bsd gene), and resistance genes for eukaryotic cell culture. Examples of suitable resistance genes include erythromycin (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culture in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of the vector construct into the host cell can be achieved 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 in Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, and 16.

[0515] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for isolation of cells modified by the disclosed compositions and methods. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the disclosed compositions or methods from cells not modified by the disclosed compositions or methods. Such cell surface markers include, for example, but are not limited to, "cluster of designation" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Further cell surface markers include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0516] The expression vector preferably, but optionally, includes at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0517] At least one protein scaffold of the present disclosure can be expressed in modified forms, such as fusion proteins, and can include not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to final preparation of the 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.

[0518] Those skilled in the art are familiar with the numerous expression systems available for expressing nucleic acid molecules encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in host cells by operating (by engineering) in a host cell containing endogenous DNA encoding the protein scaffold of the present disclosure. Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

[0519] Examples of cell cultures useful for the production of protein scaffolds, specific portions or variants thereof are bacterial, yeast, and mammalian cells known in the art. Mammalian cell systems are often in the form of a monolayer of cells, although mammalian cell suspensions or bioreactors can also be used. Numerous suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including 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-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the 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 No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0520] Expression vectors for these cells may 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 promoter, CMV promoter (U.S. Pat. Nos. 5,168,062, 5,385,839), HSY tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1α promoter (U.S. Pat. No. 5,266,491), at least one human promoter, enhancer, and / or processing information site, such as ribosome binding site, RNA splice site, polyadenylation site (e.g., SV40 large T Ag polyA addition site), and transcription termination sequence. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and / or can be found, for example, in the American Type Culture Collection Catalogue of Cell Lines and Available from Hybridomas (www.atcc.org) or other known or commercial sources.

[0521] When eukaryotic host cells are used, a polyadenylation sequence or transcription termination sequence is typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence derived from the bovine growth hormone gene. A sequence for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron derived from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Furthermore, gene sequences for regulating replication in host cells may be incorporated into the vector, as is known in the art.

[0522] Protein scaffold purification Protein scaffolds 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 used for purification. See, e.g., Colligan, *Current Protocols in Immunology*, or *Current Protocols in Protein Science*, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0523] Protein scaffolds of the present disclosure include purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, such as, for example, E. coli, yeast, higher plants, insect, and mammalian cells. Depending on the host used in a recombinant production procedure, protein scaffolds of the present disclosure may or may not be 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; Colligan, Protein Science, supra, Chapters 12-14, all of which are incorporated herein by reference in their entireties.

[0524] Amino acid code The amino acids comprising the protein scaffolds of the present disclosure are often abbreviated. Amino acid names can be indicated by the one-letter code, three-letter code, name, or three-nucleotide codon designation of the amino acid, 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). Protein scaffolds of the present disclosure can include one or more amino acid substitutions, deletions, or additions, as specified herein, that are naturally occurring or result from mutations and / or human manipulation. Amino acids in the protein scaffolds of the present 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, Chapter 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, for example, but not limited to, at least one neutralizing activity. Sites important 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)).

[0525] As will be appreciated by those skilled in the art, the present disclosure includes at least one biologically active protein scaffold of the present disclosure. A biologically active protein scaffold has a specific activity of at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 99% or more of the specific activity of a natural (non-synthetic), endogenous, or related known protein scaffold. Methods for assaying and quantifying enzyme activity and substrate specificity measurements are well known to those skilled in the art.

[0526] In another aspect, the present disclosure relates to protein scaffolds and fragments described herein that have been modified by the covalent attachment of an organic moiety. Such modifications can produce protein scaffold fragments with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain aspects, the hydrophilic polymer 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)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group can contain about 8 to about 40 carbon atoms.

[0527] The modified protein scaffolds and fragments of the present disclosure may contain one or more organic moieties covalently attached directly or indirectly to an antibody. Each organic moiety attached to a protein scaffold or fragment of the present disclosure may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic and dicarboxylic acids. As used herein, a "hydrophilic polymer group" 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, protein scaffolds modified by the covalent attachment of polylysine are encompassed by the present disclosure. Hydrophilic polymers suitable for modifying the protein scaffolds of the present disclosure may be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxypolyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers modifying the protein scaffolds of the present disclosure have a molecular weight of about 800 to about 150,000 daltons as separate molecular entities. For example, PEG5000 and PEG20,000 can be used, where the subscript represents the average molecular weight of the polymer in daltons. The hydrophilic polymer group can be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by suitable methods. For example, a polymer containing an amine group can be attached to the carboxylic acid of a fatty acid or fatty acid ester, and an activated carboxylic acid on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be attached to a hydroxyl group on the polymer.

[0528] Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of the present disclosure may be saturated or contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffolds of the present 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-Δ9-octadecanoate (C18, oleate), all cis-Δ5,8,11,14-eicosatetraenoic acid (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group, which can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0529] Modified protein scaffolds and fragments can be prepared using suitable methods, for example, by reaction with one or more modifying agents. As used herein, the term "modifying agent" refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, 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 ester (NHS), etc. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be attached to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphoimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be directly attached to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or can be attached via a linker moiety, e.g., a divalent C1-C12 group in which 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-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. A modifying agent containing 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 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylic acid.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 carboxylic acid as described, or can be reacted with maleic anhydride and the resulting product cyclized to generate an activated maleimide derivative of a fatty acid (see, for example, WO 92 / 16221 to Thompson et al., the entire teachings of which are incorporated herein by reference).

[0530] The modified protein scaffolds of the present disclosure can be generated by reacting a protein scaffold or fragment with a modifying agent. For example, organic moieties can be non-site-specifically attached to a protein scaffold by using an amine-reactive modifying agent, such as an NHS ester of PEG. Modified protein scaffolds and fragments comprising organic moieties attached to specific sites on the protein scaffolds of the present 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 methods described in Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0531] definition In the chemical formula shown herein:

[0532] [ka] indicates the point at which the functional group is attached to another part of the molecule. Definitions of certain functional groups and chemical terms are described in more detail below.

[0533] 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, racemic mixtures thereof, and other mixtures thereof, as being within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are intended to be included in the present invention.

[0534] Isomeric mixtures containing any of a variety of isomer ratios can be utilized in accordance with the present invention. For example, when 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 skilled in the art will readily appreciate that similar ratios are contemplated for more complex isomer mixtures.

[0535] For example, if a specific enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, the resulting diastereomeric mixture separated, and the auxiliary cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino, or an acidic functional group such as carboxyl, diastereomeric salts can be 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, followed by recovery of the pure enantiomer.

[0536] The "enantiomeric excess" of a substance is a measure of the purity of the desired enantiomer relative to the undesired enantiomer. Enantiomeric excess is defined as the absolute difference between the mole fractions of each enantiomer and is most often expressed as percent enantiomeric excess. For mixtures of diastereomers, there are similar definitions and uses of "diastereomeric excess" and percent diastereomeric excess.

[0537] For example, a sample with 70% R isomer and 30% S isomer has an enantiomeric excess of 40% and can be thought of as a mixture of 40% pure R and 60% racemic mixture (which gives 30% R and 30% S for the total composition).

[0538] Those skilled in the art will appreciate that the synthetic methods described herein utilize a variety of protecting groups. As used herein, the term "protecting group" means that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be selectively carried out at another reactive site in a multifunctional compound. In certain embodiments, the protecting group reacts selectively in good yield to provide a protected substrate that is stable to the planned reaction; the protecting group is selectively removable in good yield with readily available, preferably non-toxic, reagents that do not attack other functional groups; the protecting group forms an easily separable derivative (more preferably, without creating a new stereocenter); and the protecting group has minimal additional functionality to avoid further reactive sites. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.

[0539] It will be understood that the compounds described herein can be substituted with any number of substituents or functional moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, refers 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 can be substituted with more than one substituent selected from a specified group, the substituents can be the same or different at all positions. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom. 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 for the treatment of diseases or disorders. The term "stable," as used herein, preferably refers to a compound that has sufficient stability to allow for manufacture and maintains the integrity of the compound for a period of time sufficient to be detected, preferably for a period of time sufficient to be useful for the purposes detailed herein.

[0540] 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 understood by those skilled in the art, "aliphatic," as used herein, is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight-chain, branched-chain, and cyclic alkyl groups. A similar convention applies to other general 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 refer to alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 6 carbon atoms.

[0541] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-8 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-4 carbon atoms. Thus, exemplary aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, -CH-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH-cyclohexyl moieties, and the like, which may also bear one or more substituents. Alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0542] The term "alkyl," as used herein, refers to a saturated, straight- or branched-chain hydrocarbon radical derived by removing one hydrogen atom from a hydrocarbon moiety containing from 1 to 20 carbon atoms. 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.

[0543] The term "alkenyl" refers to a monovalent group derived by removing a hydrogen atom from a hydrocarbon moiety having at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0544] The term "alkynyl," as used herein, refers to a monovalent group derived by removing a hydrogen atom from a hydrocarbon containing at least one carbon-carbon triple bond. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0545] The term "alkoxy" or "thioalkyl," as used herein, refers to an alkyl group, as defined above, attached to the parent molecule via an oxygen or 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 still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-8 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6 aliphatic carbon atoms. In still 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.

[0546] The term "alkylamino" refers to a group having the structure --NHR', where R' is aliphatic, as defined herein. In certain embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In certain embodiments, alkyl, alkenyl, and alkynyl groups contain 1-15 aliphatic carbon atoms. In certain other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In still other embodiments, aliphatic groups employed in the present invention contain 1-8 aliphatic carbon atoms. In yet other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n-propylamino, isopropylamino, cyclopropylamino, n-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like.

[0547] As used herein, the term "carboxylic acid" refers to a group of formula --CO2H.

[0548] The term "dialkylamino" refers to a group having the structure --NRR', where R and R' are each an aliphatic group, as defined herein. R and R' can be the same or different in a dialkylamino moiety. 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 groups contain 1-10 aliphatic carbon atoms. In still other embodiments, the aliphatic groups used in the present invention contain 1-8 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, the aliphatic groups contain 1-4 aliphatic carbon atoms. Examples of dialkylamino groups include, but are not limited to, dimethylamino, methylethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(isopropyl)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 non-aromatic. Examples of cyclic diaminoalkyl groups include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

[0549] Some examples of substituents for the above aliphatic (and other) portions of the compounds of the invention include aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, F, --Cl, --Br, --I, --OH, --NO2, --CN, --CF3, --CH2CF3, --CHCl2, --CH2OH, --CH2CH2OH, --CH2NH2, --CH2SO2CH3, --C(O)R x , --CO2(Rx ), --CON(R x )2, --OC(O)R x , --OCO2R x , --OCON(R x )2, --N(R x )2, --S(O)2R x , -NR x (CO)R R In particular, the following compounds are included: x independently at each occurrence includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Further examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0550] In general, the terms "aryl" and "heteroaryl," as used herein, refer to stable monocyclic or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties, preferably having 3 to 14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the aforementioned substituents, i.e., those listed for aliphatic moieties or other moieties disclosed herein, that result in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a monocyclic 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 invention, the term "heteroaryl," as used herein, refers to a cyclic aromatic radical having 5 to 10 ring atoms, one ring atom of which is selected from S, O, and N, 0, 1, or 2 ring atoms are additional heteroatoms independently selected from S, O, and N, and the remaining ring atoms are carbon, the radical being attached through any of the ring atoms to the remainder of the moiety, e.g., pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, etc.

[0551] It is understood that aryl and heteroaryl groups can be unsubstituted or substituted, where substitution means replacing one, two, three, or more of the hydrogen atoms thereon, independently, with an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, --F, --Cl, --Br, --I, --OH, --NO2, --CN, --CF3, --CH2CF3, --CHCl2, --CH2OH, --CH2CH2OH, --CH2NH2, --CH2SO2CH3, --C(O)R x , --CO2(R x ), --CON(R x)2, --OC(O)R x , --OCO2R x , --OCON(R x )2, --N(R x )2, --S(O)2R x , -NR x (CO)R R and wherein R x independently at each occurrence includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Further examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0552] The term "heteroaliphatic," as used herein, refers to an aliphatic moiety that contains, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of a carbon atom. Heteroaliphatic moieties can be branched, unbranched, cyclic, or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, and the like. In certain embodiments, a heteroaliphatic moiety can replace one or more of its hydrogen atoms with an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, --F, --Cl, --Br, --I, --OH, --NO, --CN, --CF, --CHCF, --CHCl, --CHOH, --CHCHOH, --CHNH, --CHSOCH, --C(O)R x , --CO2(R x ), --CON(R x )2, --OC(O)R x , --OCO2Rx , --OCON(R x )2, --N(R x )2, --S(O)2R x , -NR x (CO)R R wherein R is substituted by independently replacing one or more moieties, including, but not limited to, x independently at each occurrence includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Further examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0553] The term "cycloalkyl," as used herein, specifically refers to groups having 3 to 7, preferably 3 to 10, carbon atoms. Suitable cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, and may optionally be, as in other aliphatic, heteroaliphatic, or heterocyclic moieties, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, --F, --Cl, --Br, --I, --OH, --NO2, --CN, --CF3, --CH2CF3, --CHCl2, --CH2OH, --CH2CH2OH, --CH2NH2, --CH2SO2CH3, --C(O)R x , --CO2(R x ), --CON(R x )2, --OC(O)R x , --OCO2R x , --OCON(R x)2, --N(R x )2, --S(O)2R x , -NR x (CO)R R and wherein R x independently at each occurrence includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Further examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0554] The term "heteroaliphatic," as used herein, refers to an aliphatic moiety that contains, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of a carbon atom. Heteroaliphatic moieties can be branched, unbranched, cyclic, or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, and the like. In certain embodiments, a heteroaliphatic moiety can replace one or more of its hydrogen atoms with an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, --F, --Cl, --Br, --I, --OH, --NO, --CN, --CF, --CHCF, --CHCl, --CHOH, --CHCHOH, --CHNH, --CHSOCH, --C(O)R x , --CO2(R x ), --CON(R x )2, --OC(O)R x , --OCO2R x , --OCON(R x )2, --N(R x )2, --S(O)2R x, -NR x (CO)R R wherein R is substituted by independently replacing one or more moieties, including, but not limited to, x independently at each occurrence includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Further examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0555] The term "haloalkyl" means an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto, and is exemplified by groups such as chloromethyl, bromoethyl, trifluoromethyl, and the like.

[0556] The term "heterocycloalkyl" or "heterocycle," as used herein, refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group, including, but not limited to, bicyclic or tricyclic groups containing fused 6-membered rings having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen atoms, where (i) each 5-membered ring has 0 to 1 double bond and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms are optionally oxidized, (iii) the nitrogen heteroatom is optionally quaternized, and (iv) any of the above heterocycles 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 group in which one, two, or three of the hydrogen atoms thereon are replaced with, but not limited to, an aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, --F, --Cl, --Br, --I, --OH, --NO, --CN, --CF, --CHCF, --CHCl, --CHOH, --CHCHOH, --CHNH, --CHSOCH, --C(O)R x , --CO2(R x ), --CON(R x )2, --OC(O)R x , --OCO2R x , --OCON(R x )2, --N(R x )2, --S(O)2R x , -NR x (CO)R R refers to a heterocycloalkyl or heterocyclic group as defined above, substituted by independent replacements with xindependently at each occurrence includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Further examples of generally applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0557] As used herein, the term "carbocycle" refers to an aromatic or non-aromatic ring in which each atom of the ring is a carbon atom.

[0558] The term "independently selected" is used herein to indicate that the R groups can be the same or different.

[0559] The terms "halo" and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0560] The term "heterocycle," as used herein, refers to a non-aromatic, partially unsaturated, or fully saturated 3- to 10-membered ring system, including monocyclic rings 3 to 8 atoms in size, as well as bicyclic and tricyclic ring systems that may contain an aromatic 6-membered aryl or heteroaromatic group fused to a non-aromatic ring. These heterocyclic rings include those having 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, where the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.

[0561] The term "heteroaryl," as used herein, refers to a cyclic aromatic radical having 5 to 10 ring atoms, one ring atom of which is selected from sulfur, oxygen, and nitrogen, 0, 1, or 2 ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen, and the remaining ring atoms are carbon, the radical being attached through any of the ring atoms to the remaining moiety, e.g., pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, etc.

[0562] The terms "substituted" (whether preceded by the term "optionally" or not) and "substituent" as used herein refer to the ability to change one functional group into another, provided that the valences of all atoms are maintained, as understood by one of skill in the art. When more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents can be the same or different at all positions. Substituents can also be further substituted (e.g., an aryl group substituent can have another substituent, such as another aryl group that is further substituted with fluorine at one or more positions).

[0563] "Effective amount": Generally, an "effective amount" of an active agent or composition refers to the amount necessary to elicit a desired biological response. As will be understood by those skilled in the art, the effective amount of an agent or device can vary depending on factors such as the desired biological endpoint, the agent being delivered, the composition of the encapsulation matrix, the target tissue, etc. For example, an effective amount of antigen-containing microparticles delivered to immunize an individual is the amount that generates an immune response sufficient to prevent infection by an organism bearing the administered antigen.

[0564] As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0565] The term "about" or "approximately" means within an acceptable error range for a 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 one 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 this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0566] The present 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 natural 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 that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide) (optimally, protein-coding sequences). For example, in various embodiments, an 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 sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. 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 a protein of the disclosure or a biologically active portion thereof is recombinantly produced, optimally, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals that are not the target protein.

[0567] The present disclosure provides fragments and variants of the disclosed DNA sequences and the proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or amino acid sequence, and thus a portion of the protein encoded thereby. Fragments of DNA sequences, including coding sequences, may encode protein fragments that retain the biological activity of the native protein, thus retaining the DNA recognition or binding activity for the target DNA sequences described herein. Alternatively, fragments of DNA sequences useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of DNA sequences can range from at least about 20 nucleotides, about 50 nucleotides, or about 100 nucleotides, to the full-length polynucleotides of the present disclosure.

[0568] The nucleic acids or proteins of the disclosure can be constructed by a modular approach, involving preassembling monomeric and / or repeating units in a target vector, which can then be assembled into a final destination vector. The polypeptides of the disclosure may comprise repeating monomers of the disclosure, which can be constructed by a modular approach by preassembling repeating units in a target vector, which can then be assembled into a final destination vector. The disclosure provides polypeptides produced by this method, as well as nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells comprising nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0569] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with polyepitopic specificity. It is also within the scope of the present specification to use natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the antibodies of the present specification as defined herein. Thus, according to certain aspects of the present specification, the term "antibodies of the present specification" in its broadest sense also encompasses such analogs. Generally, such analogs may have one or more amino acid residues substituted, deleted, and / or added compared to the antibodies of the present invention as defined herein.

[0570] As used herein, an "antibody fragment" and all grammatical variations thereof are defined as a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, which portion does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include any antibody fragment that is a polypeptide having a primary structure consisting of a single, uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments, diabodies, (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only a light-chain variable domain, or fragments thereof containing the three CDRs of a light-chain variable domain and no associated heavy-chain portion, and (3) single-chain polypeptides containing only a heavy-chain variable region, or fragments thereof containing the three CDRs of a heavy-chain variable region and no associated light-chain portion, as well as multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype), and / or may contain any hinge region sequence found in an intact antibody, and / or may contain a leucine zipper sequence fused to or located on the hinge region or constant domain sequence of the heavy chain. The term also includes single domain antibodies ("sdAB"), which generally refer to antibody fragments (e.g., derived from Camelidae) having a single monomeric variable antibody domain. Such antibody fragment types will be readily understood by those skilled in the art.

[0571] "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.

[0572] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, shall mean excluding any other element of essential importance to the combination when used for its intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants or inert carriers. "Consisting of" shall mean excluding more than trace amounts of other components and substantial method step elements. Embodiments defined by each of these transitions are within the scope of this disclosure.

[0573] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope can include three amino acids in a spatial conformation that is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, at least 8, 9, or 10 such amino acids. Methods for 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.

[0574] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0575] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs that have been modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that have been modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0576] "Alteration" or "modulation" 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.

[0577] The term "operatively linked" or its equivalents (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact to affect a function ascribed to one or both molecules, or a combination thereof.

[0578] Non-covalently linked components and methods for making and using non-covalently linked components are disclosed. The various components can take a variety of different forms as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for 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 allows for functional association only or primarily under circumstances where such association is required for the desired activity. The association may be of sufficient duration to allow for the desired effect.

[0579] Disclosed is a method for directing a protein to a specific locus in the genome of an organism, which may include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are operably linked via a non-covalent bond.

[0580] The term "scFv" refers to a single-chain variable region fragment. An scFv is a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL) connected by a linker peptide. The linker peptide can be about 5 to 40 amino acids, or about 10 to 30 amino acids, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single-chain variable region fragments lack the constant Fc region found in intact antibody molecules and therefore lack the consensus binding site (e.g., protein G) used to purify antibodies. The term also includes scFvs, which are intrabodies, that is, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0581] The term "single-domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a specific antigen. Single-domain antibodies are generally peptide chains of about 110 amino acids in length comprising one variable domain (VH) of a heavy-chain antibody, or a typical IgG, and generally have similar affinity for antigen as a whole antibody, but are more heat-resistant and stable to detergents and high concentrations of urea. Examples are those derived from camelid or fish antibodies. Alternatively, single-domain antibodies can be generated from typical mouse or human IgG, which has four chains.

[0582] The terms "specifically bind" and "specific binding," as used herein, refer to the ability of an antibody, antibody fragment, or nanobody to preferentially bind to a particular antigen present in a homogenous mixture of different antigens. In some embodiments, the specific binding interaction discriminates between desired and undesired antigens in a sample. In some embodiments, by about 10-100 fold or more (e.g., about 1000 fold or more than 10,000 fold). "Specificity" refers to the ability of an immunoglobulin or immunoglobulin fragment, such as a nanobody, to preferentially bind to one antigen target over different antigen targets and does not necessarily imply high affinity.

[0583] 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.

[0584] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can also encompass the complementary strand of a depicted single strand. Nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0585] A probe of the present disclosure may comprise a single-stranded nucleic acid capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0586] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may contain double-stranded sequences even if the majority of the molecule is single-stranded. The nucleic acids of the present disclosure may contain single-stranded sequences even if the majority of the molecule is double-stranded. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure can be synthesized to contain unnatural amino acid modifications. The nucleic acids of the present disclosure can be obtained by chemical synthesis or recombinant methods.

[0587] The nucleic acids of the present disclosure may not be naturally occurring in their entirety or in any part thereof. The nucleic acids of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, resulting in a sequence that is non-naturally occurring and making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain non-naturally occurring modified, artificial, or synthetic nucleotides, making the entire nucleic acid sequence non-naturally occurring.

[0588] Given the redundancy in the genetic code, multiple nucleotide sequences may encode any particular protein, and all such nucleotide sequences are contemplated herein.

[0589] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a spatially connected promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Changes in the distance between the promoter and the gene may be accommodated without loss of promoter function.

[0590] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repression elements, which may be located as far as several thousand base pairs from the start site of transcription. Promoters may be derived from sources such as viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate expression of genetic components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an EF-1α promoter, a CAG promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0591] As used throughout this 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.

[0592] As used throughout this disclosure, the term "substantially identical" refers to a first and second sequence that is 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, where the first sequence is substantially complementary to the complement of the second sequence.

[0593] As used throughout this 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 a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0594] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain amino acids and a combination of DNA sequences, RNA sequences, or both DNA and RNA sequences.

[0595] As used throughout this 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 amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence that is substantially identical to a reference ...

Claims

1. A compound of formula (I) 【Chemical 1】 During the ceremony, A is a substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic; Each R １ are independently hydrogen, substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, R １ at least one occurrence of is hydrogen; Each of B, C, and D is independently hydrogen, a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group, or -(CHR ６ )CH(SXLY)R ６ and X is S or CH ２ and Y is -OH, 【Chemistry 2】 where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic; Each R ６ are independently hydrogen, substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, R ６ wherein at least one occurrence of is hydrogen, or a pharmaceutically acceptable salt thereof.

2. One of B, C, and D is —(CHR ６ )CH(SXLY)R ６ 2. The compound of claim 1, wherein:

3. Two of B, C, and D are -(CHR ６ )CH(SXLY)R ６ 2. The compound of claim 1, wherein:

4. Each of B, C, and D is independently —(CHR ６ )CH(SXLY)R ６ 2. The compound of claim 1, wherein:

5. 2. The compound of claim 1, wherein X is S and Y is -OH.

6. 2. The compound of claim 1, wherein X is S.

7. X is CH ２ 2. The compound of claim 1, wherein:

8. The compound of claim 6 or 7, wherein Y is -OH.

9. The compound of claim 1, wherein L is an unsubstituted branched or unbranched C1-6 alkyl.

10. 10. The compound of claim 9, wherein L is an unsubstituted C2 alkyl.

11. R １ The compound of claim 1, wherein is a substituted or unsubstituted branched or unbranched C1-15 alkyl.

12. R １ The compound of claim 1, wherein is a substituted or unsubstituted branched or unbranched C1-10 alkyl.

13. R １ The compound of claim 1, wherein is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

14. R ６ The compound of claim 1, wherein is a substituted or unsubstituted branched or unbranched C1-15 alkyl.

15. R ６ The compound of claim 1, wherein is a substituted or unsubstituted branched or unbranched C1-10 alkyl.

16. R ６ The compound of claim 1, wherein is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

17. 【Chemical 3】 but, 【Chemistry 4】 2. The compound of claim 1, wherein:

18. 【Chemical 5】 2. The compound of claim 1, wherein:

19. 【Chemical 6】 2. The compound of claim 1, wherein:

20. 4. The compound of claim 3, wherein one of B, C, and D is methyl, X is S, and Y is -OH.

21. 21. The compound of claim 20, wherein L is an unsubstituted C2 alkyl.

22. R １ But C １２ H ２５ 21. The compound of claim 20, wherein:

23. R ６ But C １２ H ２５ 21. The compound of claim 20, wherein:

24. 【Chemical 7】 The compound of claim 20, wherein is a C3 alkyl.

25. formula: 【Chemistry 8】 Compound.

26. A compound of formula (II) 【Chemistry 9】 During the ceremony, A is a substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic; R １ , R ２ , R ３ , and R ４ is independently hydrogen, a substituted or unsubstituted branched or unbranched C1-15 aliphatic, or a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic, wherein at least one occurrence of R1 is hydrogen, and R ２ at least one occurrence of is hydrogen, and R ３ at least one occurrence of is hydrogen, and R ４ at least one occurrence of is hydrogen; X is S or CH ２ and Y is -OH, 【Chemistry 10】 where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic, or a pharmaceutically acceptable salt thereof.

27. 27. The compound of claim 26, wherein X is S and Y is -OH.

28. 27. The compound of claim 26, wherein X is S.

29. X is CH ２ 27. The compound of claim 26, wherein:

30. 30. The compound of claim 28 or 29, wherein Y is -OH.

31. 27. The compound of claim 26, wherein L is unsubstituted branched or unbranched C1-6 alkyl.

32. 32. The compound of claim 31, wherein L is an unsubstituted C2 alkyl.

33. R １ , R ２ , R ３ , and R ４ 27. The compound of claim 26, wherein each of is independently a substituted or unsubstituted branched or unbranched C1-15 alkyl.

34. R １ , R ２ , R ３ , and R ４ 27. The compound of claim 26, wherein each of is independently a substituted or unsubstituted branched or unbranched C1-10 alkyl.

35. R １ , R ２ , R ３ , and R ４ 27. The compound of claim 26, wherein each of is independently a substituted or unsubstituted branched or unbranched C1-5 alkyl.

36. R １ , R ２ , R ３ , and R ４ Each of the groups independently represents C １０ H ２１ 27. The compound of claim 26, wherein:

37. 【Catalog 11】 but, 【Chemistry 12】 27. The compound of claim 26, wherein:

38. A compound of the formula: 【Chemistry 13】

39. A compound of formula (III): 【Chemistry 14】 During the ceremony, Each of Ra and Rb independently represents 【Chemistry 15】 or 【Chemistry 16】 and X is S or CH ２ and Y is -OH, 【Chemistry 17】 where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic; R ７ is a 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 【Chemistry 18】 a compound, or a pharmaceutically acceptable salt thereof.

40. Ra 【Chemistry 19】 and Rb is 【Chemistry 20】 40. The compound of claim 39, wherein:

41. Ra 【Chemical 21】 40. The compound of claim 39, wherein:

42. Ra 【Chemical 22】 40. The compound of claim 39, wherein:

43. Ra 【Chemical 23】 40. The compound of claim 39, wherein:

44. Rb 【Chemistry 24】 40. The compound of claim 39, wherein:

45. Rb 【Chemistry 25】 40. The compound of claim 39, wherein:

46. Rb 【Chemical 26】 40. The compound of claim 39, wherein:

47. 40. The compound of claim 39, wherein x is 1 and y is 2.

48. 40. The compound of claim 39, wherein X is S and Y is -OH.

49. 40. The compound of claim 39, wherein X is S.

50. X is CH ２ 40. The compound of claim 39, wherein:

51. 51. The compound of claim 49 or 50, wherein Y is -OH.

52. 40. The compound of claim 39, wherein L is unsubstituted branched or unbranched C1-6 alkyl.

53. 53. The compound of claim 52, wherein L is an unsubstituted C2 alkyl.

54. R ７ 40. The compound of claim 39, wherein is a substituted or unsubstituted branched or unbranched C1-15 alkyl.

55. R ７ 40. The compound of claim 39, wherein is a substituted or unsubstituted branched or unbranched C1-10 alkyl.

56. R ７ 40. The compound of claim 39, wherein is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

57. R ７ But C １０ H ２１ 40. The compound of claim 39, wherein:

58. Ra 【Chemical 27】 40. The compound of claim 39, wherein:

59. Rb 【Chemical Formula 28】 40. The compound of claim 39, wherein:

60. formula: 【Chemical Formula 29】 Compound.

61. A compound of formula (IV), 【Chemistry 30】 During the ceremony, Each of Re and Rf independently represents 【Chemical 31】 and x is an integer between 1 and 10 inclusive; Each Ry and Rz is independently 【Chemical 32】 and X is S or CH ２ and Y is -OH, 【Chemical 33】 where n=1 to 3 and R″=H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic, substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic; R ７ is a substituted or unsubstituted branched or unbranched C1-15 aliphatic, substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic compound, or a pharmaceutically acceptable salt thereof.

62. Re 【Chemical 34】 62. The compound of claim 61, wherein:

63. Rf is 【Chemistry 35】 62. The compound of claim 61, wherein:

64. 62. The compound of claim 61, wherein x is 1.

65. 62. The compound of claim 61, wherein x is 2.

66. 62. The compound of claim 61, wherein x is 3.

67. 62. The compound of claim 61, wherein X is S and Y is -OH.

68. 62. The compound of claim 61, wherein X is S.

69. X is CH ２ 62. The compound of claim 61, wherein:

70. 70. The compound of claim 68 or 69, wherein Y is -OH.

71. 62. The compound of claim 61, wherein L is unsubstituted branched or unbranched C1-6 alkyl.

72. 72. The compound of claim 71, wherein L is an unsubstituted C2 alkyl.

73. R ７ is a substituted or unsubstituted branched or unbranched C1-15 alkyl.

74. R ７ 62. The compound of claim 61, wherein is a substituted or unsubstituted branched or unbranched C1-10 alkyl.

75. R ７ 62. The compound of claim 61, wherein is a substituted or unsubstituted branched or unbranched C1-5 alkyl.

76. 62. A composition comprising at least one lipid nanoparticle comprising at least one compound or salt thereof described in claim 1, 26, 39, or 61, wherein the at least one lipid nanoparticle further comprises at least one nucleic acid molecule.

77. The at least one lipid nanoparticle comprises about 54 mol% of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; the at least one nucleic acid molecule comprises at least one RNA molecule; The at least one lipid nanoparticle is about 35 mole % cholesterol; about 10 mol % DOPC; and about 1 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w).

78. The at least one lipid nanoparticle comprises about 43.3 mol% of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one RNA molecule, and the at least one lipid nanoparticle comprises: about 43.3 mol % cholesterol; about 12 mol % DOPC; and about 1.5 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w).

79. The at least one lipid nanoparticle comprises about 33.5 mol % of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one RNA molecule, and the at least one lipid nanoparticle comprises: about 33.5 mol % cholesterol and about 32 mol % DOPE; and about 1 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40:1 (w / w).

80. the at least one lipid nanoparticle comprises from about 34 mol% to about 60 mol% of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one DNA molecule, and the at least one lipid nanoparticle comprises: about 30 mol% to about 60 mol% cholesterol and about 5 mol% to about 11.9 mol% DOPC; and about 1 mol % to about 2 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is from about 80:1 (w / w) to about 120:1 (w / w).

81. the at least one lipid nanoparticle comprises from about 49.6 mol % to about 60 mol % of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one DNA molecule, and the at least one lipid nanoparticle comprises: about 30 mol% to about 45 mol% cholesterol; about 0.2 mol % to about 9.5 mol % DOPC; and about 1 mol % to about 1.5 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is from about 80:1 (w / w) to about 120:1 (w / w).

82. The at least one lipid nanoparticle comprises about 49.6 mol % of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one DNA molecule, and the at least one lipid nanoparticle comprises: about 39.9 mol % cholesterol; about 9.5 mol % DOPC; and about 1 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 120:1 (w / w).

83. The at least one lipid nanoparticle comprises about 52.1 mol % of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one DNA molecule, and the at least one lipid nanoparticle comprises: about 45 mole percent cholesterol; about 1.9 mol % DOPC; and about 1 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w).

84. The at least one lipid nanoparticle comprises about 60 mol % of at least one compound of claim 1, 26, 39, or 61, or a salt thereof; The at least one nucleic acid molecule comprises at least one DNA molecule, and the at least one lipid nanoparticle comprises: about 30 mol% cholesterol; about 9 mol % DOPC; and about 1 mol % DMG-PEG2000; 77. The composition of claim 76, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 120:1 (w / w).

85. 80. The composition of any one of claims 77 to 79, wherein the RNA molecule is an mRNA molecule.

86. 86. The composition of claim 85, wherein the mRNA molecule further comprises a 5'-CAP.

87. 80. The composition of any one of claims 77-79, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB 100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

88. 85. The composition of any one of claims 80 to 84, wherein the DNA molecule is a circular DNA molecule, a DoggyBone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule.

89. 85. The composition of any one of claims 80 to 84, wherein the at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.

90. 77. The composition of claim 76, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one therapeutic protein.

91. 77. The composition of Claim 76, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one transposon, the transposon comprising a nucleic acid sequence encoding at least one therapeutic protein.

92. the at least one therapeutic protein (a) chimeric antigen receptor (CAR), (b) ornithine transcarbamylase (OTC) polypeptide; (c) a methylmalonyl-CoA mutase (MUT1) polypeptide; (d) a factor VIII (FVIII) polypeptide, or (d) any combination thereof.

93. 77. A pharmaceutical composition comprising the composition of claim 76 and at least one pharmaceutically acceptable excipient or diluent.

94. 77. A method of delivering at least one nucleic acid to at least one cell, comprising contacting said at least one cell with at least one composition of claim 76.

95. 77. A method of genetically modifying at least one cell, comprising contacting said at least one cell with at least one composition of claim 76.

96. the at least one cell (a) hepatocytes, wherein the hepatocytes are hepatic parenchymal cells, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells; (b) T cells, wherein the T cells are activated T cells, resting T cells, or stem memory T cells (T ＳＣＭ T cells, The method of claim 94 or claim 95, wherein (c) is a hematopoietic stem cell (HSC).

97. 96. At least one cell modified according to the method of claim 95.

98. 98. A method of treating at least one disease or disorder in a subject in need thereof, the method comprising administering to said subject at least one therapeutically effective amount of a composition according to any one of claims 90 to 93 or at least one cell according to claim 97.

99. The at least one disease or disorder is a liver disease or disorder, and the liver disease or disorder is (a) metabolic liver disease; 99. The method of claim 98, wherein (b) the UCD is a urea cycle disorder, wherein the UCD is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), or any combination thereof.

100. 103. The method of claim 102, wherein the at least one disease or disorder is cancer.

101. 103. The method of claim 102, wherein the at least one disease or disorder is hemophilia A.

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