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

Abstract

A lipid nanoparticle composition (LNP); a process for preparing LNP; the use of LNP includes, but is not limited to, methods for treating certain diseases and disorders, including, but is not limited to, liver disorders; kits for delivering nucleic acids in vivo, ex vivo, and in vitro to various types of cells, including T cells and hepatocytes.

Description

Background Art

[0001] There has been a long - standing but unmet need in the art for compositions and methods for delivering nucleic acids in vivo, ex vivo, and in vitro to cells and for genetically modifying cells. Widely accepted gene delivery and genetic modification techniques, such as those using viral vectors including AAV, can cause acute toxicity and adverse side effects in patients. The present disclosure provides improved compositions, methods, and kits for delivering nucleic acids in vivo, ex vivo, and in vitro to various types of cells including T cells and hepatocytes. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods of using them. These lipid nanoparticle compositions and methods allow for the delivery of nucleic acids to cells with high efficiency and low toxicity. Thus, the compositions and methods of the present disclosure have broad applicability in a variety of fields, including gene therapy and the production of cell - based therapeutic agents. Summary of the Invention

[0002] In some aspects, novel lipid nanoparticles (“LNPs”) comprising novel compounds are provided. In one aspect, the novel compound is a compound of Formula (I) - Formula (IV).

[0003] In some aspects, pharmaceutical compositions are provided that comprise the compositions of the present disclosure and at least one pharmaceutically acceptable excipient or diluent.

[0004] In some aspects, methods for delivering at least one nucleic acid to at least one cell are provided, the methods comprising contacting the at least one cell with at least one composition of the present disclosure.

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

[0006] In some aspects, methods for treating at least one disease or disorder in a subject in need thereof are provided, the methods comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.

[0007] In some aspects, methods for delivering at least one nucleic acid to at least one cell are provided, the methods 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 one of the aspects and / or embodiments described herein can be combined with any other aspect and / or embodiment described herein.

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

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

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

[0013] The compositions of the present disclosure

[0014] The present disclosure provides a composition comprising at least one lipid nanoparticle, the at least one lipid nanoparticle comprising a compound of the present disclosure and at least one nucleic acid molecule. In some aspects, the lipid nanoparticle may further comprise at least one structural lipid. In some aspects, the lipid nanoparticle may further comprise at least one phospholipid. In some aspects, the lipid nanoparticle may further comprise at least one PEGylated lipid. In non-limiting examples, the compositions and methods of the present disclosure can be used for gene delivery.

[0015] Compound

[0016] In some embodiments, the present disclosure provides a compound of formula (I):

[0017]

[0018] A is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group;

[0019] Each R1 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, where at least one occurrence of R1 is hydrogen;

[0020] 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 -(CHR6)CH(SXLY)R6;

[0021] X is S or CH2

[0022] Y is -OH, n = 1-3, R” = H or Me;

[0023] L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group;

[0024] Each R6 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, where at least one occurrence of R6 is hydrogen;

[0025] or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments, one of B, C, and D is -(CHR6)CH(SXL Y)R6. In some embodiments, two of B, C, and D are -(CHR6)CH(SXL Y)R6. In some embodiments, each of B, C, and D is independently -(CHR6)CH(SXL Y)R6.

[0027] In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXL Y)R6, the other two variables are the same. For example, in certain embodiments, when B is -(CHR6)CH(SXL Y)R6, both C and D are the same. In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXL Y)R6, the other two variables are different. For example, in certain embodiments, when B is -(CHR6)CH(SXL Y)R6, C and D are different.

[0028] In some embodiments, two of B, C, and D are the same. In some embodiments, all of B, C, and D are the same. In some embodiments, all of B, C, and D are different.

[0029] In some embodiments, X is S and Y is -OH.

[0030] In some embodiments, X is S. In some embodiments, X is CH2. In certain of these embodiments, Y is -OH.

[0031] In some embodiments, L is an unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is an unsubstituted C2 alkyl.

[0032] In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXL Y)R6, each occurrence of R1 and R6 that is not hydrogen is the same for the two occurrences. In certain embodiments, when one of B, C, and D is -(CHR6)CH(SXL Y)R6, each occurrence of R1 and R6 that is not hydrogen is different for the two occurrences.

[0033] In certain embodiments, when two of B, C, and D are -(CHR6)CH(SXL Y)R6, each occurrence of R1 and R6 that is not hydrogen is the same for the three occurrences. In certain embodiments, when two of B, C, and D are -(CHR6)CH(SXL Y)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(SXL Y)R6, each occurrence of R1 and R6 that is not hydrogen is different for the three occurrences.

[0034] In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-15 alkyl group. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-10 alkyl group. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

[0035] In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic group. In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic group.

[0036] In some embodiments, R1 is

[0037]

[0038] In some embodiments, R1 is a substituted or unsubstituted branched or unbranched C1-15 alkenyl group. In some embodiments, R1 is

[0039]

[0040] In some embodiments, R1 is

[0041]

[0042]

[0043] In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-15 alkyl group. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-10 alkyl group. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

[0044] In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic group. In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic group.

[0045] In some embodiments, R6 is

[0046]

[0047] In some embodiments, R6 is a substituted or unsubstituted branched or unbranched C1-15 alkenyl group. In some embodiments, R6 is

[0048]

[0049] In some embodiments, R6 is

[0050]

[0051]

[0052] In some embodiments, is

[0053]

[0054] In some embodiments, is In some embodiments, is

[0055] In some embodiments, is a substituted or unsubstituted branched or unbranched aliphatic group. In some embodiments, is a substituted or unsubstituted branched or unbranched alkyl group. In some embodiments, is a substituted or unsubstituted branched or unbranched heteroaliphatic group.

[0056] In some embodiments, when one of B, C, and D is methyl, X is S and Y is -OH. In certain of these embodiments, R1 is C 12 H 25 . In certain of these embodiments, R6 is C 12 H 25 . In certain of these embodiments, L is an unsubstituted C2 alkyl group. In certain of these embodiments, is a C3 alkyl group.

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

[0058]

[0059] In some embodiments, the present disclosure provides a compound of formula (II):

[0060] (II)

[0061] A is

[0062] a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group;

[0063] Each of R1, R2, R3, and R4 is independently hydrogen, a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, or a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group, 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;

[0064] X is S or CH2;

[0065] Y is -OH, n = 1 - 3, R” = H or Me;

[0066] L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group;

[0067] or a pharmaceutically acceptable salt thereof.

[0068] In some embodiments, X is S and Y is -OH.

[0069] In some embodiments, X is S. In some embodiments, X is CH2. In certain of these embodiments, Y is -OH.

[0070] In some embodiments, L is an unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is an unsubstituted C2 alkyl.

[0071] 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.

[0072] In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-15 aliphatic group. In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-10 aliphatic group. In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-5 aliphatic group.

[0073] In some embodiments, each of R1, R2, R3, and R4 is independently:

[0074]

[0075] In some embodiments, each of R1, R2, R3, and R4 is independently a substituted or unsubstituted, branched or unbranched C1-15 alkenyl group. In some embodiments, each of R1, R2, R3, and R4 is independently:

[0076]

[0077] In some embodiments, each of R1, R2, R3, and R4 is independently:

[0078]

[0079]

[0080] In some embodiments, each of R1, R2, R3, and R4 is independently an unsubstituted, branched or unbranched C1-15 heteroaliphatic group.

[0081] In some embodiments, each of R1, R2, R3, and R4 is independently C 10 H 21 .

[0082] 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.

[0083] In some embodiments, is

[0084]

[0085] In some embodiments, is In some embodiments, is

[0086] In some embodiments, is a substituted or unsubstituted, branched or unbranched aliphatic group. In some embodiments, is a substituted or unsubstituted, branched or unbranched alkyl group. In some embodiments, is a substituted or unsubstituted, branched or unbranched heteroaliphatic group.

[0087] In some embodiments, the present disclosure provides a compound of the following formula:

[0088]

[0089] In some embodiments, the present disclosure provides a compound of formula (III):

[0090]

[0091] Wherein:

[0092] Each of Ra and Rb is independently

[0093] X is S or CH2,

[0094] Y is -OH, n = 1 - 3, R" = H or Me;

[0095] L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group;

[0096] R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group;

[0097] x is an integer from 1 to 10, including the end values;

[0098] y is an integer from 1 to 10, including the end values;

[0099] Each Ry and Rz is independently

[0100] or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments, Ra is and Rb is

[0102] In some embodiments, Ra is In some embodiments, Ra is In some embodiments, Ra is

[0103] In some embodiments, Rb is In some embodiments, Rb is In some embodiments, Rb is

[0104]

[0105] In some embodiments, x is 1 and y is 2.

[0106] In some embodiments, X is S and Y is -OH.

[0107] In some embodiments, X is S. In some embodiments, X is CH2. In certain ones of these embodiments, Y is -OH.

[0108] In some embodiments, L is an unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is an unsubstituted C2 alkyl.

[0109] 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.

[0110] In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic group. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic group.

[0111] In some embodiments, R7 is C 10 H 21 。

[0112] In some embodiments, Ra is

[0113] In some embodiments, Rb is

[0114] In some embodiments, the present disclosure provides a compound of the following formula:

[0115]

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

[0117]

[0118] Wherein:

[0119] Each of Re and Rf is independently

[0120] x is an integer from 1 to 10, including the end values;

[0121] Each Ry and Rz is independently

[0122] X is S or CH2;

[0123] Y is -OH, n = 1 - 3, R" = H or Me;

[0124] L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group;

[0125] R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group;

[0126] or a pharmaceutically acceptable salt thereof.

[0127] In some embodiments, Re is

[0128] In some embodiments, Rf is

[0129] In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3.

[0130] In some embodiments, X is S and Y is -OH.

[0131] In some embodiments, X is S. In some embodiments, X is CH2. In certain of these embodiments, Y is -OH.

[0132] In some embodiments, L is an unsubstituted branched or unbranched C1-6 alkyl. In some embodiments, L is an unsubstituted C2 alkyl.

[0133] 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.

[0134] In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-10 aliphatic group. In some embodiments, R7 is a substituted or unsubstituted branched or unbranched C1-5 aliphatic group.

[0135] It should be understood that the compounds of any formula disclosed herein and any pharmaceutically acceptable salts thereof include the stereoisomers of the compounds, mixtures of stereoisomers, and polymorphs of all isomeric forms.

[0136] It should be understood that the compounds disclosed herein may exist in non-specified configurations (e.g., not in a specific stereochemistry). Such statements are intended to encompass all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, statements of compounds without a specified configuration herein are intended to refer to each available isomer, tautomer, positional isomer, and stereoisomer of the compound, or any mixture thereof.

[0137] It should be understood that, where applicable, compounds of any formula described herein include the compounds themselves, as well as their salts and their solvates. For example, salts can be formed between an anion and a positively charged group (e.g., an amino group) 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).

[0138] General methods for preparing the compounds of formula (I) - formula (IV) of the present disclosure

[0139] The compounds of the present invention can be prepared by any method known in the art. These compounds can be prepared from commercially available starting materials, such as amines and thioepoxide compounds. These compounds can also be prepared by total synthesis from commercially available starting materials.

[0140] Generally, the first step in preparing the compounds of formulas (I) - (IV) disclosed herein is to select an appropriate amine to prepare the "A" amine nucleus precursor. Non-limiting examples of amines that can be selected to prepare the "A" amine nucleus precursor include:

[0141]

[0142]

[0143] In some embodiments, the next step in preparing the compounds of formulas (I) - (IV) is the reaction of the amine with a terminal thioepoxide to form a precursor compound. The following is an exemplary reaction scheme for the ring-opening of a thioepoxide and attachment to an amine between a primary amine and a thioepoxide to form a precursor compound:

[0144]

[0145] wherein R is an aliphatic group as defined herein.

[0146] In some embodiments, one equivalent of an amine is reacted with one equivalent of a thioepoxide compound. In other embodiments, one equivalent of an amine is reacted with one, two, three, four, five equivalents or more equivalents of a thioepoxide compound. The amount of the thioepoxide compound can be limited to prevent the functionalization of all amino groups. The resulting compound can contain secondary amino groups and / or primary amino groups, which can be further functionalized, for example, with different terminal thioepoxides or different electrophiles. Such further functionalization of the amine can produce compounds with different thioepoxide-derived tails.

[0147] In some embodiments, the amine and the thioepoxide will react at the unsubstituted carbon of the thioepoxide to produce a compound:

[0148] In some embodiments, the amine and the thioepoxide will react at the substituted carbon of the thioepoxide to produce a compound:

[0149] Thioepoxide compounds useful in the present invention include any thioepoxide compound that is racemic or its stereoisomers, all having different chain lengths and bearing functional groups with different degrees of saturation. In certain embodiments, the thioepoxide 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 following formula:

[0150] In some embodiments, the reaction of an amine with a terminal thioepoxide produces a compound having a thiol moiety attached to the carbon at the β-position of the amino group (“β-carbon”).

[0151] In certain embodiments, the preparation of the compounds of formula (I)-(IV) involves reacting an amine with a terminal thioepoxide under conditions to form an “A” amine precursor compound having a disulfide bond attached to the β-carbon of the amino group. For example, the amine can be reacted with a thioepoxide compound in the presence of methyl methanesulfonate to form a precursor compound having a methyl group attached by a disulfide bond to the β-carbon of the amino group.

[0152] In some embodiments, the disulfide can then be treated with a reducing agent to form a free thiol.

[0153] In some embodiments, the final step of preparing the compounds of formula (I)-(IV) involves further functionalization of the thiol moiety. For example, an amine precursor compound can be treated with 2-(2-(pyridin-2-yl)disulfanyl)ethanol to add a disulfide-linked hydroxy group at the β-carbon of the amino group of the amine precursor compound. In another example, an amine precursor compound can be treated with an acrylate or acrylamide to add a sulfur-linked ester or sulfur-linked amide group at the β-carbon of the amino group of the amine precursor compound.

[0154] In some embodiments, the present disclosure provides methods that include reacting one or more equivalents of an amine of one of the following formulas:

[0155]

[0156]

[0157]

[0158] with a sulfur-containing epoxy compound of the following formula: under conditions to form a compound of formula (I)-(IV):

[0159]

[0160]

[0161] wherein A, B, C, D, X, L, Y, R1, R2, R3, R4, Ra, Rb, Re, and Rf are as defined herein.

[0162] In some embodiments, the present disclosure provides a method that includes reacting one or more equivalents of an amine of the following formula: with a sulfur-containing epoxy compound of the following formula: under conditions to form a compound of the following formula:

[0163] (b) Reducing a compound of the following formula: to produce a compound of the following formula: and

[0164] (c) Reacting a compound of the following formula:

[0165] with a compound of the following formula:

[0166] to produce a compound of the following formula:

[0167] wherein A, R1, L, Y, B, C, and D are as described herein.

[0168] In some embodiments, the present disclosure provides a method that includes reacting one or more equivalents of an amine of the following formula: with a sulfur-containing epoxide compound of the following formula: under conditions to form a compound of the following formula:

[0169] (b) reducing a compound of the following formula:

[0170] to produce a compound of the following formula: and

[0171] (c) reacting a compound of the following formula:

[0172] with a compound of the following formula:

[0173] to produce a compound of the following formula:

[0174] wherein R7, L, and Y are as described herein.

[0175] In some embodiments, the preparation of the compounds of formulas (I)-(IV) includes reacting an amine with an aldehyde to form an amine precursor compound. The following is an exemplary reaction scheme for the formation of a precursor compound by reductive amination between an amine and a terminal aldehyde:

[0176]

[0177] wherein R is an aliphatic group as defined herein and Z is hydrogen or -SR.

[0178] In some embodiments, the reaction of the amine with the terminal aldehyde produces a compound having a thiol moiety attached to the β-carbon of the amino group.

[0179] In some embodiments, the preparation of the compounds of formulas (I)-(IV) includes reacting 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, the amine can react with an aldehyde compound containing a methyl disulfide group at the β-position of the aldehyde to form a precursor compound having a methyl disulfide attached to the β-carbon of the amino group.

[0180] In some embodiments, the disulfide can then be treated with a reducing agent to form a free thiol.

[0181] In some embodiments, the final step of preparing the compounds of formula (I)-(IV) includes further functionalization of the thiol moiety. For example, an amine precursor compound can be treated with 2-(2-(pyridin-2-yl)disulfanyl)ethanol to add a disulfide-linked hydroxy group at the β-carbon of the amino group of the amine precursor compound. In another example, an amine precursor compound can be treated with an acrylate or acrylamide to add a sulfur-linked ester or sulfur-linked amide group at the β-carbon of the amino group of the amine precursor compound.

[0182] In some embodiments, the present disclosure provides a method that includes the following steps:

[0183] (a) Prepare an aldehyde of the following formula: (b) Condense an aldehyde of the following formula: with an amine of the following formula: to produce a compound of the following formula: (c) Reduce a compound of the following formula: to produce a compound of the following formula: And (d) React a compound of the following formula: with a compound of the following formula: to produce a compound of the following formula: wherein A, R1, L, Y, B, C, and D are as described herein.

[0184] In some embodiments, one equivalent of an amine is reacted with one equivalent of an aldehyde compound. In other embodiments, one equivalent of an amine is reacted with one, two, three, four, five equivalents, or more equivalents of an aldehyde compound. The amount of the aldehyde compound can be limited to prevent functionalization of all amino groups. The resulting compound can contain secondary amino groups and / or primary amino groups, which can be further functionalized, for example, with different aldehydes or different electrophiles. Such further functionalization of the amine can produce compounds having tails derived from different aldehyde compounds.

[0185] In some embodiments, the preparation of the compounds of formula (I)-(IV) includes selecting or forming an amino alcohol precursor compound. For example, an exemplary amine can be reacted with an epoxide to form an amino alcohol precursor compound by ring opening of the epoxide. In some embodiments, the next step of preparing the compounds of formula (I)-(IV) includes further functionalization of the amino alcohol moiety. For example, an amine precursor compound can be treated with 2-mercaptoethanol to add a sulfur-linked ethanol group at the β-carbon of the amino group of the precursor compound.

[0186] LNP components

[0187] In some aspects, the LNPs of the present disclosure can comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% by mole of at least one compound of the present disclosure. In some aspects, the at least one compound is at least one compound of Formula (I)-(IV) as described herein.

[0188] In some aspects, the LNPs of the present disclosure can comprise about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% by mole of at least one compound of the present disclosure. In some aspects, the at least one compound is at least one compound of Formula (I)-(IV) as described herein.

[0189] In some aspects, the LNP can further comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% by mole of at least one structural lipid of the present disclosure.

[0190] In some aspects, the LNP may further comprise at least about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% by mole of at least one structural lipid.

[0191] In some aspects, the LNP may further comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% by mole of at least one phospholipid.

[0192] In some aspects, the LNP may further comprise at least about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% by mole of at least one phospholipid.

[0193] In some aspects, the LNP may further comprise at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1.0%, or at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10% by mole of PEGylated lipid.

[0194] A. Structural lipids

[0195] In some aspects, the structural lipid can be a steroid. In some aspects, the structural lipid can be a sterol. In some aspects, the structural lipid can comprise cholesterol. In some aspects, the structural lipid can comprise ergosterol. In some aspects, the structural lipid can be a phytosterol.

[0196] B. Phospholipids

[0197] As used herein, the term "phospholipid" is used in its broadest sense and refers to any amphiphilic molecule that contains a polar (hydrophilic) head group and two hydrophobic fatty acid chains, where the head group contains a phosphate ester. In some aspects, the phospholipid can include dioleoylphosphatidylethanolamine (DOPE). In some aspects, the phospholipid can include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some aspects, the phospholipid can include 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[phospho-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-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 salt / 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-phosphoethanolamine), 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), 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.,

[0198] C. PEGylated lipids

[0199] As used herein, the term "PEGylated lipid" is used to refer to any lipid that has been modified (e.g., covalently linked to) with at least one polyethylene glycol molecule. In some aspects, the PEGylated lipid can comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000, hereinafter referred to as DMG-PEG2000.

[0200] LNP compositions

[0201] In some aspects, the lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, and at least one structural lipid.

[0202] In some aspects, the lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, and at least one PEGylated lipid.

[0203] In some aspects, the at least one structural lipid is a mixture of two structural lipids.

[0204] In some aspects, the at least one PEGylated lipid is a mixture of two PEGylated lipids.

[0205] In some aspects, the lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, at least one structural lipid, at least one PEGylated lipid, or any combination thereof.

[0206] In some aspects, the lipid nanoparticle can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, and at least one PEGylated lipid.

[0207] In some aspects, the lipid nanoparticle can comprise at least one nucleic acid molecule, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, at least one PEGylated lipid, or any combination thereof.

[0208] In some aspects, the lipid nanoparticle can comprise at least one nucleic acid, at least one compound of the present disclosure, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid.

[0209] In some aspects, the at least one compound of the present disclosure is a compound of formula (I) - formula (IV).

[0210] In some aspects, the at least one compound of the present disclosure is a mixture of two or more compounds of formula (I) - formula (IV).

[0211] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) at about 54 mol%, at least one structural lipid at about 35 mol%, at least one phospholipid at about 10 mol%, and at least one PEGylated lipid at about 1 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 44 mol% to about 64 mol%, at least one structural lipid at about 25 mol% to about 45 mol%, at least one phospholipid at about 0.1 mol% to about 20 mol%, and at least one PEGylated lipid at about 0.1 mol% to about 11 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 49 mol% to about 59 mol%, at least one structural lipid at about 30 mol% to about 40 mol%, at least one phospholipid at about 5 mol% to about 15 mol%, and at least one PEGylated lipid at about 0.5 mol% to about 6 mol%.

[0212] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) at about 43.3 mol%, at least one structural lipid at about 43.3 mol%, at least one phospholipid at about 12 mol%, and at least one PEGylated lipid at about 1.5 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 33.3 mol% to about 53.3 mol%, at least one structural lipid at about 33.3 mol% to about 53.3 mol%, at least one phospholipid at about 2 mol% to about 22 mol%, and at least one PEGylated lipid at about 0.1 mol% to about 11.5 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 38.3 mol% to about 48.3 mol%, at least one structural lipid at about 38.3 mol% to about 48.3 mol%, at least one phospholipid at about 7 mol% to about 17 mol%, and at least one PEGylated lipid at about 0.5 mol% to about 6.5 mol%.

[0213] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) at about 33.5 mol%, at least one structural lipid at about 33.5 mol%, at least one phospholipid at about 32 mol%, and at least one PEGylated lipid at about 1 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 23.5% to about 43.5 mol%, at least one structural lipid at about 23.5% to about 43.5 mol%, at least one phospholipid at about 22% to about 42 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 28.5% to about 38.5 mol%, at least one structural lipid at about 28.5% to about 38.5 mol%, at least one phospholipid at about 27% to about 37 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%.

[0214] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) at about 49.6 mol%, at least one structural lipid at about 39.9 mol%, at least one phospholipid at about 9.5 mol%, and at least one PEGylated lipid at about 1 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 39.6% to about 59.6 mol%, at least one structural lipid at about 29.9% to about 49.9 mol%, at least one phospholipid at about 0.1% to about 19.5 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 44.6% to about 54.6 mol%, at least one structural lipid at about 34.9% to about 44.9 mol%, at least one phospholipid at about 4.5% to about 14.5 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%.

[0215] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) at about 52.1 mol%, at least one structural lipid at about 45 mol%, at least one phospholipid at about 1.9 mol%, and at least one PEGylated lipid at about 1 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 42.1% to about 62.1 mol%, at least one structural lipid at about 35% to about 55 mol%, at least one phospholipid at about 0.1% to about 11.9 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 47.1% to about 57.1 mol%, at least one structural lipid at about 40% to about 50 mol%, at least one phospholipid at about 0.5% to about 6.9 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%.

[0216] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) at about 60 mol%, at least one structural lipid at about 30 mol%, at least one phospholipid at about 9 mol%, and at least one PEGylated lipid at about 1 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 50% to about 70 mol%, at least one structural lipid at about 20% to about 40 mol%, at least one phospholipid at about 0.1% to about 19 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 55% to about 65 mol%, at least one structural lipid at about 25% to about 35 mol%, at least one phospholipid at about 4% to about 14 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%.

[0217] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) in an amount of about 34% to about 60% by mole, at least one structural lipid in an amount of about 30% to about 60% by mole, at least one phospholipid in an amount of about 5% to about 11.9% by mole, and at least one PEGylated lipid in an amount of about 1% to about 2% by mole. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) in an amount of about 24% to about 70% by mole, at least one structural lipid in an amount of about 20% to about 70% by mole, at least one phospholipid in an amount of about 0.1% to about 21.9% by mole, and at least one PEGylated lipid in an amount of about 0.1% to about 12% by mole. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) in an amount of about 29% to about 65% by mole, at least one structural lipid in an amount of about 25% to about 65% by mole, at least one phospholipid in an amount of about 1% to about 16.9% by mole, and at least one PEGylated lipid in an amount of about 0.5% to about 7% by mole.

[0218] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise at least one compound of formula (I)-(IV) in an amount of about 49.6% to about 60% by mole, at least one structural lipid in an amount of about 30% to about 45% by mole, at least one phospholipid in an amount of about 0.2% to about 9.5% by mole, and at least one PEGylated lipid in an amount of about 1% to about 1.5% by mole. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) in an amount of about 39.6% to about 70% by mole, at least one structural lipid in an amount of about 20% to about 55% by mole, at least one phospholipid in an amount of about 0.1% to about 19.5% by mole, and at least one PEGylated lipid in an amount of about 0.1% to about 11.5% by mole. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) in an amount of about 44.6% to about 65% by mole, at least one structural lipid in an amount of about 25% to about 50% by mole, at least one phospholipid in an amount of about 0.1% to about 14.5% by mole, and at least one PEGylated lipid in an amount of about 0.5% to about 6.5% by mole.

[0219] In some aspects, the compound of formula (I)-(IV) comprised in the LNP composition is one of Compounds 3, 6, 9, 12 - 16, and 19.

[0220] In some aspects of the foregoing LNPs, the structural lipid can be cholesterol. In some aspects of the foregoing LNPs, the phospholipid can be DOPE. In some aspects of the foregoing LNPs, the phospholipid can be DSPC. In some aspects of the foregoing LNPs, the phospholipid can be DOPC. In some aspects of the foregoing LNPs, the PEGylated lipid can be DMG-PEG2000.

[0221] In some aspects of the foregoing LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPE and the PEGylated lipid can be DMG-PEG2000.

[0222] In some aspects of the foregoing LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPC and the PEGylated lipid can be DMG-PEG2000.

[0223] In some aspects of the foregoing LNPs, the at least one nucleic acid molecule is a DNA molecule. In one aspect, the at least one DNA is a DoggyBone DNA molecule. In some aspects, the at least one DNA is a DNA nanoplasmid.

[0224] In some aspects, the lipid nanoparticle comprising at least one nucleic acid can comprise at least one nucleic acid molecule. In some aspects, the lipid nanoparticle can comprise multiple nucleic acid molecules. In some aspects, the at least one nucleic acid molecule or the multiple nucleic acid molecules can be formulated in the lipid nanoparticle.

[0225] In some aspects, the lipid nanoparticle can comprise a specified ratio (weight / weight) of lipid and nucleic acid.

[0226] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise a lipid and a nucleic acid, with a lipid:nucleic acid weight / weight ratio of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1, or about 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or about 115:1 to about 125:1, or about 120:1 to about 130:1, or about 125:1 to about 135:1, or about 130:1 to about 140:1, or about 135:1 to about 145:1, or about 140:1 to about 150:1.

[0227] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise a lipid and a nucleic acid, with a lipid:nucleic acid weight / weight ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1.

[0228] In some aspects, a lipid nanoparticle comprising at least one nucleic acid can comprise a lipid and a nucleic acid, with a lipid:nucleic acid weight / weight ratio of about 10:1, or about 25:1, or about 40:1.

[0229] In some aspects of the foregoing LNP, the at least one nucleic acid molecule is an RNA molecule. In some aspects, the RNA molecule is an mRNA molecule. In some aspects, the mRNA molecule further comprises a 5'-CAP.

[0230] Accordingly, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 54 mol%, at least one structural lipid at about 35 mol%, at least one phospholipid at about 10 mol%, and at least one PEGylated lipid at about 1 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 44% to about 64 mol%, at least one structural lipid at about 25% to about 45 mol%, at least one phospholipid at about 0.1% to about 20 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 49% to about 59 mol%, at least one structural lipid at about 30% to about 40 mol%, at least one phospholipid at about 5% to about 15 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the mRNA molecule further comprises a 5'-CAP. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100:1 (w / w).

[0231] In some aspects, a lipid nanoparticle is provided that comprises at least one compound of formula (I)-(IV) at about 43.3 mol%, at least one structural lipid at about 43.3 mol%, at least one phospholipid at about 12 mol%, and at least one PEGylated lipid at about 1.5 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 33.3 mol% to about 53.3 mol%, at least one structural lipid at about 33.3 mol% to about 53.3 mol%, at least one phospholipid at about 2 mol% to about 22 mol%, and at least one PEGylated lipid at about 0.1 mol% to about 11.5 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 38.3 mol% to about 48.3 mol%, at least one structural lipid at about 38.3 mol% to about 48.3 mol%, at least one phospholipid at about 7 mol% to about 17 mol%, and at least one PEGylated lipid at about 0.5 mol% to about 6.5 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the mRNA molecule further comprises a 5'-CAP. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 90:1 to about 110:1 (w / w), or about 95:1 to about 105:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100:1 (w / w).

[0232] In some aspects, a lipid nanoparticle is provided that comprises at least one compound of formula (I)-(IV) at about 33.5 mol%, at least one structural lipid at about 33.5 mol%, at least one phospholipid at about 32 mol%, and at least one PEGylated lipid at about 1 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 23.5% to about 43.5 mol%, at least one structural lipid at about 23.5% to about 43.5 mol%, at least one phospholipid at about 22% to about 42 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 28.5% to about 38.5 mol%, at least one structural lipid at about 28.5% to about 38.5 mol%, at least one phospholipid at about 27% to about 37 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%, wherein the lipid nanoparticle further comprises at least one RNA molecule (e.g., an mRNA molecule). In some aspects, the mRNA molecule further comprises a 5'-CAP. In some aspects, the lipid-to-nucleic acid ratio in the nanoparticle can be about 30:1 to about 50:1 (w / w) or about 35:1 to about 45:1 (w / w). In some aspects, the lipid-to-nucleic acid ratio in the nanoparticle can be about 40:1 (w / w).

[0233] In some aspects, the nucleic acid molecule is a DNA molecule. Accordingly, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 49.6 mol%, at least one structural lipid at about 39.9 mol%, at least one phospholipid at about 9.5 mol%, and at least one PEGylated lipid at about 1 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 39.6 mol% to about 59.6 mol%, at least one structural lipid at about 29.9 mol% to about 49.9 mol%, at least one phospholipid at about 0.1 mol% to about 19.5 mol%, and at least one PEGylated lipid at about 0.1 mol% to about 11 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle comprising at least one compound of formula (I)-(IV) at about 44.6 mol% to about 54.6 mol%, at least one structural lipid at about 34.9 mol% to about 44.9 mol%, at least one phospholipid at about 4.5 mol% to about 14.5 mol%, and at least one PEGylated lipid at about 0.5 mol% to about 6 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA is a DoggyBone DNA molecule. In some aspects, the at least one DNA is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be from about 110:1 to about 130:1 (w / w), or from about 115:1 to about 125:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 120:1 (w / w).

[0234] In some aspects, a lipid particle is provided that comprises at least one compound of formula (I)-(IV) at about 52.1 mol%, at least one structural lipid at about 45 mol%, at least one phospholipid at about 1.9 mol%, and at least one PEGylated lipid at about 1 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 42.1% to about 62.1 mol%, at least one structural lipid at about 35% to about 55 mol%, at least one phospholipid at about 0.1% to about 11.9 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 47.1% to about 57.1 mol%, at least one structural lipid at about 40% to about 50 mol%, at least one phospholipid at about 0.5% to about 6.9 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA is a DoggyBone DNA molecule. In some aspects, the at least one DNA is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be from about 90:1 to about 110:1 (w / w), or from about 95:1 to about 105:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 100:1 (w / w).

[0235] In some aspects, a lipid particle is provided that comprises at least one compound of formula (I)-(IV) at about 60 mol%, at least one structural lipid at about 30 mol%, at least one phospholipid at about 9 mol%, and at least one PEGylated lipid at about 1 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 50% to about 70 mol%, at least one structural lipid at about 20% to about 40 mol%, at least one phospholipid at about 0.1% to about 19 mol%, and at least one PEGylated lipid at about 0.1% to about 11 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) at about 55% to about 65 mol%, at least one structural lipid at about 25% to about 35 mol%, at least one phospholipid at about 4% to about 14 mol%, and at least one PEGylated lipid at about 0.5% to about 6 mol%, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA is a DoggyBone DNA molecule. In some aspects, the at least one DNA is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be from about 110:1 to about 130:1 (w / w), or from about 115:1 to about 125:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 120:1 (w / w).

[0236] In some aspects, a lipid particle is provided that comprises at least one compound of formula (I)-(IV) in an amount of about 34% to about 60% by mole, at least one structural lipid in an amount of about 30% to about 60% by mole, at least one phospholipid in an amount of about 5% to about 11.9% by mole, and at least one PEGylated lipid in an amount of about 1% to about 2% by mole, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) in an amount of about 24% to about 70% by mole, at least one structural lipid in an amount of about 20% to about 70% by mole, at least one phospholipid in an amount of about 0.1% to about 21.9% by mole, and at least one PEGylated lipid in an amount of about 0.1% to about 12% by mole, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) in an amount of about 29% to about 65% by mole, at least one structural lipid in an amount of about 25% to about 65% by mole, at least one phospholipid in an amount of about 1% to about 16.9% by mole, and at least one PEGylated lipid in an amount of about 0.5% to about 7% by mole, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA is a DoggyBone DNA molecule. In some aspects, the at least one DNA is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 70:1 to about 130:1 (w / w), or about 75:1 to about 125:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be about 80:1 (w / w) to about 120:1 (w / w).

[0237] In some aspects, a lipid particle is provided that comprises at least one compound of formula (I)-(IV) in an amount of about 49.6% to about 60% by mole, at least one structural lipid in an amount of about 30% to about 45% by mole, at least one phospholipid in an amount of about 0.2% to about 9.5% by mole, and at least one PEGylated lipid in an amount of about 1% to about 1.5% by mole, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) in an amount of about 39.6% to about 70% by mole, at least one structural lipid in an amount of about 20% to about 55% by mole, at least one phospholipid in an amount of about 0.1% to about 19.5% by mole, and at least one PEGylated lipid in an amount of about 0.1% to about 11.5% by mole, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the present disclosure provides a lipid nanoparticle that comprises at least one compound of formula (I)-(IV) in an amount of about 44.6% to about 65% by mole, at least one structural lipid in an amount of about 25% to about 50% by mole, at least one phospholipid in an amount of about 0.1% to about 14.5% by mole, and at least one PEGylated lipid in an amount of about 0.5% to about 6.5% by mole, wherein the at least one nucleic acid comprises at least one DNA molecule. In one aspect, the at least one DNA is a DoggyBone DNA molecule. In some aspects, the at least one DNA is a DNA nanoplasmid. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be from about 70:1 to about 130:1 (w / w), or from about 75:1 to about 125:1 (w / w). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle can be from about 80:1 (w / w) to about 120:1 (w / w).

[0238] In some aspects, the compound of formula (I)-(IV) comprised in the LNP composition is one of Compounds 3, 6, 9, 12-16, and 19.

[0239] In some aspects of the foregoing LNP, the structural lipid can be cholesterol. In some aspects of the foregoing LNP, the phospholipid can be DOPE. In some aspects of the foregoing LNP, the phospholipid can be DSPC. In some aspects of the foregoing LNP, the phospholipid can be DOPC. In some aspects of the foregoing LNP, the PEGylated lipid can be DMG-PEG2000.

[0240] In some aspects of the foregoing LNP, the structural lipid can be cholesterol, the phospholipid can be DOPE and the PEGylated lipid can be DMG-PEG2000.

[0241] In some aspects of the foregoing LNPs, the structural lipid can be cholesterol, the phospholipid can be DOPC, and the PEGylated lipid can be DMG-PEG2000.

[0242] The pharmaceutical compositions of the present disclosure

[0243] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the present disclosure and at least one second nanoparticle of the present disclosure, wherein the at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, and wherein the at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon. In some aspects, the at least one nucleic acid molecule encoding at least one transposase can be an RNA molecule (e.g., an mRNA molecule), and the at least one nucleic acid molecule encoding at least one transposon can be a DNA molecule (e.g., a DoggyBone DNA molecule or a DNA nanoplasmid).

[0244] In some aspects, the present disclosure provides a composition comprising at least one cell that has been contacted with at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using any method of the present disclosure.

[0245] In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been contacted with at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been genetically modified using at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one cell that has been genetically modified using any method of the present disclosure.

[0246] The methods of the present disclosure

[0247] The present disclosure provides a method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0248] In all of the methods, compositions, and kits of the present disclosure, at least one cell can be a liver cell. Liver cells can include, but are not limited to, hepatocytes, hepatic stellate cells, Kupffer cells, or liver sinusoidal endothelial cells. In all of the methods, compositions, and kits of the present disclosure, at least one cell can be a T cell. T cells can be resting T cells, activated T cells, stem cell memory T cells (TscM cells), central memory T cells (T CM ) or stem cell-like T cells.

[0249] 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.

[0250] The present disclosure provides a method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0251] In some aspects, genetically modifying the cell can include delivering at least one exogenous nucleic acid to the cell such that the cell expresses at least one protein that the cell does not normally express, or such that the at least one cell expresses the at least one protein at a level higher than the level at which the cell normally expresses the at least one protein, or such that the cell expresses at least one protein at a level lower than the level at which the cell normally expresses the at least one protein. In some aspects, genetically modifying the cell can include delivering at least one exogenous nucleic acid to the cell such that the at least one exogenous nucleic acid integrates into the genome of the at least one cell.

[0252] In all of the 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 aspects, standard techniques known in the art can be used to activate T cells, 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.

[0253] In some aspects, T cells can be activated at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 10 hours, or at least 11 hours, or at least 12 hours, or at least 13 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 17 hours, or at least 18 hours, or at least 19 hours, or at least 20 hours, or at least 21 hours, or at least 22 hours, or at least 23 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 60 hours or at least 72 hours before contact with at least one composition or nanoparticle of the present disclosure.

[0254] In some aspects, T cells can be activated at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 10 hours, or at least 11 hours, or at least 12 hours, or at least 13 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 17 hours, or at least 18 hours, or at least 19 hours, or at least 20 hours, or at least 21 hours, or at least 22 hours, or at least 23 hours, or at least 24 hours, or at least 36 hours, or at least 48 hours, or at least 60 hours or at least 72 hours after contact with at least one composition or nanoparticle of the present disclosure.

[0255] In some aspects of the foregoing method, step c) can be carried out 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).

[0256] In some aspects of the foregoing method, step a) can be carried out 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).

[0257] In some aspects, the methods of the present disclosure can generate 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 plurality of cells express at least one protein, and the plurality of cells express at least one protein encoded by at least one nucleic acid delivered to the plurality of cells via the nanoparticles of the present disclosure.

[0258] In some aspects, the methods of the present disclosure can generate 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 plurality of cells are stem memory T cells.

[0259] In some aspects, the methods of the present disclosure can generate 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 express stem memory T cells (TSCM ) or one or more cell surface markers of TscM-like cells and wherein the one or more cell surface markers comprise CD62L and CD45RA.

[0260] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure, the at least one composition comprising at least one nucleic acid encoding a therapeutic protein.

[0261] 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, the at least one nanoparticle comprising at least one nucleic acid encoding a therapeutic protein.

[0262] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of cells that have been contacted with at least one nanoparticle of the present disclosure, the at least one nanoparticle comprising at least one nucleic acid encoding a therapeutic protein. The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of cells that have been genetically modified using a composition and / or method of the present disclosure.

[0263] In some aspects, the at least one disease can be a malignant disease, including but not limited to cancer. In some aspects, the at least one disease can be a metabolic liver disorder (MLD). In some aspects, the at least one disease can be a urea cycle disorder (UCD). MLD and / or UCD can include but are not limited to N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthetase I deficiency (CPSI deficiency), ornithine carbamoyltransferase (OTC) deficiency, argininosuccinate synthetase deficiency (ASSD) (citrullinemia I), citrin protein deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), or any combination thereof.

[0264] In some aspects, the at least one disease can be hemophilia A

[0265] Accordingly, the present disclosure provides a method of 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 an FVIII polypeptide.

[0266] 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.

[0267] 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.

[0268] Nucleic acid molecules

[0269] In some aspects, the nucleic acid molecule can be an RNA molecule. Thus, in some aspects, the lipid nanoparticle can comprise at least one RNA molecule. The at least one RNA molecule can be encapsulated within the lipid nanoparticle. In some aspects, the RNA molecule can be an mRNA molecule. In some aspects, the lipid nanoparticle can comprise at least one mRNA molecule. The mRNA molecule can be encapsulated within the lipid nanoparticle.

[0270] In some aspects, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some aspects, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some aspects, the non-naturally occurring nucleotide can comprise at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some aspects, the nucleic acid molecule can be a modified nucleic acid molecule. In some aspects, the modified nucleic acid molecule can comprise at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0271] In some aspects, any method known in the art and / or a capping moiety can be used to cap the mRNA molecule. The mRNA molecule 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". The mRNA molecule can be capped with a moiety. The moiety can comprise an m7G(5')ppp(5')(2'OMeA)( AG) moiety. The moiety can comprise an m7G(5')ppp(5')(2'OMeG)( GG) moiety. The mRNA molecule can be capped with an anti-reverse cap analog moiety. The moiety can comprise an m7(3'-O-methyl)G(5')ppp(5')G moiety. The mRNA molecule can be capped with a 3'OMe moiety .

[0272] In some aspects, the mRNA molecule can comprise at least one modified nucleic acid.

[0273] The at least one modified nucleic acid can comprise 5-methoxyuridine (5moU). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. In some aspects, all of the uridine bases in the mRNA molecule are 5-methoxyuridine bases. Without wishing to be bound by theory, 5-methoxyuridine can improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849 - 853 and Vaidyanathan et al., Molecular Therapy - Nucleic Acids, 2018, 12, 530 - 542).

[0274] In some aspects, the mRNA molecule can comprise at least one modified nucleic acid.

[0275] The at least one modified nucleic acid can comprise N1-methylpseudouridine (me 1 Ψ) . In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA are N1-methylpseudouridine bases. In some aspects, 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).

[0276] In some aspects, the mRNA molecule can comprise at least one modified nucleic acid.

[0277] The at least one modified nucleic acid can comprise pseudouridine ('Ψ). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the uridine bases in the mRNA are pseudouridine bases. In some aspects, all of the uridine bases in the mRNA molecule are pseudouridine bases. Without wishing to be bound by theory, pseudouridine can improve protein expression and reduce immunogenicity (see Li et al., Bioconjugate Chem. 2016, 27, 3, 849 - 853 and Vaidyanathan et al., Molecular Therapy - Nucleic Acids, 2018, 12, 530 - 542).

[0278] In some aspects, the mRNA molecule can comprise at least one modified nucleic acid.

[0279] The at least one modified nucleic acid can comprise 5 - methylcytidine (5 - MeC). In some aspects, at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, at least about 90%, or at least about 95%, or at least about 99% of the cytidine bases in the mRNA are 5 - MeC bases. In some aspects, all of the cytidine bases in the mRNA molecule are 5 - MeC bases.

[0280] In some aspects, the nucleic acid molecule can comprise a DNA molecule. Thus, in some aspects, the lipid nanoparticle can comprise a DNA molecule. In some aspects, the DNA molecule can be a circular DNA molecule, such as but not limited to a DNA plasmid or a DNA nanoplasmid. Thus, in some aspects, the lipid nanoparticle can comprise a circular DNA molecule. In some aspects, the lipid nanoparticle can comprise Doggybone DNA. In some aspects, the lipid nanoparticle can comprise a DNA plasmid. In some aspects, the lipid nanoparticle can comprise a DNA nanoplasmid. In some aspects, the DNA molecule can be a linearized DNA molecule, such as but not limited to a linearized DNA plasmid or a linearized DNA nanoplasmid.

[0281] The DNA plasmid or DNA nanoplasmid can be at least about 0.25 kb in length, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb.

[0282] In some aspects, the nucleic acid molecules formulated in the lipid nanoparticles of the present disclosure can comprise at least one transgene sequence. In some aspects, the transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, the transgene sequence can comprise a nucleotide sequence encoding at least one transposase. In some aspects, the transgene sequence can comprise a nucleotide sequence encoding at least one transposon. In some aspects, the transposon can comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, the transposon can comprise a nucleotide sequence encoding at least one therapeutic protein and at least one promoter sequence, wherein the at least one therapeutic protein is operably linked to the at least one promoter sequence.

[0283] In some aspects, the therapeutic protein can be an ornithine carbamoyltransferase (OTC) polypeptide, a methylmalonyl-CoA mutase (MUT1) polypeptide, a chimeric antigen receptor, or a factor VIII (FVIII) polypeptide.

[0284] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform. In some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0285] In some aspects, the microfluidic mixing platform can produce the lipid nanoparticles of the present invention by combining a miscible solvent phase containing the lipid components of the nanoparticles and an aqueous phase containing the lipid nanoparticle cargo (e.g., nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some aspects, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow immediate mixing of the two phases. As the two phases move under laminar flow in the microfluidic channel, the microscopic features in the channel can allow controlled and uniform mixing to produce the lipid nanoparticles of the present disclosure.

[0286] In some aspects, the microfluidic mixing platform can include, but is not limited to Spark (Precision NanoSystems), Ignite TM (Precision NanoSystems), Benchtop (Precision NanoSystems), Blaze (Precision NanoSystems) or GMP system (Precision NanoSystems).

[0287] In some aspects, a microfluidic mixing platform can be used to generate the lipid nanoparticles of the present disclosure, wherein the microfluidic mixing platform mixes at a rate of at least about 2.5 mL / min, or at least about 5 mL / min, or at least about 7.5 mL / min, or at least about 10 mL / min, or at least about 12.5 mL / min, or at least about 15 mL / min, or at least about 17.5 mL / min, or at least about 20 mL / min, or at least about 22.5 mL / min, or at least about 25 mL / min, or at least about 27.5 mL / min, or at least about 30 mL / min.

[0288] In some aspects, a microfluidic mixing platform can be used to generate the lipid nanoparticles of the present disclosure, wherein the microfluidic mixing platform mixes a miscible solvent phase and an aqueous phase at a solvent phase:aqueous phase 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.

[0289] piggyBac ITR sequences

[0290] In some aspects, the nucleic acid can comprise a piggBac ITR sequence. In some aspects, the nucleic acid can comprise a first piggyBac ITR sequence and a second piggBac ITR sequence.

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

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

[0293] Promoter sequences

[0294] In some aspects, the nucleic acid can comprise a promoter sequence. In some aspects, the promoter sequence can include any promoter sequence known in the art. In some aspects, the promoter sequence can include any liver-specific promoter sequence known in the art.

[0295] In some aspects, the promoter sequence can include a hybrid liver promoter (HLP). In some aspects, the promoter sequence can include an LP1 promoter. In some aspects, the promoter sequence can include the leukocyte-specific expression of the pp52 (LSP1) long promoter. In some aspects, the promoter sequence can include a thyroxine-binding globulin (TBG) promoter.

[0296] In some aspects, the promoter sequence can include a wTBG promoter. In some aspects, the promoter sequence can include a hepatic combinatorial bundle (HCB) promoter. In some aspects, the promoter sequence can include a 2xApoE-hAAT promoter. In some aspects, the promoter sequence can include the leukocyte-specific expression of pp52 (LSP1) plus a chimeric intron promoter. In some aspects, the promoter sequence can include a cytomegalovirus (CMV) promoter.

[0297] Transgene sequences

[0298] In some aspects, the transgenic sequence can include a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide. The MUT1 polypeptide can be any MUT1 polypeptide known in the art.

[0299] In some aspects, the transgenic sequence can include a nucleic acid sequence encoding an ornithine carbamoyltransferase (OTC) polypeptide. The OTC polypeptide can be any OTC polypeptide known in the art.

[0300] In some aspects, the transgenic sequence can include a nucleic acid sequence encoding a factor VIII (FVIII) polypeptide. The FVIII polypeptide can be any FVIII polypeptide known in the art.

[0301] In some aspects, the transgenic sequence can include a nucleic acid sequence encoding an iCAS9 polypeptide.

[0302] In some aspects, the transgenic sequence can be codon-optimized according to methods known in the art.

[0303] In some aspects, at least one transgenic sequence can be operably linked to at least one promoter sequence present in the same polynucleotide.

[0304] polyA sequences

[0305] In some aspects, the nucleic acid can include a polyA sequence. In some aspects, the polyA sequence can include any polyA sequence known in the art.

[0306] Self-cleaving peptide sequences

[0307] In some aspects, the nucleic acid can comprise a self-cleaving peptide sequence. In some aspects, the self-cleaving peptide sequence can comprise any self-cleaving peptide sequence known in the art. In some aspects, the self-cleaving peptide sequence can 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.

[0308] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a T2A peptide.

[0309] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-T2A peptide.

[0310] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding an E2A peptide.

[0311] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-E2A peptide.

[0312] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding an F2A peptide.

[0313] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-F2A peptide.

[0314] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a P2A peptide.

[0315] In some aspects, the self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-P2A peptide.

[0316] Chimeric antigen receptor (CAR)

[0317] The transgenic sequence can comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises: an extracellular domain comprising at least one antigen recognition region; a transmembrane domain and an intracellular domain comprising at least one co-stimulatory domain. The CAR can further comprise a hinge region between the antigen recognition domain and the transmembrane domain.

[0318] The antigen recognition region can comprise at least one single-chain variable fragment (scFv), Centyrin, a single-domain antibody, or a combination thereof. In one aspect, the at least one single-domain antibody is a VHH. In one aspect, the at least one single-domain antibody is a VH.

[0319] scFv

[0320] In some aspects, the antigen recognition region of the CAR can comprise one or more scFvs configured to recognize and bind a specific target protein / antigen. The antigen recognition region can comprise at least two scFvs. The antigen recognition region can comprise at least three scFvs. In one aspect, the CARs of the present disclosure are bispecific CARs comprising at least two scFvs that specifically bind two different antigens.

[0321] The scFv composition can comprise the variable heavy and variable light 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, and the VH and VL domains are linked by a short peptide linker. Although the constant regions are removed and a linker is introduced, the scFv can retain the specificity of the original immunoglobulin.

[0322] Centyrin

[0323] In some aspects, the antigen recognition region of the CAR can comprise one or more Centyrins configured to recognize and bind a specific target protein / antigen. A Centyrin that specifically binds an antigen can be used to direct the specificity of a cell (e.g., a cytotoxic immune cell) to a particular antigen. A CAR comprising a Centyrin is referred to herein as a CARTyrin.

[0324] The Centyrins of the present disclosure can comprise a protein scaffold, wherein the scaffold is capable of specifically binding an antigen. The Centyrins of the present disclosure can comprise a protein scaffold comprising a consensus sequence of at least one type III fibronectin (FN3) domain, wherein the scaffold is capable of specifically binding an antigen. The at least one type III fibronectin (FN3) domain can be derived from a human protein. The human protein can be tenascin-C.

[0325] The consensus sequence can be modified at one or more positions within the following structures: (a) within the A-B loop at positions 13-16 of the consensus sequence; (b) within the B-C loop at positions 22-28 of the consensus sequence; (c) within the C-D at positions 38-43 of the consensus sequence; (d) within the D-E loop at positions 51-54 of the consensus sequence; (e) within the E-F loop at positions 60-64 of the consensus sequence; (f) within the F-G loop at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). The Centyrins of the present disclosure can comprise a consensus sequence of at least 5 type III fibronectin (FN3) domains, at least 10 type III fibronectin (FN3) domains, or at least 15 type III fibronectin (FN3) domains.

[0326] 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 a naturally occurring antibody. Antibody mimetics can include proteins, nucleic acids, or small molecules. The target sequence specifically bound by the antibody mimetics of the present disclosure can be an antigen. Antibody mimetics can offer properties superior to antibodies, including but not limited to, excellent solubility, tissue permeability, stability to heat and enzymes (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), Fynomomers, Kunitz domain peptides, and monobodies.

[0327] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one affibody molecule. The affibody molecule of the present disclosure comprises a protein scaffold that comprises one or more α-helices or consists of one or more α-helices without any disulfide bridges. Preferably, the affibody molecule of the present disclosure comprises three α-helices or consists of three α-helices. For example, the affibody molecule of the present disclosure can comprise an immunoglobulin-binding domain. The affibody molecule of the present disclosure can comprise the Z domain of protein A.

[0328] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one affilin molecule. The affilin molecule of the present disclosure comprises a protein scaffold produced by modifying the exposed amino acids of, for example, γ-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 for preparing affilins, those amino acids that are accessible to the solvent or possible binding partners in the correctly folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids can be modified to specifically bind to a target sequence or antigen.

[0329] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one affimer molecule. The affimer molecule of the present disclosure comprises a protein scaffold that comprises a highly stable protein that has been engineered to display a peptide loop that provides a high-affinity binding site for a specific target sequence. Exemplary affimer molecules of the present disclosure comprise a protein scaffold based on the cystatin protein or its tertiary structure. Exemplary affimer molecules of the present disclosure can have a common tertiary structure that comprises an α-helix located above an antiparallel β-sheet.

[0330] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one affitin molecule. The affitin molecules of the present disclosure comprise an artificial protein scaffold, the structure of which can be derived from, for example, a DNA-binding protein (e.g., the DNA-binding protein Sac7d). The affitins of the present disclosure selectively bind to a target sequence, which can be the whole or a part of an antigen. Exemplary affitins of the present disclosure are made by randomizing one or more amino acid sequences on the binding surface of a DNA-binding protein and subjecting the resulting proteins to ribosome display and selection. The target sequences of the affitins of the present disclosure can be found, for example, in the genome or on the surface of a peptide, protein, virus, or bacterium. In some aspects, affitins can be used as specific inhibitors of enzymes. The affitin molecules of the present disclosure can comprise a heat-resistant protein or a derivative thereof.

[0331] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one Alphabody molecule. The Alphabody molecules of the present disclosure can also be referred to as cell-penetrating Alphabodies (CPABs). The Alphabody molecules of the present disclosure comprise small proteins (generally 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-chain α helix (similar to a naturally occurring coiled-coil structure). The Alphabody molecules of the present disclosure can comprise a protein scaffold that contains one or more amino acids that have been modified to specifically bind to a target protein. Regardless of the binding specificity of the molecule, the alphabody molecules of the present disclosure maintain proper folding and thermal stability.

[0332] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one Anticalin molecule. The Anticalin molecules of the present disclosure comprise artificial proteins that bind to a target sequence or target site in a protein or small molecule. The Anticalin molecules of the present disclosure can comprise artificial proteins derived from human lipocalins. The Anticalin molecules can be used in place of, for example, monoclonal antibodies or fragments thereof. The Anticalin molecules can demonstrate superior tissue penetration and thermal stability compared to monoclonal antibodies or fragments thereof. Exemplary anticalin molecules of the present disclosure can comprise approximately 180 amino acids and have a mass of approximately 20 kDa. Structurally, the anticalin molecules of the present disclosure comprise a barrel structure that contains eight antiparallel β-strands paired by loops and attached α-helices. In some aspects, the anticalin molecules of the present disclosure comprise a barrel structure that contains eight antiparallel β-strands paired by loops and attached α-helices.

[0333] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one Avimer molecule. The Avimer molecules of the present disclosure comprise artificial proteins that specifically bind to a target sequence (which can also 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 more than one target, the avimers mimic the function of bispecific antibodies. The artificial protein avimer can comprise two or more peptide sequences each about 30 - 35 amino acids in length. These peptides can be linked via one or more linker peptides. The amino acid sequence of one or more peptides of the avimer can be derived from the A domain of a membrane receptor. The avimer has a rigid structure that can optionally comprise disulfide bonds and / or calcium. Compared to antibodies, the avimers of the present disclosure can exhibit higher thermal stability.

[0334] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one DARPin. The DARPin (designed ankyrin repeat protein) of the present disclosure comprises a genetically engineered, recombinant, or chimeric protein that has high specificity and high affinity for a target sequence. In some aspects, the DARPin of the present disclosure is derived from ankyrin and optionally comprises at least three repeat motifs (also referred to as repeat units) of ankyrin. Ankyrin mediates high-affinity protein-protein interactions. The DARPin of the present disclosure comprises a large target interaction surface.

[0335] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one Fynomer. The Fynomer of the present disclosure comprises a small binding protein (about 7 kDa) derived from the human Fyn SH3 domain and is engineered to bind to a target sequence and molecule with an affinity and specificity equal to that of an antibody.

[0336] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one Kunitz domain peptide. The Kunitz domain peptides of the present disclosure comprise a protein scaffold containing a Kunitz domain. The Kunitz domain contains an active site that inhibits protease activity. Structurally, the Kunitz domain of the present disclosure comprises an α + β fold rich in disulfide bonds. This structure is exemplified by bovine pancreatic trypsin inhibitor. The Kunitz domain peptide recognizes a specific protein structure and acts as a competitive protease inhibitor. The Kunitz domain of the present disclosure can comprise ecallantide (derived from human lipoprotein-associated coagulation inhibitor (LACI)).

[0337] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding at least one monomer. The monomers of the present disclosure are small proteins (comprising approximately 94 amino acids and having a mass of approximately 10 kDa) that are comparable in size to single-chain antibodies. These genetically engineered proteins specifically bind to target sequences including antigens. The monomers of the present disclosure can specifically target one or more different proteins or target sequences. In some aspects, the monomers of the present disclosure comprise a protein scaffold that mimics the structure of human fibronectin, and more preferably, the structure of the tenth extracellular type III domain of fibronectin. The tenth extracellular type III domain of fibronectin, and its monomeric mimics, contain seven beta-sheets that form a barrel and three exposed loops on each side corresponding to the three complementarity-determining regions (CDRs) of an antibody. In contrast to the structure of the antibody variable domain, the monomer lacks any binding sites for metal ions and the central disulfide bond. Multispecific monomers can be optimized by modifying loops BC and FG. The monomers of the present disclosure can comprise adnectin.

[0338] VHH

[0339] In some aspects, the antigen recognition region of the CAR can comprise at least one single-domain antibody (SdAb) to recognize and bind to a specific target protein / antigen. In one aspect, the single-domain antibody is a VHH. A VHH is a heavy-chain antibody found in camelids. A VHH that specifically binds an antigen can be used to direct the specificity of a cell (e.g., a cytotoxic immune cell) to a specific antigen. The antigen recognition region can comprise at least two VHHs. The antigen recognition region can comprise at least three VHHs. In one aspect, the CAR of the present disclosure is a bispecific CAR comprising at least two VHHs that specifically bind two different antigens. A CAR comprising a VHH is referred to herein as a VCAR.

[0340] At least one VHH protein or VCAR of the present disclosure can optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987 - 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994 - 2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997 - 2001).

[0341] The amino acids of the VHH protein can be altered, added, and / or deleted to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, binding rate, dissociation rate, avidity, specificity, half-life, stability, solubility, or any other suitable characteristic known in the art.

[0342] Optionally, the VHH protein can be engineered to retain high affinity for the antigen and other favorable biological properties. To achieve this, the VHH protein can optionally be prepared by methods that analyze the parental sequence and various conceptual engineered products using three-dimensional models of the parental sequence and the engineered sequences. Three-dimensional models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of the selected candidate sequences and can measure possible immunogenicity (e.g., the Immunofilter program, Xencor, Inc., Monrovia, Calif). Examination of these displays allows analysis of the possible role of residues in the function of the candidate sequence, i.e., analysis of the residues that affect the ability of the candidate VHH protein to bind its antigen. In this way, residues can be selected and combined from the parental sequence and reference sequences to obtain 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 for VHHs that specifically bind to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, such as in vitro display. Competitive assays can be performed with the VHH or VCAR of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the VHH or VCAR of the present disclosure for binding to the target protein and / or share epitope regions. These assays, which are readily known to those of ordinary skill in the art, evaluate the competition between antagonists or ligands for a limited number of binding sites on the protein.

[0343] VH

[0344] In some aspects, the antigen recognition region of the CAR can comprise at least one single-domain antibody (SdAb) to recognize and bind a specific target protein / antigen. In one aspect, the single-domain antibody is a VH. A VH is a single-domain binder derived from common IgG. A VH that specifically binds an antigen can be used to direct the specificity of a cell (e.g., a cytotoxic immune cell) to a specific antigen. The antigen recognition region can comprise at least two VHs. The antigen recognition region can comprise at least three VHs. In one aspect, the CAR of the present disclosure is a bispecific CAR comprising at least two VHs that specifically bind two different antigens.

[0345] The VH can be isolated from 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 aspects, the VH is a fully humanized VH. In some aspects, the VH is neither a naturally occurring antibody nor a fragment of a naturally occurring antibody. In some aspects, the VH is not a fragment of a monoclonal antibody. In some aspects, the VH is a UniDab antibody (TeneoBio). In some aspects, the VH is modified to remove the Fc domain or a portion thereof. In some aspects, the framework sequence of the VH is modified to, for example, improve expression, reduce immunogenicity, or improve function.

[0346] The VH can be fully engineered using the UniRat (TeneoBio) system and "NGS-based discovery" to generate the VH. Using this method, a particular VH is not naturally occurring but is generated using a fully engineered system. The VH is not derived from a naturally occurring monoclonal antibody (mAb) isolated directly from a host (e.g., mouse, rat, or human) or directly from a single clone (hybridoma) of a cell or cell line. These VHs are not subsequently cloned from the cell line. Instead, the UniRat system is used as a transgene to fully engineer the VH sequence, the transgene comprising a human variable region (VH domain) with a rat Fc domain and thus being a human / rat chimera without a light chain and different from the standard mAb format. The native rat gene is knocked out, and the only antibodies expressed in the rat are from the transgene with a VH domain linked to a rat Fc (UniAb). These are proprietary Abs expressed in UniRat. Next-generation sequencing (NGS) and bioinformatics are used to identify the complete antigen-specific repertoire of heavy-chain antibodies generated by UniRat after immunization. Then, using a unique gene assembly method, the antibody library sequence information is converted into a large collection of fully human heavy-chain antibodies that can be screened for multiple functions in vitro. In some aspects, fully humanized VHs are generated by fusing human VH domains with human Fcs in vitro (to generate non-naturally occurring recombinant VH antibodies). In some aspects, the VHs are fully humanized, but they are expressed in vitro as human / rat chimeras without light chains (human VH, rat Fc). The fully humanized VHs are expressed in vivo because the human / rat chimera without a light chain (human VH, rat Fc) is approximately 80 kDa (versus 150 kDa).

[0347] The CARs of the present disclosure can bind to a human antigen with at least one affinity selected from the following K D less than or equal to 10 -9 M, less than or equal to 10 -10 M, less than or equal to 10 -11 M, less than or equal to 10 -12 M, less than or equal to 10 -13 M, less than or equal to 10-14 M and less than or equal to 10 -15 M.K D It can be measured by any means, including but not limited to surface plasmon resonance.

[0348] In one aspect, the antigen recognition region of the disclosed CAR comprises at least one anti-BCMA Centyrin. A CAR comprising anti-BCMA Centyrin is referred to herein as BCMA CARTyrin.

[0349] In some aspects, the nanoparticles of the present disclosure can comprise a nucleic acid sequence encoding BCMA CARTyrin.

[0350] In one aspect, the antigen recognition region of the disclosed CAR comprises at least one anti-PSMA Centyrin. A CAR comprising anti-PSMA Centyrin is referred to herein as PSMA CARTyrin.

[0351] In some aspects, the nanoparticles of the present disclosure can comprise a nucleic acid sequence encoding PSMA CARTyrin.

[0352] In one aspect, the antigen recognition region of the disclosed CAR comprises at least one anti-BCMA VH. A CAR comprising anti-BCMA VH is referred to herein as BCMA VCAR.

[0353] In some aspects, the nanoparticles of the present disclosure can comprise a nucleic acid sequence encoding BCMA VCAR.

[0354] The extracellular domain can comprise a signal peptide. The signal peptide can comprise a sequence encoding the signal peptide of human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR. In a preferred aspect, the signal peptide comprises, consists essentially of, or consists of: the human CD8 alpha (CD8α) signal peptide (SP) or a portion thereof.

[0355] The hinge domain or hinge region can comprise the sequence of human CD8α, IgG4, CD4 or a combination thereof. In a preferred aspect, the hinge can comprise, consist essentially of, or consist of: the human CD8 alpha (CD8α) hinge or a portion thereof.

[0356] The transmembrane domain can comprise, consist essentially of, or consist of the following: a sequence encoding the transmembrane domain of human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR. Preferably, the transmembrane domain can comprise, consist essentially of, or consist of the human CD8 alpha (CD8α) transmembrane domain or a portion thereof.

[0357] The at least one co-stimulatory domain can comprise, consist essentially of, or consist of the following: human 4-1BB, CD28, CD3 zeta (CD3ζ), CD40, ICOS, MyD88, the intracellular domain of OX-40, or any combination thereof. Preferably, the at least one co-stimulatory domain comprises CD3ζ, the 4-1BB co-stimulatory domain, or a combination thereof.

[0358] Transposon systems

[0359] In some aspects, the nucleic acid can comprise a transposon or a nano-transposon, which comprises: (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) a sequence within the ITR or a sequence encoding the sequence within the ITR, wherein the sequence within the ITR comprises a transposon sequence or a sequence encoding a transposon.

[0360] In some aspects, the nucleic acid can comprise a transposon or a nano-transposon, which comprises: (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) a sequence within the ITR or a sequence encoding the sequence within the ITR, wherein the sequence within the ITR comprises a transposon sequence or a sequence encoding a transposon, and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding the inter-ITR sequence, wherein the length of the inter-ITR sequence is equal to or less than 700 nucleotides.

[0361] The transposon or nano-transposon of the present disclosure comprises a protein scaffold (e.g., a CAR comprising at least one scFv, a single-domain antibody, or a Centyrin). The transposon or nano-transposon can be a plasmid DNA transposon, which comprises a sequence encoding a protein scaffold (e.g., a CAR comprising at least one scFv, a single-domain antibody, or a Centyrin), flanked by two cis-regulatory insulating elements. The transposon or nano-transposon can further comprise a plasmid containing a sequence encoding a transposase. The sequence encoding the transposase can be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0362] The transposons or minitransposons of the present disclosure may be piggyBac TM (PB) transposons. In some aspects, when the transposon is a PB transposon, the transposase is piggyBac TM (PB) transposase, piggyBac-like (PBL) transposase or SuperpiggyBac TM (SPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[0363] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and PCT Publication No. WO 2010 / 099296.

[0364] PB, PBL and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of the transposon and insert the contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) within a chromosomal locus or at the sequence 5'-TTAA-3' (TTAA target sequence) within a chromosomal locus. The target sequences of PB or PBL transposons may comprise or consist of: 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3'5'-TTCT-3' and 5'-TTTT-3'. The PB or PBL transposon system has no payload limitation on the gene of interest that may be contained between the ITRs.

[0365] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; and U.S. Patent No. 8,399,643. In a preferred aspect, the PB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:1.

[0366] The PB or PBL transposase can comprise or consist of an amino acid sequence having amino acid substitutions at two or more positions, three or more positions, or at each position among positions 30, 165, 282, or 538 of the sequence of SEQ ID NO:1. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of the sequence of SEQ ID NO:1, wherein the amino acid substitution at position 30 can be a valine (V) substitution for isoleucine (I), the amino acid substitution at position 165 can be a serine (S) substitution for glycine (G), the amino acid substitution at position 282 can be a valine (V) substitution for methionine (M), and the amino acid substitution at position 538 can be a lysine (K) substitution for asparagine (N).

[0367] In certain aspects where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases can further comprise amino acid substitutions at one or more positions among positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591, described in more detail in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816.

[0368] The PB, PBL, or SPB transposase can be isolated from or derived from an insect, vertebrate, crustacean, or urochordate, as described in more detail in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816. In a preferred aspect, the PB, PBL, or SPB transposase is isolated from or derived from the insect Trichoplusia ni (GenBank accession number AAA87375) or the silkworm Bombyx mori (GenBank accession number BAD11135).

[0369] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred aspect, the hyperactive PB or PBL transposase is isolated from or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0370] In some aspects, the PB or PBL transposase is integration-deficient. An integration-deficient PB or PBL transposase is a transposase that can excise its corresponding transposon but 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 No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0371] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO 2019 / 173636.

[0372] The transposon or minitransposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Patent No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0373] The transposon or minitransposon of the present disclosure can be a Helraiser transposon. Exemplary Helraiser transposons include Helibat1. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO 2019 / 173636).

[0374] The transposon or minitransposon of the present disclosure can be a Tol2 transposon. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO 2019 / 173636).

[0375] The transposons or minitransposons of the present disclosure can be TcBuster transposons. In some aspects, 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 can comprise a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. The polynucleotide encoding the TcBuster transposase can comprise a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[0376] In some aspects, as described in more detail in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816, the mutant TcBuster transposase contains one or more sequence variations when compared to the wild-type TcBuster transposase.

[0377] The cell delivery compositions (e.g., transposons) disclosed herein can comprise nucleic acid molecules encoding a therapeutic protein or a therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publication No. WO 2019 / 173636 and PCT / US2019 / 049816.

[0378] The cells and modified cells of the present disclosure

[0379] The cells and modified cells of the present disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of the present disclosure can be immune cells. The immune cells of the present disclosure can include lymphoid progenitors, 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 progenitors, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, red blood cells, erythrocytes (RBCs), megakaryocytes, or osteoclasts.

[0380] Immune progenitor cells can include any cells that can differentiate into one or more types of immune cells. Immune progenitor cells can include pluripotent stem cells that can self-renew and develop into immune cells. Immune progenitor cells can include hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells can include precursor cells that can develop into immune cells. Immune progenitor cells can include hematopoietic progenitor cells (HPCs).

[0381] Hematopoietic stem cells (HSCs) are pluripotent self-renewing cells. All differentiated blood cells from lymphoid and myeloid lineages are derived from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

[0382] HSCs can be isolated from or derived from primary or cultured stem cells. HSCs can be isolated from or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0383] Immune progenitor cells can include HSCs or HSC progeny cells. Non-limiting examples of HSC progeny cells include pluripotent stem cells, lymphoid progenitors, natural killer (NK) cells, T lymphocytes (T-cells), B lymphocytes (B-cells), myeloid progenitors, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0384] HSCs generated by the disclosed methods can retain the characteristics of "primitive" stem cells that are isolated from or derived from adult stem cells and share the characteristics of embryonic stem cells while committing to a single lineage. For example, "primitive" HSCs generated by the disclosed methods maintain their "stemness" and do not differentiate after division. Thus, as adoptive cell therapy, "primitive" HSCs generated by the disclosed methods not only replenish their numbers but also expand in vivo. When administered as a single dose, "primitive" HSCs generated by the disclosed methods can be therapeutically effective.

[0385] 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+.

[0386] Primitive HSCs, HSCs, and / or HSC progeny cells can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). Modified primitive HSCs, modified HSCs, and / or modified HSC progeny cells can differentiate forward to produce modified immune cells, including but not limited to modified T cells, modified natural killer cells, and / or modified B cells.

[0387] The modified immune cells or immune precursor cells can be NK cells. NK cells can be cytotoxic lymphocytes differentiated from lymphoid progenitor cells. The modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some aspects, the non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0388] The modified immune cells or immune precursor cells can be B cells. B cells are a type of lymphocyte that express B cell receptors on the cell surface. The B cell receptors bind to specific antigens. The modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0389] The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Different from traditional biologics and chemotherapeutic agents, the disclosed modified T cells have the ability to rapidly replicate upon antigen recognition, thereby potentially eliminating the need for repeated treatments. To achieve this, in some aspects, the modified T cells not only drive an initial response but also persist in the patient as a stable population of surviving memory T cells to prevent potential relapse. Alternatively, in some aspects, the modified T cells do not persist in the patient when not desired.

[0390] Considerable efforts have been focused on developing antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as modified T cell products containing early memory T cells, especially stem cell memory (T SCM ) or stem cell-like T cells. The stem cell-like modified T cells of the present disclosure exhibit the strongest self-renewal ability and the pluripotent ability to give rise to 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 modified T cell engraftment. A linear differentiation pathway may be responsible for generating these cells: naive T cells (T) N >T SCM >T CM >T EM >T E >T TE , whereby T N is the parental precursor cell that directly gives rise to T SCM , and T SCM then directly gives rise to T CM and so on. The composition of the T cells of the present disclosure can include one or more of each parental T cell subset, T SCMCells are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0391] Immune cell precursors can differentiate into or be capable of differentiating into 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 . Immune cell precursors can be the original HSCs, HSCs or HSC progeny cells of the present disclosure. 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 .

[0392] The methods of the present disclosure can modify and / or generate 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 the 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 comprises a plurality of modified T SCM cells. The population of modified early memory T cells comprises a plurality of modified T CM cells.

[0393] The methods of the present disclosure can modify and / or generate 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 the plurality of modified T cells in the population express one or more cell surface markers of stem cell-like T cells. The population of modified stem cell-like T cells comprises a plurality of modified T SCM cells. The population of modified stem cell-like T cells comprises a plurality of modified T CM cells.

[0394] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% or any percentage therebetween of the plurality of modified T cells in the population express one or more cell surface markers of naïve memory T cells (T SCM ) or T SCM -like cells; and wherein the one or more cell surface markers include CD45RA and CD62L. The cell surface markers can include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. The cell surface markers can include one or more of CD45RA, CD95, IL-2Rβ, CCR7 and CD62L.

[0395] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the plurality of modified T cells in the population express one or more cell surface markers of central memory T cells (T CM ) or T CM -like cells; and wherein the one or more cell surface markers include CD45RO and CD62L. The cell surface markers can include one or more of CD45RO, CD95, IL-2Rβ, CCR7 and CD62L.

[0396] The methods of the present disclosure can modify and / or generate 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 the plurality of modified T cells in the population express one or more cell surface markers of naïve T cells (T N ). The cell surface markers can include one or more of CD45RA, CCR7 and CD62L.

[0397] The methods of the present disclosure can modify and / or generate 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 the plurality of modified T cells in the population express one or more cell surface markers of effector T cells (modified T EFF ). The cell surface markers can include one or more of CD45RA, CD95, and IL-2Rβ.

[0398] The methods of the present disclosure can modify and / or generate 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 the plurality of modified T cells in the population express one or more cell surface markers of stem cell-like T cells, stem memory T cells (T SCM ) or central memory T cells (T CM ).

[0399] The plurality of modified cells of the population contain a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of cells in the population contain the transgene or the sequence encoding the transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the modified cell population express one or more cell surface markers comprising CD34, or wherein at least about 70% to about 99%, about 75% to about 95% or about 85% to about 95% of the modified cell population express one or more cell surface markers comprising CD34 (e.g., comprising the cell surface marker phenotype CD34+).

[0400] Multiple modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the multiple cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the modified cell population express one or more cell surface markers comprising CD34 and do not express one or more cell surface markers comprising CD38, or wherein at least about 45% to about 90%, about 50% to about 80% or about 65% to about 75% of the modified cell population express one or more cell surface markers comprising CD34 and do not express one or more cell surface markers comprising CD38 (e.g., comprising the cell surface marker phenotype CD34+ and CD38-).

[0401] Multiple modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the multiple cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38 (e.g., comprising the cell surface marker phenotype CD34+, CD38−, and CD90+).

[0402] Multiple modified cells of the population contain a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the multiple cells of the population contain the transgene or the sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38 and CD45RA, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD38 and CD45RA (e.g., comprising the cell surface marker phenotype CD34+, CD38-, CD90+, CD45RA-).

[0403] Multiple modified cells of the population contain a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the multiple cells of the population contain the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the modified cell population express one or more cell surface markers comprising CD34, CD90 and CD49f and do not express one or more cell surface markers comprising CD38 and CD45RA, or wherein at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2% or about 0.04% to about 1% of the modified cell population express one or more cell surface markers comprising CD34, CD90 and CD49f and do not express one or more cell surface markers comprising CD38 and CD45RA (e.g., comprising the cell surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+).

[0404] Multiple modified cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the multiple cells of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD45RA, or wherein at least about 0.2% to about 5%, about 0.2% to about 3%, or about 0.4% to about 3% of the modified cell population express one or more cell surface markers comprising CD34 and CD90 and do not express one or more cell surface markers comprising CD45RA (e.g., comprising the cell surface marker phenotype CD34+, CD90+ and CD45RA-).

[0405] Compositions and methods for generating and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified T cells) and buffers for maintaining or enhancing the cell viability and / or level of a dry-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells) are disclosed elsewhere herein and are disclosed in more detail in U.S. Patent No. 10,329,543 and PCT Publication No. WO 2019 / 173636.

[0406] 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 cells or allogeneic cells. The allogeneic cells are engineered to prevent adverse reactions to implantation after administration to a subject. The allogeneic cells can be any type of cell. The allogeneic cells can be stem cells or can be derived from stem cells. The allogeneic cells can be differentiated somatic cells.

[0407] Methods for expressing chimeric antigen receptors

[0408] The present disclosure provides a method for expressing a CAR on the cell surface. The method includes (a) obtaining a cell population; (b) contacting the cell population with a composition of the present disclosure comprising a CAR or a sequence encoding a CAR under conditions sufficient to transfer 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 integrating the sequence encoding the CAR; and (d) amplifying and / or selecting at least one cell from the modified cell population that expresses the CAR on the cell surface.

[0409] In some aspects, the cell population can comprise white blood cells and / or CD4+ and CD8+ white blood cells. The cell population can comprise an optimized ratio of CD4+ and CD8+ white blood cells. The optimized ratio of CD4+ to CD8+ white blood cells does not occur naturally in vivo. The cell population can comprise tumor cells.

[0410] In some aspects, the conditions sufficient to transfer a CAR or a sequence encoding a CAR, a transposon, or a vector across the cell membrane of at least one cell in the cell population include at least one of the following: applying one or more electrical pulses at a specified voltage, a buffer, and one or more supplementary factors. In some aspects, the conditions suitable for integrating the sequence encoding the CAR include at least one of a buffer and one or more supplementary factors.

[0411] The buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more supplementary factors can include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, minerals, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cell DNA sensing, metabolism, differentiation, signal transduction, one or more apoptosis pathways, or combinations thereof; and (e) reagents for modifying or stabilizing one or more nucleic acids. Recombinant human cytokines, chemokines, interleukins, or any combination thereof can 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-1α / IL-1F1, IL-1β / IL-1F2, IL-12p70, IL-12 / IL-35p35, 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-α / TNF-β, TGF-β, TNF-α, TRANCE / TNFSFI I / RANK L, or any combination thereof. Salts, minerals, metabolites, or any combination thereof can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacement, antibiotics, pH regulators, Earle salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, nucleofection 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 can 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 cell DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof include TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-κB, type I interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1β, PBK, Akt, Wnt3A, glycogen synthase kinase-3β (GSK-3β) inhibitors (such as TWS119), or any combination thereof. Examples of such inhibitors can include Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof. Reagents for modifying or stabilizing one or more nucleic acids include pH regulators, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA-binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

[0412] The amplification and selection steps can occur in parallel or sequentially. Amplification can occur before selection. Amplification can occur after selection, and optionally, a further (i.e., second) selection can occur after amplification. Parallel amplification and selection can be simultaneous. The amplification and / or selection steps can be carried out for a period of 10 to 14 days, including the end values.

[0413] Amplification can 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 amplified cell population. The antigen can be presented on the surface of a substrate. The substrate can have any form, including but not limited to surfaces, wells, beads, or multiples thereof, and matrices. The substrate can also contain paramagnetic or magnetic components. The antigen can be presented on the surface of a substrate, where the substrate is a magnetic bead, and where a magnet can be used to remove or separate the magnetic bead from the modified and amplified cell population. The antigen can be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells can include but are not limited to tumor cells and stem cells.

[0414] In some aspects in which the transposon or vector contains a selectable gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selectable gene confers resistance, thereby identifying cells that express the selectable gene as surviving the selection and identifying cells that fail to express the selectable gene as failing to survive the selection step.

[0415] The present disclosure provides a composition comprising a modified, expanded, and selected cell population comprising the methods described herein.

[0416] A more detailed description of the methods for expressing CAR on the cell surface is disclosed in PCT Publication No. WO 2019 / 049816 and PCT / US2019 / 049816.

[0417] The present disclosure provides a cell or cell population, wherein the cell comprises a composition comprising (a) an inducible transgenic 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 a foreign receptor such as CAR, wherein, after the constructs of (a) and (b) are integrated into the genomic sequence of the cell, the foreign receptor is expressed, and wherein, after binding a ligand or antigen, the foreign receptor transduces an intracellular signal that directly or indirectly targets the inducible promoter regulating the expression of the inducible transgene (a) to alter gene expression.

[0418] The composition can alter gene expression by reducing gene expression. The composition can alter gene expression by transiently altering gene expression (e.g., for the duration of ligand binding to the foreign receptor). The composition can acutely alter gene expression (e.g., ligand binds reversibly to the foreign receptor). The composition can alter gene expression long-term (e.g., ligand binds irreversibly to the foreign receptor).

[0419] In some aspects, the nucleic acid can comprise a transgene comprising a nucleic acid molecule encoding at least one foreign receptor. The foreign receptor can include an endogenous receptor with respect to the genomic sequence of the cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell surface receptors, transmembrane receptors, ligand-gated ion channels, and G protein-coupled receptors.

[0420] An exogenous receptor can include a non-naturally occurring receptor. The non-naturally occurring receptor can be a synthetic, modified, recombinant, mutated, or chimeric receptor. The non-naturally occurring receptor can comprise one or more sequences isolated from or derived from a T cell receptor (TCR). The non-naturally occurring receptor can comprise one or more sequences isolated from or derived from a scaffold protein. In some aspects, including those in which the non-naturally occurring receptor does not comprise a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane receptor, a membrane-bound receptor, and / or an intracellular receptor, which transduces an intracellular signal upon contact with the non-naturally occurring receptor. The non-naturally occurring receptor can comprise a transmembrane domain. The non-naturally occurring receptor can interact with an intracellular receptor that transduces an intracellular signal. The non-naturally occurring receptor can comprise an intracellular signaling domain. The non-naturally occurring receptor can be a chimeric ligand receptor (CLR). The CLR can be a chimeric antigen receptor (CAR).

[0421] The sequence encoding an inducible promoter comprises a sequence encoding an NFK13 promoter, a sequence encoding an interferon (IFN) promoter, or a sequence encoding an interleukin-2 promoter. In some aspects, the IFN promoter is an IFNγ promoter. The inducible promoter can be isolated from or derived from a promoter of a cytokine or chemokine. The cytokine or chemokine can include IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor β (TGFβ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon γ (IFNγ), tumor necrosis factor α (TNFα), LTα, perforin, granzyme C (Gzmc), granzyme B (Gzmb), C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 4 (Ccl4), C-C motif chemokine ligand 3 (Ccl3), X-C motif chemokine ligand 1 (Xcl1), or leukemia inhibitory factor (Lif).

[0422] The inducible promoter can be isolated from or derived from a promoter of a gene comprising a surface protein involved in cell differentiation, activation, exhaustion, and function. In some aspects, the gene includes CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0423] Inducible promoters can be isolated from or derived from the promoters of genes involved in CD metabolism and differentiation. Inducible promoters can be isolated from or derived from the promoters of Nr4al, Nr4a3, Tnfrsf9 (4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6, and Neto2.

[0424] In some aspects, the inducible transgenic construct comprises or drives the expression of signaling components, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins conferring sensitivity to cancer therapies, and oncogenes or tumor suppressor genes downstream of inhibitory checkpoint signals. Non-limiting examples of inducible transgenic constructs are disclosed in PCT Publication No. WO 2019 / 173636 and PCT Application No. PCT / US2019 / 049816.

[0425] Armor cells

[0426] The modified cells (e.g., CART cells) of the present disclosure can be further modified to enhance their therapeutic potential. Alternatively or additionally, the modified cells can be further modified to be less sensitive to immune and / or metabolic checkpoints. This type of modification is cell "arming", and these cells can be referred to herein as "armed" cells (e.g., armed T cells) after modification. Armed cells can be generated, for example, by naturally blocking and / or attenuating specific checkpoint signals (e.g., checkpoint inhibition) delivered to the cells in the tumor immunosuppressive microenvironment.

[0427] The armored cells of the present disclosure can be derived from any cell, such as T cells, NK cells, hematopoietic progenitor cells, T cells derived from peripheral blood (PB) (including T cells isolated from or derived from G-CSF mobilized peripheral blood) or T cells derived from umbilical cord blood (UCB). The armored cells (e.g., armored T cells) can comprise one or more of the following: chimeric ligand receptor (CLR comprising a protein scaffold, antibody, ScFv or antibody mimetic) / chimeric antigen receptor (CAR comprising a protein scaffold, antibody, ScFv or antibody mimetic), CARTyrin (CAR comprising Centyrin), and / or VCAR (CAR comprising a camelid VHH or single domain VH). The armored cells (e.g., armored T cells) can comprise the inducible pro-apoptotic polypeptides disclosed herein. The armored cells (e.g., armored T cells) can comprise an exogenous sequence. The exogenous sequence can comprise a sequence encoding a therapeutic protein. Exemplary therapeutic proteins can be nuclear, cytoplasmic, intracellular, transmembrane, cell surface-bound or secreted proteins. The exemplary therapeutic proteins expressed by the armored cells (e.g., armored T cells) can alter the activity of the armored cells or can alter the activity of a second cell. The armored cells (e.g., armored T cells) can comprise a selectable gene or selectable marker. The armored cells (e.g., armored T cells) can comprise a synthetic gene expression cassette (also referred to herein as an inducible transgenic construct).

[0428] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding receptors for inhibitory checkpoint signals to produce armored cells (e.g., armored CAR T cells). The receptors for inhibitory checkpoint signals are expressed on the cell surface or within the cytoplasm of the cell. Silencing or reducing the expression of the genes encoding receptors for inhibitory checkpoint signals results in the loss of protein expression of the inhibitory checkpoint receptors on the surface or within the cytoplasm of the armored cells. Thus, the armored cells in which the expression of one or more genes encoding the inhibitory checkpoint receptors is silenced or reduced are resistant, non-responsive or insensitive to the checkpoint signals. The resistance or reduced sensitivity of the armored cells to the inhibitory checkpoint signals enhances the therapeutic potential of the armored cells in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immunosuppression) are disclosed in PCT Publication No. WO 2019 / 173636. Preferred examples of inhibitory checkpoint signals that can be silenced include, but are not limited to, PD-1 and TGFβRII.

[0429] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding intracellular proteins involved in checkpoint signaling to produce armored cells (e.g., armored CAR T cells). The activity of the modified cells can be enhanced by targeting any intracellular signaling protein involved in the checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference of one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in PCT Publication No. WO 2019 / 173636.

[0430] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding transcription factors that impede therapeutic efficacy to produce armored cells (e.g., armored CAR T cells). The activity of the modified cells can be enhanced or regulated by silencing or reducing the expression of transcription factors that impede therapeutic efficacy (or repressing the function of transcription factors). Non-limiting examples of transcription factors that can be modified to silence or reduce expression or repress their function include, but are not limited to, the exemplary transcription factors disclosed in PCT Publication No. WO 2019 / 173636.

[0431] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding cell death or apoptosis receptors to produce armored cells (e.g., armored CAR T cells). The interaction of death receptors with their endogenous ligands triggers the initiation of apoptosis. Disrupting the expression, activity, or interaction of cell death and / or apoptosis receptors and / or ligands renders the modified cells less susceptible to death signals and, thus, makes the armored cells more effective in the tumor environment. Non-limiting examples of cell death and / or apoptosis receptors and ligands are disclosed in PCT Publication No. WO 2019 / 173636. A preferred example of a cell death receptor that can be modified is Fas (CD95).

[0432] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding metabolic sensing proteins to produce armored cells (e.g., armored CAR T cells). Disruption of metabolic sensing of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose, and other molecules) in the modified cells results in long-term retention of T-cell function, and thus each cell kills more tumor cells. Non-limiting examples of metabolic sensing genes and metabolic sensing proteins are disclosed in PCT Publication No. WO 2019 / 173636. In a preferred example, HIF1a and VHL play a role in T-cell function while in a hypoxic environment. The armored T cells can have silenced or reduced expression of one or more genes encoding HIF1a or VHL.

[0433] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding proteins (including monoclonal antibodies) that confer sensitivity to cancer therapies to produce armored cells (e.g., armored CAR T cells). Thus, in the presence of cancer therapies (e.g., chemotherapy, monoclonal antibody therapy, or another anti-tumor treatment), the armored cells can function and can exhibit excellent function or efficacy. Non-limiting examples of proteins involved in conferring sensitivity to cancer therapies are disclosed in PCT Publication No. WO 2019 / 173636.

[0434] The modified (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 growth advantage factors to produce armored cells (e.g., armored CART cells). Silencing or reducing the expression of oncogenes can confer a growth advantage to the cells. For example, silencing or reducing the expression of the TET2 gene (e.g., disrupting the expression) during CART cell manufacturing results in the production of armored CAR T cells with significant expansion capacity, and subsequent eradication of tumors compared to non-armored CAR T cells lacking such expansion capacity. This strategy can be combined with a safety switch (e.g., the iC9 safety switch described herein), which allows for targeted destruction of armored CART cells in the case of adverse reactions from the subject or uncontrolled growth of the armored CART cells. Non-limiting examples of growth advantage factors are disclosed in PCT Publication No. WO 2019 / 173636.

[0435] The modified cells of the present disclosure (e.g., CAR T cells) can be further modified to express a modified / chimeric checkpoint receptor to produce the armored T cells of the present disclosure.

[0436] The modified / chimeric checkpoint receptor can include a null receptor, a decoy receptor, or a dominant negative receptor. The null receptor, decoy receptor, or dominant negative receptor can be a modified / chimeric receptor / protein. The null receptor, decoy receptor, or dominant negative receptor can be truncated to express an intracellular signaling domain. Alternatively, or additionally, the null receptor, decoy receptor, or dominant negative receptor can be mutated at one or more amino acid positions within the intracellular signaling domain that are determined or required for efficient signaling. Truncation or mutation of the null receptor, decoy receptor, or dominant negative receptor can result in loss of the receptor's ability to transmit or transduce checkpoint signals to or within the cell.

[0437] For example, attenuation or blockade of immunosuppressive checkpoint signals from the PD-L1 receptor expressed on the surface of tumor cells can be achieved by expressing a modified / chimeric PD-1 null receptor on the surface of armored cells (e.g., armored CAR T cells), which effectively competes with the endogenous (unmodified) PD-1 receptor also expressed on the surface of the armored cells to reduce or inhibit transduction of immunosuppressive checkpoint signals through the endogenous PD-1 receptor of the armored cells. In this non-limiting example, competition between two different receptors for binding to PD-L1 expressed on tumor cells reduces or weakens the level of efficient checkpoint signaling, thereby enhancing the therapeutic potential of the armored cells expressing the PD-1 null receptor.

[0438] The modified / chimeric checkpoint receptor can include a null receptor, a decoy receptor, or a dominant negative receptor, which is a transmembrane receptor, a membrane-associated or membrane-linked receptor / protein, or an intracellular receptor / protein. Exemplary null, decoy, or dominant negative intracellular receptor / proteins include, but are 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 conferring sensitivity to cancer therapies, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication No. WO 2019 / 173636.

[0439] An engineered / chimeric checkpoint receptor can include a switch receptor. Exemplary switch receptors include engineered / chimeric receptors / proteins in which a native or wild-type intracellular signaling domain is converted or replaced with a different intracellular signaling domain that is non-native to the protein and / or not a wild-type domain. For example, replacing an inhibitory signaling domain with a stimulatory signaling domain will convert an immunosuppressive signal to an immunostimulatory signal. Alternatively, replacing an inhibitory signaling domain with a different inhibitory domain can decrease or enhance the level of inhibitory signal transduction. Expression or overexpression of a switch receptor can cause attenuation and / or blockade of a cognate checkpoint signal by competing with an endogenous wild-type checkpoint receptor (which is not a switch receptor) for binding to cognate checkpoint receptors expressed within an immunosuppressive tumor microenvironment. Armored cells (e.g., armored CAR T cells) can include a sequence encoding a switch receptor, resulting in the expression of one or more switch receptors and thus altering the activity of the armored cells. Armored cells (e.g., armored CAR T cells) can express a switch receptor that targets a checkpoint receptor, a transcription factor, a cytokine receptor, a death receptor, a metabolic sensing molecule, a cancer therapy, an oncogene, and / or a protein expressed intracellularly downstream of a tumor suppressor protein or gene.

[0440] Exemplary switch receptors can comprise or can 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 conferring sensitivity to cancer therapies, and oncogenes or tumor suppressor genes.

[0441] 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 armored T cells. The combination of the CLR / CAR and the conditional gene expression system in the nucleus of the armored T cells constitutes a synthetic gene expression system that is conditionally activated upon binding of a cognate ligand to the CLR or a cognate antigen to the CAR. This system can help "armor" or enhance the therapeutic potential of the engineered T cells by reducing or restricting synthetic gene expression at the ligand or antigen binding site (e.g., at or within the tumor environment).

[0442] The present disclosure provides gene editing compositions and / or cells comprising the gene editing compositions. The gene editing compositions can comprise nanoparticles containing nucleic acids, wherein the nucleic acids comprise a sequence encoding a DNA-binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. The sequence encoding the nuclease protein or the sequence encoding its nuclease domain can comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain can comprise one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0443] The nuclease or its nuclease domain can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing compositions can comprise a fusion protein. The fusion protein can comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing compositions can further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0444] The transgene can comprise a nucleic acid sequence encoding a small Cas9 (Cas9) operably linked to an effector. The present disclosure provides a fusion protein comprising a DNA targeting component and an effector molecule, consisting essentially of or consisting of them, wherein the effector comprises a small Cas9 (Cas9). The small Cas9 construct of the present disclosure can comprise an effector containing a type IIS endonuclease.

[0445] The transgene can comprise a nucleic acid sequence encoding an inactivated small Cas9 (dSaCas9) operably linked to an effector. The transgene can comprise a nucleic acid sequence encoding a fusion protein comprising a DNA targeting component and an effector molecule, consisting essentially of or consisting of them, wherein the effector comprises a small, inactivated Cas9 (dSaCas9). The small, inactivated Cas9 (dSaCas9) construct of the present disclosure can comprise an effector containing a type IIS endonuclease.

[0446] The transgene can comprise a nucleic acid sequence encoding an inactivated Cas9 (dCas9) operably linked to an effector. The transgene can comprise a nucleic acid sequence encoding a fusion protein comprising a DNA targeting component and an effector molecule, consisting essentially of or consisting of them, wherein the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) construct of the present disclosure can comprise an effector containing a type IIS endonuclease.

[0447] dCas9 can be isolated from or derived from Streptoccocus pyogenes. dCas9 can include dCas9 having substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A.

[0448] Cells comprising a gene editing composition can stably or transiently express the gene editing composition. Preferably, the gene editing composition is transiently expressed. The guide RNA can comprise a sequence complementary to a target sequence within a genomic DNA sequence. The target sequence within the genomic DNA sequence can be a target sequence within a safe harbor site of the genomic DNA sequence.

[0449] Gene editing compositions comprising Cas-CLOVER and methods of using such compositions for gene editing are described in detail in U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U.S. Patent No. 10,415,024.

[0450] Chimeric stimulatory receptors and recombinant HLA-E polypeptides

[0451] 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 of the present disclosure (e.g., allogeneic cells) can be modified to disrupt the expression or function of the T cell receptor (TCR) and / or class I major histocompatibility complex (MHC). The TCR mediates the graft-versus-host (GvH) reaction, while the MHC mediates the host-versus-graft (HvG) reaction. In a preferred aspect, any expression and / or function of the TCR is eliminated to prevent T cell-mediated GvH that can lead to the death of the subject. Thus, in one preferred aspect, the present disclosure provides a pure TCR-negative allogeneic I cell composition (e.g., each cell of the composition expresses at a level as low as undetectable or absent).

[0452] Reduce or eliminate the expression and / or function of MHC class I (MHC-I, particularly HLA-A, HLA-B, and HLA-C) to prevent HvG and thus improve engraftment of the cells in the subject. Improved engraftment results in longer cell persistence and thus a greater therapeutic window for the subject. Specifically, reduce or eliminate the expression and / or function of the structural element β-2-microglobulin (B2M) of MHC-I.

[0453] The above strategies induce further challenges. Knockout (KO) of the T cell receptor (TCR) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), which is part of the TCR complex. The loss of CD3ζ in TCR-KO T cells significantly reduces the ability to optimally activate and expand these cells using standard stimulation / activation reagents, including but not limited to agonist anti-CD3 mAb. When the expression or function of any one component of the TCR complex is disrupted, all components of the complex are lost, 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 expansion. Agonist anti-CD3 mAb typically recognizes CD3ε and may recognize another protein within the complex, which in turn transduces a signal to CD3ζ. CD3ζ provides the primary stimulus (along with secondary co-stimulatory signals) for T cell activation for optimal activation and expansion. Under normal conditions, full T cell activation depends on the engagement of the TCR with a second signal mediated by one or more co-stimulatory receptors that enhance the immune response, such as CD28, CD2, 4-1BBL. However, in the absence of the TCR, T cell expansion is severely reduced when stimulated with standard activation / stimulation reagents, including agonist anti-CD3 mAb. In fact, when stimulated with standard activation / stimulation reagents, including agonist anti-CD3 mAb, T cell expansion is reduced to only 20%-40% of normal expansion levels.

[0454] Accordingly, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising an activating component, wherein the activating component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not the same.

[0455] In some aspects, the transgenic sequence can comprise a nucleic acid sequence encoding a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising an activating component, wherein the activating component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not the same.

[0456] The activating component can comprise a part of one or more of the following: T cell receptor (TCR) components, TCR complex components, TCR coreceptor components, TCR costimulatory protein components, TCR inhibitory protein components, cytokine receptors, and chemokine receptors to which an agonist of the activating component binds. The activating component can comprise an agonist-bound CD2 extracellular domain or a part thereof.

[0457] The signal transduction domain can comprise one or more of the following: human signal transduction domain components, T cell receptor (TCR), TCR complex components, TCR coreceptor components, TCR costimulatory protein components, TCR inhibitory protein components, cytokine receptors, and chemokine receptors. The signal transduction domain can comprise a CD3 protein or a part thereof. The CD3 protein can comprise a CD3ζ protein or a part thereof.

[0458] The intracellular domain can further comprise a cytoplasmic domain. The cytoplasmic domain can be isolated from or derived from a third protein. The first protein and the third protein can be the same. The extracellular domain can further comprise a signal peptide. The signal peptide can be derived from a fourth protein. The first protein and the fourth protein can be the same. The transmembrane domain can be isolated from or derived from a fifth protein. The first protein and the fifth protein can be the same.

[0459] In some aspects, the activating component does not bind to a naturally occurring molecule. In some aspects, the activating component binds to a naturally occurring molecule, but CSR does not transduce a signal when the activating component binds to the naturally occurring molecule. In some aspects, the activating component binds to a non-naturally occurring molecule. In some aspects, the activating component does not bind to a naturally occurring molecule but binds to a non-naturally occurring molecule. When the activating component binds to a non-naturally occurring molecule, CSR can selectively transduce a signal.

[0460] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) that comprises: (a) an extracellular domain comprising a signal peptide and an activating component, wherein the signal peptide comprises a CD2 signal peptide or a part thereof, and wherein the activating component comprises an agonist-bound CD2 extracellular domain or a part thereof; (b) a transmembrane domain, wherein the transmembrane domain comprises a CD2 transmembrane domain or a part thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a part thereof, and wherein the at least one signal transduction domain comprises a CD3ζ protein or a part thereof.

[0461] The present disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR), wherein the extracellular domain comprises a modification. The modification can include a mutation or truncation of the amino acid sequence of the activating component or the first protein when compared to the wild-type sequence of the activating component or the first protein. The mutation or truncation of the amino acid sequence of the activating component can include a mutation or truncation of the agonist-binding CD2 extracellular domain or a portion thereof. The mutation or truncation of the CD2 extracellular domain can reduce or eliminate binding to naturally occurring CD58.

[0462] In a preferred aspect, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising a signal peptide and an activating component, wherein the signal peptide comprises the CD2 signal peptide or a portion thereof, and wherein the activating component comprises the agonist-binding CD2 extracellular domain or a portion thereof, and wherein the agonist-binding CD2 extracellular domain or a 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 intracellular domain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises the CD2 cytoplasmic domain or a portion thereof, and wherein the at least one signal transduction domain comprises the CD3ζ protein or a portion thereof.

[0463] The present disclosure provides a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a transposon or vector comprising a nucleic acid sequence encoding any CSR disclosed herein.

[0464] The present disclosure provides a cell comprising any CSR disclosed herein. The present disclosure provides a cell comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein.

[0465] The modified cells disclosed herein can be allogeneic cells or autologous cells. In some preferred aspects, the modified cells are allogeneic cells. In some aspects, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred aspects, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0466] The present disclosure provides a composition comprising any CSR disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any CSR disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein or a composition comprising a plurality of modified cells disclosed herein.

[0467] The present disclosure provides a modified T lymphocyte (T cell) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR; and (b) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain comprising an activating component, wherein the activating component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not the same.

[0468] The modified T cell may further comprise an inducible pro-apoptotic polypeptide. The modified T cell may further comprise a modification of an endogenous sequence encoding β-2-microglobulin (B2M), wherein the modification reduces or eliminates the expression or activity level of major histocompatibility complex (MHC) class I (MHC-I).

[0469] The modified 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 the HLA-E polypeptide may further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising the HLA-E polypeptide may further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising the HLA-E polypeptide may further comprise a linker, wherein the linker is located between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising the 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 located between the B2M signal peptide and the peptide, and a second linker located between the B2M polypeptide and the peptide encoding HLA-E.

[0470] The modified T cell 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 include a chimeric antigen receptor (CAR).

[0471] CSR can be transiently expressed in modified T cells. CSR can be stably expressed in modified T cells. A polypeptide comprising an HLA-E polypeptide can be transiently expressed in modified T cells. A polypeptide comprising an HLA-E polypeptide can be stably expressed in modified T cells. An inducible pro-apoptotic polypeptide can be transiently expressed in modified T cells. An inducible pro-apoptotic polypeptide can be stably expressed in modified T cells. A non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be transiently expressed in modified T cells. A non-naturally occurring antigen receptor or a sequence encoding a therapeutic protein can be stably expressed in modified T cells.

[0472] Gene editing compositions, including but not limited to RNA-guided fusion proteins comprising dCas9-Clo051 as described in detail herein, can be used to target and reduce or eliminate the expression of endogenous T cell receptors. In preferred aspects, the gene editing compositions target genes encoding endogenous T cell receptors, portions of genes, or regulatory elements of genes (such as promoters) and delete them. 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), targeting and deleting TCR-beta (TCR-β), and targeting and deleting beta-2 microglobulin (β2M) are disclosed in PCT application number PCT / US2019 / 049816.

[0473] 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 MHCI, MHCII, or MHC activators. In preferred aspects, the gene editing compositions target genes encoding endogenous MHCI, MHCII, or MHC activators, portions of genes, or regulatory elements of genes (such as promoters) and delete them. Non-limiting examples of guide RNA (gRNA) for targeting and deleting MHC activators are disclosed in PCT application number PCT / US2019 / 049816.

[0474] Detailed descriptions of non-naturally occurring chimeric stimulatory receptors, genetic modifications of endogenous sequences encoding TCR-alpha (TCR-α), TCR-beta (TCR-β), and / or beta-2 microglobulin (β2M), and non-naturally occurring polypeptides comprising HLA class I histocompatibility antigens, alpha chain E (HLA-E) polypeptides are disclosed in PCT application number PCT / US2019 / 049816.

[0475] Formulations, dosages, and modes of administration

[0476] The present disclosure provides formulations, dosages, and methods for administering the compositions described herein.

[0477] The disclosed compositions and pharmaceutical compositions may also include at least one of any suitable adjuvants, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples 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 ed., Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference", 52nd ed., Medical Economics (Montvale, N.J.) 1998. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility, and / or stability of the composition may be routinely selected as is well known in the art or as described herein.

[0478] For example, the LNP compositions disclosed by the present invention may also include a diluent. In some compositions, the diluent may be phosphate buffered saline ("PBS").

[0479] Non-limiting examples of pharmaceutically acceptable excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derived sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination and may account for 1% - 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / protein components that may also exert 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.

[0480] The composition may also include a buffer or pH regulator; generally, the buffer is a salt prepared from an organic acid or organic base. Representative buffers include salts of organic acids, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are salts of organic acids, such as citrates.

[0481] A number of 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 rapid bolus, oral, infusion, intra-articular, intra-bronchial, intra-abdominal, intra-capsular, intra-cartilaginous, intracavitary, intracoelomic, intracerebellar, intraventricular, intra-colonic, intra-cervical, intra-gastric, intra-hepatic, intralesional, intramuscular, intra-myocardial, intranasal, intra-ocular, intraosseous, intraosteal, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal, intra-synovial, intra-thoracic, intra-uterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or vaginal routes.

[0482] The compositions of the present disclosure can be formulated for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in a semi-solid form, such as but not limited to creams and suppositories; for oral or sublingual administration, such as but not limited to in the form of tablets or capsules; or for intranasal administration, such as but not limited to in the form of powders, nasal drops or aerosols or certain medicaments; or for transdermal administration, such as but not limited to gel, ointment, lotion, suspension or patch delivery systems, with chemical enhancers such as dimethyl sulfoxide to alter skin structure or increase drug concentration in transdermal patches (Junginger et al. “Drug Permeation Enhancement;” Hsieh, D.S. ed., pp. 59-90 (Marcel Dekker, Inc. New York 1994)), or by applying an electric field to create transient transport pathways, such as electroporation, or increasing the mobility of charged drugs through the skin, such as iontophoresis, or by applying ultrasound, such as sonophoresis (U.S. Patent Nos. 4,309,989 and 4,767,402) (the above disclosures and patents are incorporated herein by reference in their entirety).

[0483] For parenteral administration, any of the compositions disclosed herein can be formulated as a solution, suspension, emulsion, granule, powder, or lyophilized powder, provided alone or in combination with a pharmaceutically acceptable parenteral vehicle. Preparations for parenteral administration may contain sterile water or saline, polyalkylene glycols (such as polyethylene glycol), oils of vegetable origin, hydrogenated naphthalene, etc., as common excipients. Aqueous or oily suspensions for injection can be prepared according to known methods by using suitable emulsifying or wetting agents and suspending agents. Medicaments for injection may be non-toxic, non-oral diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Water, Ringer's solution, isotonic saline, etc., are allowed as usable vehicles or solvents; sterile non-volatile oils can be used as common solvents or suspending solvents. For these purposes, any kind of non-volatile oils and fatty acids can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic monoglycerides or diglycerides or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection methods, such as the gas-pressure needleless injection device described in U.S. Patent No. 5,851,198 and the laser perforator device described in U.S. Patent No. 5,839,446.

[0484] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered in a particle size effective to reach the lower airways of the lung or sinuses. The composition or pharmaceutical composition can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices capable of depositing the nebulized preparation in the patient's sinus cavity or alveoli include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, atomizers, etc. All such devices can be used with a formulation suitable for administering to dispense the compositions or pharmaceutical compositions described herein in an aerosol. Such aerosols can consist of solutions (aqueous and non-aqueous) or solid particles. Additionally, a spray 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 a metered-dose inhaler (MDI), the propellants, compositions or pharmaceutical compositions, and any excipients or other additives described herein are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations, and related devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0485] For absorption through the mucosal surface, the composition comprises an emulsion containing multiple submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through the mucosal surface by effecting mucoadhesion of the emulsion particles (U.S. Patent No. 5,514,670). Mucosal surfaces suitable for administering the disclosed emulsions can include corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal administration routes. Formulations for vaginal or rectal administration (e.g., suppositories) can contain, for example, polyalkylene glycols, petrolatum, cocoa butter, etc. as excipients. Formulations for intranasal administration can be solid and contain, for example, lactose as an excipient, or can be an aqueous or oily solution of a nasal drop. For oral administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816.

[0486] For transdermal administration, the disclosed composition or pharmaceutical composition is encapsulated in a delivery device, such as liposomes or polymer nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made of synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid, and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, and 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 PCT Publication No. WO 2019 / 049816.

[0487] It may be desirable to deliver the disclosed compound to a subject over an extended period of time, for example, from a single administration for a period of one week to one year. Various sustained-release, depot, or implantable dosage forms can be utilized.

[0488] Suitable dosages are well known in the art. See, e.g., Wells et al., Pharmacotherapy Handbook, 2nd ed., Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, hardcover ed., Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st ed., Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, eds. Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J. Preferred dosages can optionally include about 0.1 - 99 mg / kg per administration and / or 100 - 500 mg / kg per administration, or any range, value or fraction thereof, or a serum concentration of about 0.1 μg / ml - 5000 μg / ml per single or multiple administrations, or any range, value or fraction of such serum concentration. The preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is from about 1 mg / kg to at most about 3 mg / kg, about 6 mg / kg or about 12 mg / kg of the subject's body weight.

[0489] Alternatively, the dosage administered can vary according to known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health status and weight of the recipient; the nature and degree of the symptoms, the type of concurrent treatment, the frequency of treatment and the desired effect.

[0490] As a non-limiting example, treatment of a human or animal can be provided with a single dose or a periodic dose of the compositions or pharmaceutical compositions disclosed herein at about 0.1 mg / kg to 100 mg / kg per day, or any range, value or fraction thereof, on at least one day of days 1 - 40, or alternatively or additionally, on at least one week of weeks 1 - 52, or alternatively or additionally, on at least one year of years 1 - 20, or any combination thereof, using single, infusion or repeated doses.

[0491] In aspects where the composition administered to a subject in need is a modified cell as disclosed herein, the cells can be at about 1×10 3 to 1×10 15 cells; 1×10 3 to 1×10 15 cells, about 1×104 to 1×l0 12 cells; about 1×l0 5 to 1×l0 10 cells; about 1×10 6 to 1×10 9 cells; about 1×10 6 to 1×10 8 cells; about 1×10 6 to 1×10 7 cells; or about 1×10 6 to 25×10 6 cells are administered. In one aspect, the cells are administered at about 5×10 6 to 25×10 6 cells.

[0492] 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 PCT Publication No. WO 2019 / 04981.

[0493] The present disclosure provides the use of the disclosed compositions or pharmaceutical compositions for treating a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, by administering or contacting the cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0494] The present disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or disorders include leukemic, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myelocytic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, Kaposi sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / malignant hypercalcemia, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis cancer, Hodgkin 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, cancer-related bone pain, etc.

[0495] In a preferred aspect, the treatment of a malignant disease or disorder includes adoptive cell therapy. For example, in one aspect, the present disclosure provides modified cells expressing at least one disclosed protein scaffold and / or CARs comprising protein scaffolds (e.g., scFv, single-domain antibodies, Centyrins delivered to cells with the compositions of the present disclosure), the modified cells having been selected and / or expanded for administration to a subject in need. 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 an indicator 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 with one or more reagents at a defined density to inhibit further expansion and / or prevent cell death.

[0496] Any method can include administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods can optionally also include co-administration or combination therapy for treating such disease or disorder, wherein administering any composition or pharmaceutical composition disclosed herein further includes administering at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before, concurrently, and / or after.

[0497] In some aspects, the subject does not develop graft-versus-host (GvH) and / or host-versus-graft (HvG) after administration. In one aspect, the administration is systemic. Systemic administration can be in any manner known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or intravenous infusion. In one aspect, the administration is local. Local administration can be in any manner known in the art and described in detail herein. Preferably, local administration is by intratumoral injection or infusion, intrathecal injection or infusion, intraventricular injection or infusion, intravitreal injection or infusion, or intraosseous injection or infusion.

[0498] In some aspects, the therapeutically effective dose is a single dose. In some aspects, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number therebetween that are manufactured simultaneously. In some aspects, where the composition is autologous cells or allogeneic cells, the dose is an amount sufficient to allow the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0499] In one example, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising a protein scaffold or a CAR comprising a protein scaffold (e.g., scFv, single domain antibody, Centyrin), the antibody or CAR specifically binding an antigen on a tumor cell. In aspects where the composition comprises a modified cell or cell population, the cell or cell population can be autologous or allogeneic.

[0500] In some aspects of the treatment methods described herein, treatment can be modified or terminated. Specifically, in cases where the composition for treatment comprises an inducible pro-apoptotic polypeptide, apoptosis can be selectively induced in the cells by contacting the cells with an inducer. Treatment can be modified or terminated in response to, for example, signs of recovery or signs of reduced disease severity / progression, signs of disease remission / cessation, and / or the occurrence of an adverse event. In some aspects, the method comprises the step of administering an inhibitor of the inducer to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (e.g., when signs or symptoms of the disease recur or increase in severity and / or an adverse event subsides).

[0501] Generation, screening, and purification of protein scaffolds

[0502] At least one protein scaffold of the present disclosure (e.g., monoclonal antibody, chimeric antibody, single domain antibody, VHH, VH, single chain variable fragment (scFv), Centyrin, antigen-binding fragment (Fab) or Fab fragment) can optionally be produced by a cell line, mixed cell line, immortalized cell or clonal population of immortalized cells, as is well known in the art. See, e.g., Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

[0503] The amino acids of the protein scaffold can be altered, added, and / or deleted to reduce immunogenicity or to decrease, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, stability, solubility, or any other suitable characteristic known in the art.

[0504] Optionally, the protein scaffold can be engineered to retain high affinity for the antigen and other favorable biological properties. To achieve this, the scaffold protein can optionally be prepared by methods that analyze the parent sequence and various conceptual engineering products using three-dimensional models of the parent sequence and the engineered sequences. Three-dimensional models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., the Immunofilter program, Xencor, Inc., Monrovia, Calif.). Examination of these displays allows analysis of the possible role of residues in the function of the candidate sequence, i.e., analysis of the residues that affect the ability of the candidate protein scaffold to bind its antigen. In this way, residues can be selected and combined from the parent sequence and reference sequences to obtain desired characteristics such as affinity for the target antigen. Alternatively, or in addition to the above procedures, other suitable engineering methods can be used.

[0505] Screening for protein scaffolds that specifically bind to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, such as in vitro display. This method involves screening individual members of a large collection of peptides for a desired function or structure. The length of the displayed nucleotide or peptide sequence can be from 3 to 5000 or more nucleotides or amino acids, typically 5 - 100 amino acids long, and often about 8 to 25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of peptide sequences on the surface of phage or cells. Each phage or cell contains a nucleotide sequence encoding a specific displayed peptide sequence. Such methods are described in PCT patent publication numbers WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0506] Other systems for generating peptide libraries have aspects of in vitro chemical synthesis and recombinant methods. See, PCT Patent Publication Nos. WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also U.S. 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, Calif.) 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, 5856456 assigned to Enzon; U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 5,837,500 assigned to Dyax; U.S. Patent Nos. 5,427,908, 5,580,717 assigned to Affymax; U.S. Patent No. 5,885,793 assigned to Cambridge Antibody Technologies; U.S. Patent No. 5,750,373 assigned to Genentech; U.S. Patent Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417 assigned to Xoma, Colligan (see above); Ausubel, see above; or Sambrook, see above.

[0507] The protein scaffolds of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred aspect, at least one protein scaffold of the present disclosure can optionally bind to a target protein with high affinity, e.g., KD equal to or less than about 10 -7 M, such as but not limited to 0.1 - 9.9 (or any range or value therein) × 10 -8 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13 、10 -14 、10 -15 or any range or value of KD therein, as determined by surface plasmon resonance or Kinexa methods as practiced by those skilled in the art.

[0508] The affinity or avidity of a protein scaffold for an antigen can be experimentally determined using any suitable method. (See, e.g., Berzofsky et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W.E. ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and the methods described herein). If measured under different conditions (e.g., salt concentration, pH), the affinity of a particular protein scaffold-antigen interaction can vary. Thus, it is preferred to measure the affinity and other antigen binding parameters (e.g., KD, Kon, Koff) using a standardized solution of the protein scaffold and antigen and a standardized buffer (such as the buffers described herein).

[0509] Competitive assays can be performed with the protein scaffold to determine which proteins, antibodies, and other antagonists compete with the protein scaffold for binding to the target protein and / or share epitope regions. Such assays, which are readily known to those of ordinary skill in the art, evaluate the competition between antagonists or ligands for a limited number of binding sites on the protein. The protein and / or antibody is immobilized or made insoluble either before or after the competition, and samples that have bound to the target protein are separated from unbound samples, e.g., by decantation (where the protein / antibody is pre-made insoluble) or by centrifugation (where the protein / antibody precipitates after the competition reaction). In addition, competitive binding can be determined by whether the binding or lack of binding of the protein scaffold to the target protein alters a function, e.g., whether the protein scaffold inhibits or enhances, e.g., labeled enzyme activity. As is well known in the art, ELISAs and other functional assays can be used.

[0510] Nucleic acid molecules

[0511] The nucleic acid molecules encoding the protein scaffolds of the present disclosure 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 it can be the non-coding strand, also known as the antisense strand.

[0512] The isolated nucleic acid molecules of the present disclosure can include nucleic acid molecules comprising an open reading frame (ORF) that optionally has one or more introns (such as, but not limited to, at least one specified portion of at least one protein scaffold); nucleic acid molecules comprising a coding sequence of a protein scaffold or a loop region that binds to a target protein; and nucleic acid molecules comprising nucleotide sequences that are substantially different from those described above, but 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, generating such degenerate nucleic acid variants encoding a particular protein scaffold of the present disclosure will be routine for those skilled in the art. See, for example, Ausubel et al., supra, and such nucleic acid variants are included in the present disclosure.

[0513] As pointed out herein, the nucleic acid molecules of the present disclosure that comprise nucleic acid molecules encoding a protein scaffold can include, but are not limited to, those nucleic acid molecules themselves that encode an amino acid sequence of a fragment of a protein scaffold; the coding sequence of a complete protein scaffold or a portion thereof; the coding sequence of a protein scaffold, fragment, or portion, and additional sequences, such as the coding sequence of at least one signal leader peptide or fusion peptide, with or without the foregoing additional coding sequences, such as at least one intron, together with additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, such as transcriptional, untranslated sequences that function in transcription, mRNA processing (including splicing) and polyadenylation signals (such as ribosome binding and stability of mRNA); additional coding sequences encoding additional amino acids, such as those that provide additional functionality. Thus, the sequence encoding a protein scaffold can be fused to a tag sequence, such as a sequence encoding a peptide that facilitates the purification of a fusion protein scaffold comprising a fragment or portion of a protein scaffold.

[0514] Polynucleotides that selectively hybridize with the polynucleotides described herein

[0515] The present disclosure provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides can be used to isolate, detect, and / or quantify nucleic acids comprising 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 isolated or genomic sequences or cDNA sequences that are complementary to cDNA from a human or mammalian nucleic acid library.

[0516] Preferably, the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are generally but not exclusively used for sequences having reduced sequence identity relative to a complementary sequence. Medium and high stringency conditions are optionally used for sequences of higher identity. Low stringency conditions permit selective hybridization of sequences having about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0517] Optionally, the polynucleotide will encode at least a portion of a protein scaffold encoded by a polynucleotide described herein. The polynucleotide includes nucleic acid sequences that can be used to selectively hybridize to a polynucleotide encoding a protein scaffold of the present disclosure. See, e.g., Ausubel, supra; Colligan, supra, each incorporated herein by reference in its entirety.

[0518] Construction of nucleic acids

[0519] The isolated nucleic acids of the present disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are well known in the art.

[0520] The nucleic acid can conveniently comprise sequences other than the polynucleotides of the present disclosure. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. In addition, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotides of the present disclosure. For example, a hexahistidine tag sequence provides a convenient means for purifying the proteins of the present disclosure. In addition to the coding sequence, the nucleic acids of the present disclosure are optionally vectors, linkers, or adapters for cloning and / or expressing the polynucleotides of the present disclosure.

[0521] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to facilitate isolation of the polynucleotide, or to improve introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, linkers, and adapters is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

[0522] Recombinant methods for constructing nucleic acids

[0523] The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using many cloning methods known to those skilled in the art. In some aspects, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify desired sequences in cDNA or genomic DNA libraries. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

[0524] Screening and isolation methods for nucleic acids

[0525] cDNA or genomic libraries can be screened using probes based on the polynucleotide sequences of the present disclosure. The probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will understand that different degrees of hybridization stringency can be employed in the assay; and the hybridization or wash medium can be stringent. When the hybridization conditions become more stringent, a greater degree of complementarity between the probe and the target is required for duplex formation. The stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently altered by changing the polarity of the reaction solution, e.g., by controlling the concentration of formamide in the range of 0% to 50%. The degree of complementarity required for detectable binding (sequence identity) will vary depending on the stringency of the hybridization medium and / or wash medium. The optimal degree of complementarity will be 100% or 70%-100%, or any range or value therein. However, it should be understood that smaller sequence variations in the probes and primers can be compensated for by decreasing the stringency of the hybridization and / or wash medium.

[0526] Methods for amplifying RNA or DNA are well known in the art and can be used according to the present disclosure based on the teachings and guidance presented herein without undue experimentation.

[0527] Known DNA or RNA amplification methods include, but are not limited to, polymerase chain reaction (PCR) and related amplification methods (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al.; U.S. Patent Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Patent No. 5,142,033 to Innis; U.S. Patent No. 5,122,464 to Wilson et al.; U.S. Patent No. 5,091,310 to Innis; U.S. Patent No. 5,066,584 to Gyllensten et al.; U.S. Patent No. 4,889,818 to Gelfand et al.; U.S. Patent No. 4,994,370 to Silver et al.; U.S. Patent No. 4,766,067 to Biswas; U.S. Patent No. 4,656,134 to Ringold), and RNA-mediated amplification, which uses antisense RNA to a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek et al., commercially available as NASBA), the entire contents of these references are incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra.)

[0528] For example, polymerase chain reaction (PCR) technology can be used to directly amplify the polynucleotides of the present disclosure and the sequences of related genes from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, to use the nucleic acids as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide a person skilled in the art through in vitro amplification methods can be found in Berger (supra), Sambrook (supra), and Ausubel (supra), as well as Mullis et al., U.S. Patent No. 4,683,202 (1987); and Innis et al., PCR Protocols A Guide to Methods and Applications, edited by Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to increase the yield of long PCR products.

[0529] Synthetic methods for constructing nucleic acids

[0530] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel et al., supra). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization using the single strand as a template with DNA polymerase. Those skilled in the art will recognize that while chemical synthesis of DNA may be limited to sequences of about 100 bases or more, longer sequences can be obtained by ligating shorter sequences.

[0531] Recombinant expression cassettes

[0532] The present disclosure also provides recombinant expression cassettes comprising the nucleic acids of the present disclosure. The nucleic acid sequences of the present disclosure, such as cDNA or genomic sequences encoding the protein scaffolds of the present disclosure, can be used to construct recombinant expression cassettes, which can be introduced into at least one desired host cell. The recombinant expression cassette will typically comprise a polynucleotide of the present disclosure operably linked to a transcriptional initiation regulatory sequence that will direct the transcription of the polynucleotide in the intended host cell. Heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of the nucleic acids of the present disclosure.

[0533] In some aspects, an isolated nucleic acid used as a promoter, enhancer, or other element can be introduced into an appropriate position (upstream, downstream, or within an intron) of a non-heterologous form of the polynucleotide of the present disclosure to upregulate or downregulate the expression of the polynucleotide of the present disclosure. For example, the endogenous promoter can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0534] Expression vectors and host cells

[0535] 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 of which is incorporated herein by reference in its entirety.

[0536] The polynucleotide can optionally be ligated to a vector containing a selectable marker for propagation in a host. Typically, plasmid vectors are introduced into precipitates (such as calcium phosphate precipitates) or into complexes with charged lipids. If the vector is viral, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[0537] The DNA insert should be operably linked to a suitable promoter. The expression construct will further contain sites for transcription initiation and termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation start at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, where UAA and UAG are preferably used for mammalian or eukaryotic cell expression.

[0538] The expression vector will preferably but optionally include at least one selectable marker. Such markers include, for example but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes and ampicillin, zeocin (Sh 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 full texts of the above patents are incorporated herein by reference). Suitable media and conditions for the above host cells are known in the art. Suitable vectors will be apparent to those skilled in the art. Introduction of the vector construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0539] The expression vector will preferably but optionally include at least one optional cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. Optional cell surface markers of the present disclosure include surface proteins, glycoproteins, or a group of proteins that distinguish a cell or cell subset from another defined cell subset. Preferably, the optional cell surface marker will distinguish those cells modified by the compositions or methods of the present disclosure from those cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example but not limited to, "cluster of differentiation" 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. The cell surface marker also includes the suicide gene marker RQR8 (Philip B et al. Blood. August 21, 2014; 124(8):1277-87).

[0540] The expression vector will preferably but optionally include at least one optional drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. Optional 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.

[0541] At least one protein scaffold of the present disclosure can be expressed in a modified form, such as a fusion protein, and can include not only a secretion signal but also additional heterologous functional regions. For example, a region of additional amino acids (especially 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. In addition, a peptide moiety can be added to the protein scaffolds of the present disclosure to facilitate purification. Such regions can be removed prior to the 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.

[0542] One of ordinary skill in the art is aware of the various expression systems that can be used to express nucleic acid molecules encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in a host cell by turning on (by manipulation) the endogenous DNA containing the protein scaffold of the present disclosure encoded therein. Such methods are well known in the art and are described, for example, in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are hereby incorporated by reference in their entirety.

[0543] Exemplary cell cultures that can be used to generate protein scaffolds, specified portions thereof, or variants are bacteria, yeast, and mammalian cells known in the art. Mammalian cell systems are often in the form of cell monolayers, but mammalian cell suspensions or bioreactors can also be used. Many suitable host cell lines capable of expressing fully 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, etc., which can be readily obtained from, for example, American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include lymph-derived cells such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0544] Expression vectors for these cells can include one or more of the following: expression control sequences such as, but not limited to, origins of replication; promoters (e.g., late or early SV40 promoter, CMV promoter (U.S. Patent Nos. 5,168,062; 5,385,839), HSY tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1α promoter (U.S. Patent No. 5,266,491), at least one human promoter; enhancers, and / or processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g., SV40 large T Ag polyA addition site), and transcription terminator sequences. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells that can be used to generate the nucleic acids or proteins of the present disclosure are known and / or available from, for example, the American Type Culture Collection cell line and hybridoma catalog (www.atcc.org) or other known or commercial sources.

[0545] When using eukaryotic host cells, a polyadenylation or transcription terminator sequence is typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences that control replication in the host cell can be incorporated into the vector, as is known in the art.

[0546] Protein scaffold purification

[0547] The protein scaffolds of the present disclosure 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, hydroxyapatite 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, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each incorporated herein by reference in its entirety.

[0548] The protein scaffolds of the present disclosure include purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts including, for example, Escherichia coli, yeast, higher plants, insects, and mammalian cells. Depending on the host employed in the recombinant production procedure, the protein scaffolds of the present disclosure may be glycosylated or may be non-glycosylated. Such methods are described in many standard laboratory manuals such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18, and 20, Colligan, Protein Science, supra, Chapters 12-14, all incorporated herein by reference in their entirety.

[0549] Amino acid codes

[0550] The amino acids that make up the protein scaffolds of the present disclosure are often abbreviated. As is well known in the art, amino acid names can be represented by naming the amino acid by its single-letter code, its three-letter code, its name, or its trinucleotide codon (see Alberts, B. et al., Molecular Biology of The Cell, Third Edition, Garland Publishing, Inc., New York, 1994). As specified herein, the protein scaffolds of the present disclosure can include one or more amino acid substitutions, deletions, or additions from spontaneous or mutant and / or artificial manipulations. Amino acids essential for function in the protein scaffolds of the present disclosure can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue of the molecule. The biological activity of the resulting mutant molecules is then tested, such as but not limited to at least one neutralizing activity. Sites crucial 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)).

[0551] As will be understood by those skilled in the art, the present disclosure includes at least one bioactive protein scaffold of the present disclosure. The specific activity of the bioactive protein scaffold is at least 20%, 30%, or 40% of the specific activity of a native (non-synthetic), endogenous, or related and known protein scaffold, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-99% or higher. Methods for measuring and quantifying measures of enzyme activity and substrate specificity are well known to those skilled in the art.

[0552] In another aspect, the present disclosure relates to protein scaffolds and fragments as described herein, which are modified by covalently attaching an organic moiety. Such modifications can result in 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 polymeric group, a fatty acid group, or a fatty acid ester group. In a particular aspect, the hydrophilic polymer group can have a molecular weight of from about 800 to about 120,000 daltons and can be a polyalkylene 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 from about eight to about forty carbon atoms.

[0553] The modified protein scaffolds and fragments of the present disclosure can include one or more organic moieties covalently bonded, directly or indirectly, to an antibody. Each organic moiety bonded to a protein scaffold or fragment of the present disclosure can independently be a hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic acids and dicarboxylic acids. The term "hydrophilic polymeric group" as used herein refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, the present disclosure encompasses protein scaffolds modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying the protein scaffolds of the present disclosure can be linear or branched and include, for example, polyalkylene glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkylene oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers that modify the protein scaffolds of the present disclosure have a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG5000 and PEG20,000 can be used, where the subscript is the average molecular weight of the polymer in daltons. The hydrophilic polymeric 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 employing suitable methods. For example, a polymer containing an amine group can be coupled to the carboxylic ester of a fatty acid or fatty acid ester, and an activated carboxylic ester on the fatty acid or fatty acid ester (e.g., activated with N,N'-carbonyldiimidazole) can be coupled to a hydroxyl group on the polymer.

[0554] The fatty acids and fatty acid esters suitable for modifying the protein scaffolds of the present disclosure can be saturated or can contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffolds of the present disclosure include, for example, n-dodecanoic acid (C12, lauric acid), n-tetradecanoic acid (C14, myristic acid), n-octadecanoic acid (C18, stearic acid), n-eicosanoic acid (C20, arachidic acid), n-docosanoic acid (C22, behenic acid), n-triacontanoic acid (C30), n-tetracontanoic acid (C40), cis-Δ9-octadecenoic acid (C18, oleic acid), all-cis-Δ5,8,11,14-eicosatetraenoic acid (C20, arachidonic acid), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc. Suitable fatty acid esters include monoesters of dicarboxylic acids containing straight-chain or branched lower alkyl groups. The lower alkyl group can contain one to about twelve, preferably one to about six carbon atoms.

[0555] Modified protein scaffolds and fragments can be prepared using suitable methods, such as by reacting with one or more modifiers. As used herein, the term "modifier" refers to a suitable organic group containing an activating group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester). An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions to form a covalent bond between the modifier and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halogens (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimide esters (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 coupled to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form aminophosphonate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). The activating group can be directly bonded to the organic group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester), or bonded through a linker moiety such as a divalent C1-C12 group, where one or more carbon atoms can be replaced by heteroatoms 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—. Modifiers containing linker moieties can be produced, for example, by reacting a mono-Boc-alkyl diamine (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 carboxylate ester. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose the primary amine, which can be coupled to another carboxylate ester, as described, or can react with maleic anhydride, and the resulting product is cyclized to produce an activated maleimide derivative of the fatty acid. (See, e.g., Thompson et al., WO 92 / 16221, the entire teachings of which are incorporated herein by reference.)

[0556] The modified protein scaffolds of the present disclosure can be produced by reacting a protein scaffold or fragment with a modifier. For example, an organic moiety can be attached to a protein scaffold in a non-site-specific manner by employing an amine-reactive modifier such as the NHS ester of PEG. Modified protein scaffolds and fragments containing an organic moiety that binds to a specific site of a protein scaffold of the present disclosure can be prepared using suitable methods such as retroproteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg.Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol.Bioeng., 56(4):456-463 (1997)) and the methods described in Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0557] Definitions

[0558] In the chemical formulas shown herein, the label indicates the position at which a functional group is bonded to another part of the molecule. Definitions of specific functional groups and chemical terms are described in more detail below.

[0559] Certain compounds of the invention can exist in specific geometric or stereoisomeric forms. The invention contemplates all such compounds, including cis and trans isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, their racemic mixtures and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are intended to be included in the invention.

[0560] According to the invention, isomer mixtures containing any of various isomer ratios can be utilized. For example, in the case of combining only two isomers, the invention contemplates all mixtures containing isomer ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1 or 100:0. Those of ordinary skill in the art will readily understand that similar ratios are contemplated for more complex isomer mixtures.

[0561] For example, if a particular enantiomer of a compound of the invention is desired, it may be prepared by asymmetric synthesis or by derivation with a chiral auxiliary, where the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, in the case where the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with a suitable optically active acid or base, and the diastereomers thus formed are then resolved by fractional crystallization or chromatographic means well known in the art, followed by recovery of the pure enantiomer.

[0562] 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, most commonly expressed as a percentage of enantiomeric excess. For a mixture of diastereomers, “diastereomeric excess” and percentage diastereomeric excess have similar definitions and uses.

[0563] For example, a sample having 70% of the R isomer and 30% of the S will have an enantiomeric excess of 40%. This can also be considered a mixture of 40% pure R and 60% racemic mixture (which contributes 30% R and 30% S to the overall composition).

[0564] One of ordinary skill in the art will understand 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 the reaction can proceed selectively at another reaction site in a multifunctional compound. In certain embodiments, the protecting group reacts selectively in good yield to give a protected substrate that is stable to the planned reaction; the protecting group should be selectively removed in good yield by readily available, preferably non-toxic reagents that do not attack other functional groups; the protecting group forms a derivative that is easily separable (more preferably without creating new stereocenters); and the protecting group has a minimum of additional functional groups to avoid further reaction sites. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups can be utilized.

[0565] It should be understood that the compounds described herein can be substituted by many substituents or functional moieties. In general, the term "substituted" (whether or not preceded by the term "optionally") and the substituents contained in the formulas of the present invention refer to the replacement of a hydrogen atom group in a given structure by an atom group of a designated substituent. When more than one position in any given structure can be substituted by more than one substituent selected from the designated groups, at each position, the substituents can be the same or different. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For the purposes of the present invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the valence of the heteroatom. In addition, the present invention is not intended to be limited in any way by the permissible substituents of organic compounds. Combinations of substituents and variables contemplated by the present invention are preferably those that result in the formation of stable compounds useful for treating diseases or disorders. As used herein, the term "stable" preferably refers to compounds having sufficient stability to allow for manufacture and maintaining the integrity of the compound for a period of time sufficient to be detected and preferably for a period of time useful for the purposes detailed herein.

[0566] As used herein, the term "aliphatic" includes saturated and unsaturated straight-chain (i.e., unbranched), branched, acyclic, cyclic or polycyclic aliphatic hydrocarbons, which are optionally substituted by one or more functional groups. As will be understood by those of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight-chain, branched-chain and cyclic alkyls. Similar conventions apply to other general terms, such as "alkenyl", "alkynyl", etc. In addition, as used herein, the terms "alkyl", "alkenyl", "alkynyl", etc. encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups having 1-6 carbon atoms (cyclic, acyclic, substituted, unsubstituted, branched or unbranched).

[0567] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1 to 20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1 to 15 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1 to 10 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1 to 8 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1 to 6 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1 to 4 carbon atoms. Exemplary aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, --CH2-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, --CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, --CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, --CH2-cyclohexyl moieties, etc., which moieties may likewise bear one or more substituents. Alkenyl groups include, but are not limited to, for example, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, etc. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, etc.

[0568] As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain hydrocarbon moiety derived by removing a single hydrogen atom from a hydrocarbon containing from one to twenty carbon atoms. Examples of alkyl moieties 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.

[0569] The term "alkenyl" denotes a monovalent group derived by removing a single hydrogen atom from a hydrocarbon moiety having at least one carbon-carbon double bond. Alkenyl groups include, for example, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, etc.

[0570] As used herein, the term "alkynyl" refers to a monovalent group derived by removing a single hydrogen atom from a hydrocarbon having at least one carbon-carbon triple bond. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, etc.

[0571] As used herein, the term "alkoxy" or "thioalkyl" refers to an alkyl group as previously defined attached to the parent molecule through 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 other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-8 aliphatic carbon atoms. In other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6 aliphatic carbon atoms. In 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.

[0572] The term "alkylamino" refers to a group having the structure --NHR', where R' is an aliphatic group as defined herein. In certain embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-15 aliphatic carbon atoms. In certain other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic group employed in the present invention contains 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n-propylamino, isopropylamino, cyclopropylamino, n-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like.

[0573] As used herein, the term "carboxylic acid" refers to a group of the formula --CO2H.

[0574] The term "dialkylamino" refers to a group having the structure --NRR', where R and R' are each aliphatic groups as defined herein. R and R' in the dialkylamino moiety can be the same or different. In certain embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-15 aliphatic carbon atoms. In certain other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic groups employed in the present invention contain 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 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 can 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-triazolyl, and tetrazolyl.

[0575] Some examples of substituents of the above aliphatic (and other) moieties of the compounds of the present invention include, but are not limited to, aliphatic groups; heteroaliphatic groups; aryl groups; heteroaryl groups; aralkyl groups; heteroarylalkyl groups; alkoxy groups; aryloxy groups; heteroalkoxy groups; heteroaryloxy groups; alkylthio groups; arylthio groups; heteroalkylthio groups; heteroarylthio groups; 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 , where each occurrence of R xIndependently include but are not limited to aliphatic groups, heteroaliphatic groups, aryl, heteroaryl, arylalkyl or heteroarylalkyl, wherein any of the aliphatic groups, heteroaliphatic groups, arylalkyl or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of commonly applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0576] Generally, as used herein, the terms "aryl" and "heteroaryl" refer to stable monocyclic or polycyclic, heterocyclic, polycyclic and polyheterocyclic unsaturated moieties having preferably 3 - 14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include but are not limited to any of the foregoing substituents that result in the formation of a stable compound, i.e., the substituents recited for the aliphatic moiety or for other moieties as disclosed herein. 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, etc. In certain embodiments of the present invention, as used herein, the term "heteroaryl" refers to a cyclic aromatic moiety having five to ten ring atoms, wherein one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, and the moiety is attached to the remainder of the molecule via any of the ring atoms, such as pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thienyl, furyl, quinolinyl, isoquinolinyl, etc.

[0577] It should be understood that aryl and heteroaryl groups may be unsubstituted or substituted, wherein substitution includes replacement of one, two, three or more hydrogen atoms thereon independently by any one or more of the following moieties, which include but are not limited to: aliphatic groups; heteroaliphatic groups; aryl; heteroaryl; aralkyl; 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(Rx )2; --S(O)2R x ; --NR x (CO)R R , where each occurrence of R x independently includes but is not limited to an aliphatic group, a heteroaliphatic group, an aryl, a heteroaryl, an arylalkyl or a heteroarylalkyl, wherein any one of the aliphatic group, heteroaliphatic group, arylalkyl or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any one of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of commonly applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0578] As used herein, the term "cycloalkyl" specifically refers to a group having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc., and in the case of other aliphatic, heteroaliphatic or heterocyclic moieties, may optionally be substituted with substituents including but not limited to: aliphatic groups; heteroaliphatic groups; aryls; heteroaryls; aralkyls; heteroarylalkyls; alkoxys; aryloxys; heteroalkoxys; heteroaryloxys; alkanethiols; arylthiols; heteroalkanethiols; heteroarylthiols; --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 , where each occurrence of R x independently includes but is not limited to an aliphatic group, a heteroaliphatic group, an aryl, a heteroaryl, an arylalkyl or a heteroarylalkyl, wherein any one of the aliphatic group, heteroaliphatic group, arylalkyl or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any one of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of commonly applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0579] As used herein, the term "heteroaliphatic" refers to an aliphatic moiety containing one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms (e.g., in place of carbon atoms). The heteroaliphatic moiety can be branched, unbranched, cyclic, or acyclic, and includes saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, the heteroaliphatic moiety is substituted by independently replacing one or more hydrogen atoms thereon with one or more moieties including, but not limited to: aliphatic groups; heteroaliphatic groups; aryl; heteroaryl; aralkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkanethiol; arylthiol; heteroalkanethiol; heteroarylthiol; --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 , where each occurrence of R x independently includes, but is not limited to, aliphatic groups, heteroaliphatic groups, aryl, heteroaryl, aralkyl, or heteroarylalkyl, where any of the aliphatic groups, heteroaliphatic groups, aralkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and where any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Other examples of commonly applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0580] The term "haloalkyl" denotes an alkyl group as defined above having one, two, or three halogen atoms attached thereto, and examples are groups such as chloromethyl, bromoethyl, trifluoromethyl, etc.

[0581] As used herein, the term "heterocycloalkyl" or "heterocycle" refers to a non-aromatic 5-, 6- or 7-membered ring or polycyclic group, including but not limited to bicyclic or tricyclic groups, comprising a fused six-membered ring having one to three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) nitrogen heteroatoms may optionally be 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 tetrahydrofuranyl. In certain embodiments, "substituted heterocycloalkyl or heterocycle" groups are utilized, and as used herein, refers to a heterocycloalkyl or heterocycle group as defined above that is substituted by independently replacing one, two or three hydrogen atoms thereon with, but not limited to, the following groups: aliphatic group; heteroaliphatic group; aryl; heteroaryl; aralkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkanethiol; arylthiol; heteroalkanethiol; heteroarylthiol; --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 , where each occurrence of R x independently includes but is not limited to an aliphatic group, heteroaliphatic group, aryl, heteroaryl, aralkyl or heteroarylalkyl, wherein any of the aliphatic group, heteroaliphatic group, aralkyl or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of commonly applicable substituents are illustrated by the specific embodiments shown in the examples described herein.

[0582] As used herein, the term "carbocycle" refers to an aromatic or non-aromatic ring wherein each atom of the ring is a carbon atom.

[0583] As used herein, the term "independently selected" indicates that the R groups can be the same or different.

[0584] As used herein, the terms "halo" and "halogen" refer to atoms selected from fluorine, chlorine, bromine, and iodine.

[0585] As used herein, the term "heterocycle" refers to a 3- to 10-membered ring system that is non-aromatic and partially unsaturated or fully saturated, including monocyclic, bicyclic, and tricyclic ring systems having 3 to 8 atoms in size, and may include an aromatic six-membered aryl or aromatic heterocyclic group fused to a non-aromatic ring. These heterocycles include heterocycles having one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.

[0586] As used herein, the term "heteroaryl" refers to a cyclic aromatic moiety having five to ten ring atoms, wherein one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, and the moiety is attached to the remainder of the molecule through any one of the ring atoms, such as pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thienyl, furyl, quinolinyl, isoquinolinyl, etc.

[0587] As used herein, the terms "substituted" (whether or not preceded by the term "optionally") and "substituent" refer to the ability to change one functional group to another as understood by one of ordinary skill in the art, provided that the valence of all atoms is maintained. When more than one position in any given structure can be substituted with more than one substituent selected from the designated groups, at each position, the substituents can be the same or different. Substituents can also be further substituted (e.g., an aryl group substituent can have another substituent, such as another aryl group, which is further substituted with fluorine at one or more positions).

[0588] "Effective amount": Generally, an "effective amount" of an active agent or composition is the amount necessary to elicit a desired biological response. As will be understood by one of ordinary skill in the art, the effective amount of a medicament or device can vary depending on factors such as the desired biological endpoint, the medicament to be delivered, the composition of the encapsulating matrix, the target tissue, etc. For example, the effective amount of microparticles containing an antigen to be delivered to immunize an individual is the amount that elicits an immune response sufficient to prevent infection of an organism with the administered antigen.

[0589] As used throughout the disclosure, unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those of ordinary skill in the art, and the like.

[0590] The terms “about” or “approximately” mean 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 1 or more standard deviations. Alternatively, “about” can mean within a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, especially for biological systems or processes, the term can mean within an order of magnitude of the value, preferably within 5-fold, more preferably within 2-fold. Where a particular value is described in this application and the claims, unless otherwise stated, it should be assumed that the term “about” means within an acceptable error range for the particular value.

[0591] 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 of components that are normally associated with or interact with the polynucleotide or protein in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an “isolated” polynucleotide is free of sequences (preferably protein-coding sequences) that naturally flank the polynucleotide in genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various aspects, 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. A protein that is substantially free of cellular material includes a protein preparation having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein or biologically active portion thereof of the present disclosure is produced recombinantly, preferably the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[0592] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence and thus refers to the protein encoded thereby. A DNA sequence fragment that contains a coding sequence may encode a protein fragment that retains the biological activity of the native protein and thus retains the DNA recognition or binding activity for a target DNA sequence as described herein. Alternatively, a DNA sequence fragment that can be used as a hybridization probe generally does not encode a protein that retains biological activity or promoter activity. Thus, fragments of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides up to the full-length polynucleotide of the present disclosure.

[0593] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach that includes pre-assembling monomeric units and / or repeating units in a target vector, which can subsequently be assembled into a final vector of interest. The polypeptides of the present disclosure can contain repeating monomers of the present disclosure and can be constructed by a modular approach by pre-assembling repeating units in a target vector, which can subsequently be assembled into a final vector of interest. The present disclosure provides polypeptides produced by this method and nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding polypeptides produced by such a modular approach.

[0594] The term "antibody" is used in the broadest sense and specifically encompasses a single monoclonal antibody (including agonist and antagonist antibodies) and antibody compositions having multiple epitope specificities. Also within its scope are natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of antibodies as defined herein. Thus, in one of its aspects, the term "antibody thereof" also encompasses such analogs in its broadest sense. Generally, in such analogs, one or more amino acid residues may have been substituted, deleted, and / or added as compared to an antibody as defined herein.

[0595] As used herein, "antibody fragment" and all grammatical variants thereof are defined as a portion of a whole antibody that contains the antigen-binding site or variable region of the whole antibody, where the portion lacks the constant heavy chain domains of the Fc region of the whole antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of an 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 (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain, or fragments thereof containing the three CDRs of that light chain variable domain without an associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable region, or fragments thereof containing the three CDRs of that heavy chain variable region without an associated light chain portion; and multispecific or multivalent structures formed from antibody fragments. In antibody fragments that contain one or more heavy chains, the heavy chain may contain any constant domain sequence found in the non-Fc region of the whole antibody (e.g., CH1 in the IgG isotype), and / or may contain any hinge region sequence found in the whole antibody, and / or may contain a leucine zipper sequence fused to or located within the hinge region sequence or constant domain sequence of the heavy chain. The term also includes single-domain antibodies ("sdABs"), which generally refer to antibody fragments having a single monomeric variable antibody domain (e.g., from camelids). Such types of antibody fragments will be readily understood by those of ordinary skill in the art.

[0596] "Binding" refers to sequence-specific, non-covalent interactions 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 the DNA backbone), so long as the interaction as a whole is sequence-specific.

[0597] 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, should mean excluding other elements that have any essential significance for the use of the combination for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants or inert carriers. "Consisting of" shall mean excluding elements other than trace amounts of other components and substantial method steps. Aspects defined by each of these transitional terms are within the scope of the present invention.

[0598] The term "epitope" refers to the antigenic determinant of a polypeptide. An epitope can contain three amino acids in a spatial conformation that is unique to that epitope. Typically, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more typically of 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.

[0599] As used herein, "expression" refers to the process of transcription of a polynucleotide into mRNA and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0600] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. The gene product can be the direct transcriptional 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 the mRNA. Gene products also include RNAs modified by methods such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0601] "Regulation" or "regulation" of gene expression refers to a change in gene activity. Regulation of expression can include, but is not limited to, gene activation and gene repression.

[0602] The term "operably linked" or its equivalent terms (e.g., "operably connected") means that two or more molecules are positioned relative to each other such that they can interact to affect the function attributable to one molecule or both molecules or a combination thereof.

[0603] Non-covalently linked components and methods for preparing 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 transient interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate is achieved only or primarily in cases where such association is required for the desired activity. The linkage can persist for a time sufficient to allow the desired effect.

[0604] Methods for directing a protein to a specific locus in a biological genome are disclosed. The method can include the steps of providing a DNA targeting component and providing an effector molecule, wherein the DNA targeting component and the effector molecule are capable of being operably linked via a non-covalent bond.

[0605] The term "scFv" refers to a single-chain variable fragment. An scFv is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are joined by a linker peptide. The length of 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. The single-chain variable fragment lacks the constant Fc region found in the intact antibody molecule and thus lacks a common binding site (e.g., protein G) for antibody purification. The term also includes scFvs that are intracellular antibodies, i.e., antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0606] The term "single-domain antibody" means an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a specific antigen. Single-domain antibodies are typically peptide chains about 110 amino acids in length, containing one variable domain (VH) of a heavy-chain antibody or a common IgG, and generally having an affinity for the antigen similar to that of the intact antibody, but being 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 made from common murine or human IgG having four chains.

[0607] As used herein, the terms "specifically bind" and "specific binding" refer to the ability of an antibody, antibody fragment, or nanobody to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens. In some aspects, the specific binding interaction will distinguish the desired antigen from the undesired antigens in a sample. In some aspects, by more than about ten- to 100-fold or more (e.g., by more than about 1000-fold or 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 relative to different antigen targets and does not necessarily imply high affinity.

[0608] 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 that sufficient binding conditions exist.

[0609] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. 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 the depicted single strand. The nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complementary sequences that retain the same structure or encode the same protein.

[0610] The probes of the present disclosure can comprise single-stranded nucleic acids that hybridize to a target sequence under stringent hybridization conditions. Thus, the nucleic acids of the present disclosure can refer to probes that hybridize under stringent hybridization conditions.

[0611] The nucleic acids of the present disclosure can be single-stranded or double-stranded. Even when most of the molecules are single-stranded, the nucleic acids of the present disclosure can contain double-stranded sequences. Even when most of the molecules are double-stranded, the nucleic acids of the present disclosure can contain single-stranded sequences. The nucleic acids of the present disclosure can include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure can contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure can contain a base combination including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure can be synthesized to contain non-natural amino acid modifications. The nucleic acids of the present disclosure can be obtained by chemical synthesis methods or by recombinant methods.

[0612] The nucleic acids of the present disclosure (in whole or in any part thereof) can be non-naturally occurring. The nucleic acids of the present disclosure can contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions such that the entire nucleic acid sequence is non-naturally occurring. The nucleic acids of the present disclosure can contain one or more duplicated, inverted, or repeated sequences that result in a sequence that is not naturally occurring such that the entire nucleic acid sequence is non-naturally occurring. The nucleotides of the present disclosure can contain modified, artificial, or synthetic nucleotides that are not naturally occurring such that the entire nucleotide sequence is non-naturally occurring.

[0613] Given the redundancy of the genetic code, multiple nucleotide sequences can encode any particular protein. All such nucleotide sequences are contemplated herein.

[0614] As used throughout the disclosure, the term "operably linked" refers to the expression of a gene under the control of a promoter that is spatially linked thereto. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be substantially the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Variations in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0615] As used throughout the disclosure, the term "promoter" refers to a molecule, synthetic or of natural origin, that is capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance its expression and / or to alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements that may be located up to several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate the expression of a genetic component constitutively or differentially, relative to the cell, tissue, or organ in which expression occurs, or relative 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 the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operon-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the EF-1α promoter, the CAG promoter, the SV40 early promoter or the SV40 late promoter and the CMV IE promoter.

[0616] As used throughout the disclosure, the term "substantially complementary" means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complementary sequence of the 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 the two sequences hybridize under stringent hybridization conditions.

[0617] As used throughout the disclosure, the term "substantially identical" means that the first sequence and the second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or, with respect to nucleic acids, if the first sequence is substantially complementary to the complementary sequence of the second sequence.

[0618] As used throughout the disclosure, when used to describe a nucleic acid, the term "variant" means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a sequence that is substantially identical thereto under stringent conditions.

[0619] As used throughout the disclosure, the term "vector" means a nucleic acid sequence that contains an origin of replication. A vector can be a viral vector, a phage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be an extrachromosomal self-replicating vector, and preferably is a DNA plasmid. A vector can contain a combination of amino acids and a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0620] As used throughout the disclosure, the term "variant" when used to describe a peptide or polypeptide means a peptide or polypeptide that is different in amino acid sequence by an insertion, deletion, or conservative substitution of an amino acid but retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially identical to the amino acid sequence of a reference protein having an amino acid sequence that retains at least one biological activity.

[0621] Conservative substitution of an amino acid, i.e., the replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered to involve relatively minor changes in the art. As understood in the art, these relatively minor changes can be identified in part by considering the hydrophilicity index of the amino acids. Kyte et al., J. Mol. Biol.) 157:105-132 (1982). The hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids having similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in a protein that retains biological function. Consideration of the hydrophilicity of amino acids in the case of a peptide allows calculation of the highest local average hydrophilicity of the peptide, which is a useful measure that has been reported to be well correlated with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.

[0622] Substitution of amino acids having similar hydrophilicity values can result in a peptide that retains biological activity, such as immunogenicity. Substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. The hydrophobicity index and hydrophilicity value of an amino acid are both affected by the particular side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0623] As used herein, "conservative" amino acid substitutions can be defined as shown in Table A, B, or C below. In some aspects, the fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions that have been introduced by modifying the polynucleotides encodi...

Claims

1. A compound of formula (I): Wherein: A is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group; Each R1 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, wherein 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 group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group, or -(CHR6)CH(SXLY)R6; X is S or CH2; Y is -OH, n = 1 - 3, R" = H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group; Each R6 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, wherein at least one occurrence of R6 is hydrogen; Or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein one of B, C, and D is -(CHR6)CH(SXLY)R6.

3. The compound according to claim 1, wherein two of B, C, and D are -(CHR6)CH(SXLY)R6.

4. The compound according to claim 1, wherein each of B, C, and D is independently -(CHR6)CH(SXLY)R6.

5. The compound according to claim 1, wherein X is S and Y is -OH.

6. The compound according to claim 1, wherein X is S.

7. The compound according to claim 1, wherein X is CH2.

8. The compound according to any one of claims 6 or 7, wherein Y is -OH.

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

10. The compound according to claim 9, wherein L is an unsubstituted C2 alkyl group.

11. The compound according to claim 1, wherein R1 is a substituted or unsubstituted branched or unbranched C1-15 alkyl group.

12. The compound according to claim 1, wherein R1 is a substituted or unsubstituted branched or unbranched C1-10 alkyl group.

13. The compound according to claim 1, wherein R1 is a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

14. The compound according to claim 1, wherein R6 is a substituted or unsubstituted branched or unbranched C1-15 alkyl group.

15. The compound according to claim 1, wherein R6 is a substituted or unsubstituted branched or unbranched C1-10 alkyl group.

16. The compound according to claim 1, wherein R6 is a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

17. The compound according to claim 1, wherein is 18. The compound according to claim 1, wherein is 19. The compound according to claim 1, wherein is 20. The compound according to claim 3, wherein one of B, C, and D is methyl, X is S and Y is -OH.

21. The compound according to claim 20, wherein L is an unsubstituted C2 alkyl group.

22. The compound according to claim 20, wherein R1 is C 12 H 25 .

23. The compound according to claim 20, wherein R6 is C 12 H 25 .

24. The compound according to claim 20, wherein is a C3 alkyl group.

25. A compound of the following formula:

26. A compound of formula (II): Wherein: A is 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, a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, or a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group, 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, n = 1 - 3, R" = H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group; Or a pharmaceutically acceptable salt thereof.

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

28. The compound according to claim 26, wherein X is S.

29. The compound according to claim 26, wherein X is CH2.

30. The compound according to any one of claims 28 or 29, wherein Y is -OH.

31. The compound according to claim 26, wherein L is an unsubstituted branched or unbranched C1-6 alkyl group.

32. The compound according to claim 31, wherein L is an unsubstituted C2 alkyl group.

33. The compound according to claim 26, wherein each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-15 alkyl group.

34. The compound according to claim 26, wherein each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-10 alkyl group.

35. The compound according to claim 26, wherein each of R1, R2, R3, and R4 is independently a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

36. The compound according to claim 26, wherein each of R1, R2, R3 and R4 is independently C 10 H 21 .

37. The compound according to claim 26, wherein is 38. A compound of the following formula:

39. A compound of formula (III): Wherein: Each of Ra and Rb is independently X is S or CH2; Y is -OH, n = 1 - 3, R" = H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group; 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 from 1 to 10, including the end values; y is an integer from 1 to 10, including the end values; Each Ry and Rz is independently Or a pharmaceutically acceptable salt thereof.

40. The compound according to claim 39, wherein Ra is and Rb is 41. The compound according to claim 39, wherein Ra is 42. The compound according to claim 39, wherein Ra is 43. The compound according to claim 39, wherein Ra is 44. The compound according to claim 39, wherein Rb is 45. The compound according to claim 39, wherein Rb is 46. The compound according to claim 39, wherein Rb is 47. The compound according to claim 39, wherein x is 1 and y is 2.

48. The compound according to claim 39, wherein X is S and Y is -OH.

49. The compound according to claim 39, wherein X is S.

50. The compound according to claim 39, wherein X is CH2.

51. The compound according to any one of claims 49 or 50, wherein Y is -OH.

52. The compound according to claim 39, wherein L is an unsubstituted branched or unbranched C1-6 alkyl group.

53. The compound according to claim 52, wherein L is an unsubstituted C2 alkyl group.

54. The compound according to claim 39, wherein R7 is a substituted or unsubstituted branched or unbranched C1-15 alkyl group.

55. The compound according to claim 39, wherein R7 is a substituted or unsubstituted branched or unbranched C1-10 alkyl group.

56. The compound according to claim 39, wherein R7 is a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

57. The compound according to claim 39, wherein R7 is C 10 H 21 .

58. The compound according to claim 39, wherein Ra is 59. The compound according to claim 39, wherein Rb is 60. A compound of the following formula:

61. A compound of formula (IV): Wherein: Each of Re and Rf is independently x is an integer from 1 to 10, including the end values; Each Ry and Rz is independently X is S or CH2; Y is -OH, n = 1 - 3, R" = H or Me; L is a substituted or unsubstituted branched or unbranched C1-6 aliphatic group, a substituted or unsubstituted branched or unbranched C1-6 heteroaliphatic group; R7 is a substituted or unsubstituted branched or unbranched C1-15 aliphatic group, a substituted or unsubstituted branched or unbranched C1-15 heteroaliphatic group; or a pharmaceutically acceptable salt thereof.

62. The compound according to claim 61, wherein Re is 63. The compound according to claim 61, wherein Rf is 64. The compound according to claim 61, wherein x is 1.

65. The compound according to claim 61, wherein x is 2.

66. The compound according to claim 61, wherein x is 3.

67. The compound according to claim 61, wherein X is S and Y is -OH.

68. The compound according to claim 61, wherein X is S.

69. The compound according to claim 61, wherein X is CH2.

70. The compound according to any one of claims 68 or 69, wherein Y is -OH.

71. The compound according to claim 61, wherein L is an unsubstituted branched or unbranched C1-6 alkyl group.

72. The compound according to claim 71, wherein L is an unsubstituted C2 alkyl group.

73. The compound according to claim 61, wherein R7 is a substituted or unsubstituted branched or unbranched C1-15 alkyl group.

74. The compound according to claim 61, wherein R7 is a substituted or unsubstituted branched or unbranched C1-10 alkyl group.

75. The compound according to claim 61, wherein R7 is a substituted or unsubstituted branched or unbranched C1-5 alkyl group.

76. A composition comprising at least one lipid nanoparticle, said at least one lipid nanoparticle comprising at least one compound according to claim 1, 26, 39 or 61, or a salt thereof, wherein said at least one lipid nanoparticle further comprises at least one nucleic acid molecule.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

91. The composition according to claim 76, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one transposon, wherein the transposon comprises a nucleic acid sequence encoding at least one therapeutic protein.

92. The composition according to claim 90 or 91, wherein the at least one therapeutic protein is: (a) a chimeric antigen receptor (CAR); (b) Ornithine carbamoyltransferase (OTC) polypeptide; (c) Methylmalonyl-CoA mutase (MUT1) polypeptide; (d) Factor VIII (FVIII) polypeptide; or (d) Any combination thereof.

93. A pharmaceutical composition, the pharmaceutical composition comprising the composition according to claim 76 and at least one pharmaceutically acceptable excipient or diluent.

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

95. A method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one composition according to claim 76.

96. The method according to claim 94 or claim 95, wherein the at least one cell is: (a) A liver cell, wherein the liver cell is a hepatocyte, hepatic stellate cell, Kupffer cell or hepatic sinusoidal endothelial cell; (b) T cells, wherein the T cells are activated T cells, resting T cells or stem cell memory T cells (T SCM cells); (c) Hematopoietic stem cell (HSC).

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

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

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

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

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

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