Transfection compositions comprising cationic polymers, cationic lipids, and stabilizing agents

Abstract

Pegylated lipids are combined with cationic amine-containing polymers and copolymers and amphipathic compounds to form stabilized transfection complexes for sustained delivery of nucleic acids to cells in biological systems, specifically for in vitro cell transfection research.

Description

Attorney Docket No.: P24-229-SEC-WO01TRANSFECTION COMPOSITIONS COMPRISING CATIONIC POLYMERS,CATIONIC LIPIDS, AND STABILIZING AGENTSCross-Reference To Related Applications

[0001] This application claims the benefit of priority to U. S. Provisional Application No. 63 / 726,549, filed November 30, 2024, which is incorporated by reference herein for all purposes.Field of the Invention

[0002] The field of the present invention is compositions comprising cationic copolymers cationic lipids, and stabilizing agents, and the use of such compositions for delivering nucleic acids to a cell.Background

[0003] Nucleic acids play a central role in living organisms. Hence, they make an ideal therapeutic target. It is thought that many diseases could be controlled by the manipulation of nucleic acids in living organisms.

[0004] The key factor limiting therapies based on nucleic acid manipulation is the ability to deliver nucleic acids to the appropriate compartment of the cells. Nucleic acids are highly charged molecules and may not cross the lipid membranes surrounding the cell or readily escape from endosomal compartments involved in the uptake of macromolecules into cells. Chemical transfection of nucleic acids may provide a convenient and robust alternative to viral, liposomal encapsulation and electroporative delivery.

[0005] In chemical transfections, a transfection complex is formed when nucleic acids are combined with polycation transfection reagents. Through a combination of electrostatic and other noncovalent interactions, the cationic formulations bind the negatively charged nucleic acids to form transfection complexes, enabling cell surface binding. Such cationic non-viral, non-liposomal formulations may also help producing viral particles on a large scale.

[0006] It is an object of the invention to provide better transfection complexes to deliver nucleic acid molecules.Attorney Docket No.: P24-229-SEC-WO01Summary

[0007] In one aspect, provided herein is a composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formula (I),X-L1Formula (I)or a salt or stereoisomer thereof, wherein:A is O, CH-L2-Y or N-L2-Y;B is H, -OH, -NH2, -OCH3, or -CH3;each LI and L2 is independently a linker;L3 is a PEG linker;each X and Y is independently alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein said alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; and q is an integer from 3 to 125.

[0008] In some embodiments, the pegylated lipid is according to Formula (la):X— LL L3j OlL2JqFormula (la)or a salt or stereoisomer thereof, wherein:OAttorney Docket No.: P24-229-SEC-WO01LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently a bond, -CH?-, -(CH?)?-, or -(CH?)?-; and * denotes a connection leading to A.

[0009] In some embodiment, pegylated lipid is according to Formula (lb):Formula (lb)or a salt or stereoisomer thereof, wherein:Attorney Docket No.: P24-229-SEC-WO01LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0010] In another aspect, provided herein is a composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formulas (Ila) or (lib),Formula (Ila)Attorney Docket No.: P24-229-SEC-WO01Formula (lib)or a salt or stereoisomer thereof, wherein:D is H, -OH, -NH2, -OCH3, or -CH3;Lcis a PEG linker;X is alkyl, alkenyl, or alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; andq is an integer from 3 to 125.

[0011] In another aspect, provided herein is a composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formulas (Illa) or (Illb),or a salt or stereoisomer thereof, wherein:D is H, -OH, -NH2, -OCH3, or -CH3;E is H or C1-C4 alkyl;Lcis a PEG linker;each X and Y is independently alkyl, alkenyl, or alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groupsAttorney Docket No.: P24-229-SEC-WO01independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; andq is an integer from 3 to 125.

[0012] In another aspect, provided herein is a composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formula (IV),L1Formula (IV)or a salt or stereoisomer thereof, wherein:B is H, -OH, -NH2, -OCH3, or -CH3;LI is a bond or a linker;X is a cholesterol or a derivative thereof, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein said alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; andq is an integer from 3 to 125.

[0013] In some embodiments, for all the compositions comprising the pegylated lipid disclosed herein, the cationic polymer comprises Formula (V);R1R2R21R22Formula (V)wherein:each R1and R2is independently hydrogen or methyl;W is -NH- or -O-;R21comprises a structure of:Attorney Docket No.: P24-229-SEC-WO01RLRRXNZwherein:each RLand RRis independently -(CH2)r-;each r is independently 1, 2, 3, 4, 5, or 6;R23is H or CH3;R22is independently alkyl, alkenyl, or heteroalkyl, wherein said alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0014] In some embodiments, for all the compositions comprising the pegylated lipid disclosed herein, the cationic polymer comprises Formula (VI);R1R2Formula (VI)wherein:each R1and R2is independently hydrogen or methyl;W is -NH- or -O-;each R3and R4is independently alkyl, alkenyl, or heteroalkyl, wherein said alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, Ci- C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0015] In some embodiments, for all the compositions comprising the pegylated lipid disclosed herein and the cationic polymer disclosed herein, the amphipathic compound is according to Formula (VII);Attorney Docket No.: P24-229-SEC-WO01R13 / \ R14R1< " L4-N\ _ J\l-L5" XR12Formula (VII)wherein each R11and R12is independently alkyl or alkenyl, wherein each said alkyl and alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;each R13and R14is independently hydrogen, alkyl, alkenyl, alkoxy, alkyl-C(O)-alkylene, alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-C(O)-, heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alky 1-heterocy cl e-alkeny 1 ene-C (O)-,alkenyl-heterocycle-alkenylene-C(O)-, heteroaryl-C(O)-,heteroaryl-alkylene-C(O)-, heteroaryl-alkenylene-C(O)-,alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-, alkyl-heteroaryl-alkenylene-C(O)-, oralkenyl-heteroaryl-alkenylene-C(O)-, wherein each said alkyl, alkenyl, alkylene, alkenylene, heterocycle, an heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;each L4 and L5 is independently C2-C8 alkylene, optionally substituted with one or two groups independently selected from the group consisting of halide, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andp is 0 or 1.

[0016] In another aspect, provided herein is a transfection reagent comprising a composition of any one of those disclosed here, and a nucleic acid.

[0017] In still another aspect, provided herein is a method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of those disclosed herein with the nucleic acid to produce a mixed solution, and contacting the cell with the mixed solution, thereby transfecting the nucleic acid into the cell.Attorney Docket No.: P24-229-SEC-WO01

[0018] In another aspect, provided herein is a method of forming and stabilizing a transfection complex, comprising mixing a composition of any one of those disclosed herein with the nucleic acid, thereby producing a plurality of transfection complexes comprising the pegylated lipid, the cationic polymer, the amphipathic compound, and the nucleic acid

[0019] In still another aspect, provided herein is a method of preparing a transfection reagent mixture, comprising: (a) adding a pegylated lipid to a mixture of a cationic polymer and an amphipathic compound, thereby obtaining another mixture; and (b) mixing the another mixture, thereby obtaining the transfection reagent mixture.

[0020] In some embodiments, each of the pegylated lipid, the cationic polymer, and the amphipathic compound is according to any one of those disclosed herein.

[0021] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative instances of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different instances, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0022] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.Brief Description of the Drawings

[0023] The novel features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the presentAttorney Docket No.: P24-229-SEC-WO01invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0024] FIG. 1 shows a schematic diagram showing the changing size of a transfection complex over time in the absence of a stabilizing agent.

[0025] FIG. 2 shows a schematic diagram showing the changing size of a transfection complex over time in the presence of a stabilizing agent.

[0026] FIG. 3 shows changes in average particle size and AAV titers in an example viral production process when the three-component transfection complex is added at 5% culture volume.

[0027] FIG. 4 shows changes in average particle size and AAV titers in an example viral production process when the three-component transfection complex is added at 2% culture volume.

[0028] FIG. 5 shows changes in average particle size and AAV titers in an example viral production process when the four-component transfection complex is added at 5% culture volume.

[0029] FIG. 6 shows changes in average particle size and AAV titers in an example viral production process when the four-component transfection complex is added at 2% culture volume.

[0030] FIG. 7 shows changes in LV titers in an example viral production process when different concentrations of the stabilizing agent are used.

[0031] FIG. 8 shows genome titers and percentages of full capsids in AAV particles harvested from example viral production processes when using different stabilizing agents.

[0032] FIG. 9 shows genome titers and percentages of full capsids in AAV particles harvested from example viral production processes when using different transfection reagents.DETAILED DESCRIPTION

[0033] The present disclosure pertains to the use of compositions comprising synthetic cationic copolymers and amphipathic compounds as nucleic acid transfection agents.Attorney Docket No.: P24-229-SEC-WO01

[0034] While various embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0035] Compounds are generally described herein using standard nomenclature. For compounds having asymmetric centers, it should be understood that (unless otherwise specified) all of the optical isomers and mixtures thereof are encompassed. In addition, compounds with carbon-carbon double bonds may occur in Z- and E-forms, with all isomeric forms of the compounds being included in the present invention unless otherwise specified. Where a compound exists in various tautomeric forms, a recited compound is not limited to any one specific tautomer, but rather is intended to encompass all tautomeric forms.

[0036] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0037] When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range is encompassed within the scope of the present disclosure. Where the stated range includes upper or lower limits, ranges excluding either of those included limits are also included in the present disclosure. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulas, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.

[0038] As used herein, the term “about” or “nearly” when referring to a number or a numerical range means that the number or numerical range generally referred to is within + / - 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the stated number or numerical range.

[0039] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.Attorney Docket No.: P24-229-SEC-WO01

[0040] As used herein, the term “alkyl” generally refers to an optionally substituted straight-chain or branched alkyl radical having 1 to 50 carbon atoms.

[0041] As used herein, the term “Ci-Ce alkyl” generally refers to an optionally substituted straight or branched hydrocarbon chain (or alkyl radical) having from 1 to 6 carbon atoms, and the straight or branched hydrocarbon chain is attached to the rest of the molecule by a single bond. Likewise, an alkyl group comprising up to 3 carbon atoms is a C1-C3 alkyl group, and an alkyl group comprising up to 4 carbon atoms is a C1-C4 alkyl group. Examples of a Ci-Ce alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1 -ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3 -methylpentyl, 2-methylpentyl, 1 -methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -dimethylbutyl, 1,2-dimethylbutyl, 1,3 -dimethylbutyl, 2,3-dimethylbutyl, and 2-ethylbutyl. In some instances, a substituent of an alkyl group is specifically indicated. For example, “cyanoalkyl” refers to an alkyl group substituted with at least one cyano substituent.

[0042] The Ci-Ce alkyl group may be optionally substituted with a C1-C3 alkoxy group. Examples include, but are not limited to, methoxyethyl, methoxypropyl, methoxyisopropyl, ethoxyethyl, ethoxypropyl, ethoxyisopropyl, propoxyethyl, propoxypropyl, and propoxyisopropyl.

[0043] The Ci-Ce alkyl group may be optionally substituted with a C3- Ce cycloalkyl group. Examples include, but are not limited to, 1 -methylcyclopropyl, 1 -methylcyclobutyl, and 1 -methylcyclohexyl.

[0044] As used herein, the term “C1-C15 alkyl” generally refers to an optionally substituted straight-chain or branched hydrocarbon chain (or alkyl radical) having 1 to 15 carbon atoms, and the straight or branched hydrocarbon chain is attached to the rest of the molecule by a single bond. The following may be mentioned by way of example and by way of preference: methyl; ethyl; n-propyl; isopropyl; n-butyl; isobutyl; sec-butyl; tert-butyl; n-pentyl; isopentyl; n-hexyl; 2-methylpentyl; n-heptyl; 2-m ethylhexyl; n-octyl; 2-ethylheptyl; n-nonyl; 3-methyloctyl; n-decyl; 2-propylnonyl; n-undecyl; 3-methyldecyl; n-dodecyl; 2-ethyldecyl; n-tridecyl; 3-methylundecyl; n-tetradecyl; 2-propyltridecyl; n-pentadecyl; and 3 -methyltetradecyl. C1-C20 alkyl represents an optionally substituted straight-chain or branched alkylAttorney Docket No.: P24-229-SEC-WO01radical having 1 to 20 carbon atoms. The following may be mentioned by way of example and by way of preference: methyl; ethyl; n-propyl; isopropyl; n-butyl; isobutyl; sec-butyl; tert-butyl; n-pentyl; isopentyl; n-hexyl; 2-methylpentyl; n-heptyl; 2-m ethylhexyl; n-octyl; 2-ethylheptyl; n-nonyl; 3-methyloctyl; n-decyl; 2-propylnonyl; n-undecyl; 3-methyldecyl; n-dodecyl; 2-ethyldecyl; n-tridecyl; 3-methylundecyl; n-tetradecyl; 2-propyltridecyl; n-pentadecyl; 3 -methyltetradecyl; n-hexadecyl; 2-ethylpentadecyl; n-heptadecyl; 3 -methylhexadecyl; n-octadecyl; 2-propylheptadecyl; n-nonadecyl; and 3-methylnonadecyl; n-icosyl; 2-ethylnonadecyl; and 3-methylicosyl. C4-C48 alkyl represents an optionally substituted straight-chain or branched alkyl radical having 4 to 48 carbon atoms. The following may be mentioned by way of example and by way of preference: n-butyl; isobutyl; sec-butyl; tert-butyl; n-pentyl; isopentyl; n-hexyl; 2-methylpentyl; n-heptyl; 2-m ethylhexyl; n-octyl; 2-ethylheptyl; n-nonyl; 3-methyloctyl; n-decyl; 2-propylnonyl; n-undecyl; 3-methyldecyl; n-dodecyl; 2-ethyldecyl; n-tridecyl; 3 -methylundecyl; n-tetradecyl; 2-propyltri decyl; n-pentadecyl; and 3 -methyltetradecyl; n-hexadecyl; n-heptadecyl; n-octadecyl; n-nonadecyl; n-icosyl; n-docosyl; n-tricosyl; n-tetracosyl; n-pentacosyl; n-hexacosyl; n-heptacosyl; n-octacosyl; n-nonacosyl; n-triacontyl; n-hentriacontyl; n-dotriacontyl; n-tritriacontyl; n-tetratriacontyl; and n-pentatriacontyl.

[0045] As used herein, the term “alkoxy” generally refers to an optionally substituted straight-chain or branched alkoxy radical. A straight-chain or branched alkoxy radical having 1 to 6 carbon atoms is preferred. The following radicals may be mentioned by way of example and by way of preference: methoxy; ethoxy; n-propoxy; isopropoxy; n-butoxy; tert-butoxy; n-pentoxy and n-hexoxy. As used herein, the term “Ci-Ce alkoxy” generally refers to a radical of the formula -OR wherein R is a Ci-Ce alkyl group as defined. Likewise, an alkoxy group comprising up to 3 carbon atoms is a C1-C3 alkoxy group. Examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, n-pentoxy, isopentoxy, 2-methylbutoxy, neopentoxy, 1 -ethylpropoxy, n-hexyloxy, isohexyloxy, 4-methylpentoxy, 3 -methylpentoxy, 2-methylpentoxy, 1 -methylpentoxy, 3.3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1 -dimethylbutoxy, 1,2-dimethylbutoxy, 1.3-dimethylbutoxy, 2,3 -dimethylbutoxy, and 2-ethylbutoxy.Attorney Docket No.: P24-229-SEC-WO01

[0046] The C1-C3 alkoxy group may be optionally substituted with a C1-C3 alkoxy group. Examples include, but are not limited to, methoxymethoxy, methoxyethoxy, methoxypropoxy, methoxyisopropoxy, ethoxymethoxy, ethoxy ethoxy, ethoxypropoxy, ethoxyisopropoxy, propoxymethoxy, propoxy ethoxy, propoxypropoxy, and propoxyisopropoxy.

[0047] As used herein, the term “hydroxyalkyl” generally refers to an alkyl group as defined above that contains one or more hydroxyl (-OH) functional groups.

[0048] As used herein, the term “cycloalkyl” generally refers to an optionally substituted saturated or partially saturated cycloalkyl group, which can be either monocyclic or polycyclic, having 3 to 8 atoms per ring, whereby the polycyclic cycloalkyl group can be fused ring structures, spirocycles, bridged rings or a cycloalkyl group fused with one or more aromatic rings. The following may be mentioned by way of example and by way of preference: cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; cycloheptyl; cyclohexenyl; cyclopentenyl; cyclobutenyl; cycloheptenyl; cyclooctenyl; bicyclofl.1.0]butyl; bicyclo[2.2.0]hexyl;bicyclo[3.3.0]octyl; bicyclo[3,2.0]heptyl; bicyclo[2.2.0]hexenyl; bicyclo[3.3.0]octenyl; decalyl; norbornyl; cubyl; bicyclo[4.2.0]octan-7-ylidene; decahydronaphthalen-2-ylidene; bicyclo[2.2.0]hexan-2-ylidene; bicyclo[4.2.0]octa-l(6);2;4-trien-7-ylidene; 2;3-dihydro-lH-inden-l-ylidene; 2;3-dihydro-lH-inden-2-ylidene; spiro[3.3]heptan-2-ylidene; spiro[3.5]nonan-7-ylidene; spiro[3.5]nonan-2-ylidene; spiro[5.5]undecan-3-ylidene; adamantan-2-ylidene; bicyclo[3.1.1]heptan-3-ylidene; bicyclo[2.2.2]octan-2-ylidene; bicyclo[2.1. l]hexan-2-ylidene; bicyclo[2.2.1]heptan-2-ylidene; cholesteryl; cholesteryl-derivatives and adamantyl.

[0049] As used herein, the term “C3-C6 cycloalkyl” generally refers to a monocyclic non-aromatic radical having from 3 to 6 ring atoms, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “cycloalkenyl” as used herein generally refers to a group that comprises one or more unsaturated rings in which all ring members are carbon. Polycyclic cycloalkyls or carbocycles include, for example, adamantyl, norbomyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane,Attorney Docket No.: P24-229-SEC-WO01bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless stated otherwise specifically in the specification, a cycloalkyl is optionally substituted.

[0050] As used herein, the term “alkenyl” generally refers to an optionally substituted straight-chain or branched alkenyl radical, which comprise at least one unsaturated carbon-carbon double bond (preferably, one to five double bonds and 2 to 50 carbon atoms). Alkenyl groups include C2-8 alkenyl, C2-6 alkenyl and C2-4 alkenyl groups, which have from 2 to 8, 2 to 6, or 2 to 4 carbon atoms, respectively, including, for example, ethenyl, allyl or isopropenyl. C2-6 alkenyl represents an optionally substituted straight-chain or branched alkenyl radical having one or two double bonds and 2 to 6 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; allyl; n-prop-l-en-l-yl; iso-propenyl; n-but-l-en-l-yl; n-but-2-en-l-yl; n-but-3-en-l-yl; 2-methylprop-l-en-l-yl; 2-methylprop-2-en-l-yl; n-pent-1-en-l-yl; and 2-methylbut-2-en-l-yl. C2-15 alkenyl represents an optionally substituted straight-chain or branched alkenyl radical having one to five double bonds and 2 to 15 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; allyl; n-prop-l-en-l-yl; iso-propenyl; n-but-l-en-l-yl; n-but-2-en-l-yl; n-but-3-en-l-yl; 2-methylprop-l-en-l-yl; 2-methylprop-2-en-l-yl; n-pent-l-en-l-yl; 2-methylbut-2-en-l-yl; n-hex-l-en-l-yl; 3 -methylpent- 1-en-l-yl; n-hept-l-en-l-yl; 2-methylhex-2-en-l-yl; n-oct- 1-en-l-yl; 3 -methylhept- 1-en-l-yl; n-non- 1-en-l-yl; 2-methyloct-2-en-l-yl; n-dec- 1-en-l-yl; 3 -methylnon- 1-en-l-yl; n-undec- 1-en-l-yl; 2-methyldec-2-en-l-yl; n-dodec- 1-en-l-yl; 3 -methylundec- 1-en-l-yl; n-tridec-l-en-l-yl; 2-methyldodec-2-en-l-yl; n-tetradec- 1-en-l-yl; 3-methyltridec- 1-en-l-yl; n-pentadec- 1-en-l-yl; and 2-methyl-tetradec-2-en-l-yl. C2-20 alkenyl represents an optionally substituted straight-chain or branched alkenyl radical having one to five double bonds and 2 to 20 carbon atoms. The following may be mentioned by way of example and by way of preference: vinyl; allyl; n-prop-l-en-l-yl; isopropenyl; n-but-l-en-l-yl; n-but-2-en-l-yl; n-but-3-en-l-yl; 2-methylprop-l-en-l-yl; 2-methylprop-2-en-l-yl; n-pent- 1-en-l-yl; 2-methylbut-2-en-l-yl; n-hex-l-en-l-yl; 3-methylpent- 1-en-l-yl; n-hept- 1-en-l-yl; 2-methylhex-2-en-l-yl; n-oct- 1-en-l-yl; 3-methylhept- 1-en-l-yl; n-non- 1-en-l-yl; 2-methyloct-2-en-l-yl; n-dec-l-en-l-yl; 3-methylnon- 1-en-l-yl; n-undec- 1-en-l-yl; 2-methyldec-2-en-l-yl; n-dodec- 1-en-l-yl;Attorney Docket No.: P24-229-SEC-WO013 -methylundec- 1-en-l-yl; n-tridec-l-en-l-yl; 2-methyldodec-2-en-l-yl; n-tetradec-1-en-l-yl; 3-methyltridec-l-en-l-yl; n-pentadec- 1-en-l-yl; 2-methyltetradec-2-en-l-yl; n-hexadec-l-en-l-yl; 3 -methylpentadec- 1-en-l-yl; n-heptadec- 1-en-l-yl; 2-methylhexadec-2-en-l-yl; n-octadec- 1-en-l-yl; 3 -methylheptadec- 1-en-l-yl; n-nonadec- 1-en-l-yl; 2-methyloctadec-2-en-l-yl; n-icos-l-en-l-yl; and 3-methylnonadec- 1-en-l-yl. C4-48 alkenyl represents an optionally substituted straightchain or branched alkenyl radical having one to five double bonds and 4 to 48 carbon atoms. The following may be mentioned by way of example and by way of preference: n-but- 1-en-l-yl; n-but-2-en-l-yl; n-but-3-en-l-yl; 2-methylprop- 1-en-l-yl; 2-methylprop-2-en-l-yl; n-pent- 1-en-l-yl; 2-methylbut-2-en-l-yl; n-hex- 1-en-l-yl; 3-methylpent- 1-en-l-yl; n-hept- 1-en-l-yl; 2-methylhex-2-en-l-yl; n-oct- 1-en-l-yl; 3-methylhept- 1-en-l-yl; n-non- 1-en-l-yl; 2-methyloct-2-en-l-yl; n-dec- 1-en-l-yl; 3-methylnon- 1-en-l-yl; n-undec- 1-en-l-yl; 2-methyldec-2-en-l-yl; n-dodec- 1-en-l-yl; 3 -methylundec- 1-en-l-yl; n-tridec-l-en-l-yl; 2-methyldodec-2-en-l-yl; n-tetradec-1-en-l-yl; 3-methyltridec-l-en-l-yl; n-pentadec- 1-en-l-yl; 2-methyl-tetradec-2-en-l-yl; n-hexadec-l-en-l-yl; n-heptadec- 1-en-l-yl; n-octadec- 1-en-l-yl; n-nonadec- 1-en-l-yl; n-icos-l-en-l-yl; n-docos- 1-en-l-yl; n-tricos- 1-en-l-yl; n-tetracos- 1-en-l-yl; n-pentacos- 1-en-l-yl; n-h exacos- 1-en-l-yl; n-heptacos- 1-en-l-yl; n-octacos- 1-en-l-yl; n-nonacos- 1-en-l-yl; n-triacont-l-en-l-yl; and n-hentriacont- 1-en-l-yl.

[0051] As used herein, the term “alkynyl” as used herein generally refers to straight or branched chain alkyne groups (preferably, an alkynyl radical having one to five triple bonds and 2 to 50 carbon atoms), which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond. Alkynyl groups include C2-8 alkynyl, C2-6 alkynyl and C2-4 alkynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively. C2-6 alkynyl represents an optionally substituted straight-chain or branched alkynyl radical having one or two triple bonds and 2 to 6 carbon atoms. The following may be mentioned by way of example and by way of preference: ethynyl; prop-l-yn-l-yl; 2-propynyl; n-but- 1-yn-l-yl; 2-butynyl; and n-pent-l-yn-l-yl. C2-15 alkynyl represents an optionally substituted straight-chain or branched alkynyl radical having one to five triple bonds and 2 to 15 carbon atoms. The following may be mentioned by way of example and by way of preference: ethynyl; prop-l-yn-l-yl; 2-propynyl; n-but- 1-yn-l-yl; 2-butynyl; n-pent- 1-yn-l-yl; 3-Attorney Docket No.: P24-229-SEC-WO01methylbut-l-yn-l-yl; n-hex-l-yn-l-yl; 3 -methylpent- 1-yn-l-yl; n-hept-l-yn-l-yl; 2-methylhex-l-yn-l-yl; n-oct- 1-yn-l-yl; 3 -methylhept- 1-yn-l-yl; n-non- 1-yn-l-yl; 2-methyloct-l-yn-l-yl; n-dec- 1-yn-l-yl; 3 -methylnon- 1-yn-l-yl; n-undec- 1-yn-l-yl; 2-methyldec-l-yn-l-yl; n-dodec- 1-yn-l-yl; 3 -methylundec- 1-yn-l-yl; n-tridec- 1-yn-l-yl; 2-methyldodec-l-yn-l-yl; n-tetradec- 1-yn-l-yl; 3-methyltridec-l-yn-l-yl; n-pentadec- 1-yn-l-yl; and 2-methyltetradec- 1-yn-l-yl. C2-C20 alkynyl represents an optionally substituted straight-chain or branched alkynyl radical having one to five triple bonds and 2 to 20 carbon atoms. The following may be mentioned by way of example and by way of preference: ethynyl; prop- 1-yn-l-yl; 2-propynyl; n-but- 1-yn-l-yl; 2-butynyl; n-pent- 1-yn-l-yl; 3 -methylbut-l-yn-l-yl; n-hex-l-yn-l-yl; 3-methylpent- 1-yn-l-yl; n-hept-l-yn-l-yl; 2-methylhex-l-yn-l-yl; n-oct- 1-yn-l-yl; 3-methylhept- 1-yn-l-yl; n-non- 1-yn-l-yl; 2-m ethyl oct- 1-yn-l-yl; n-dec- 1-yn-l-yl; 3-methylnon- 1-yn-l-yl; n-undec- 1-yn-l-yl; 2-methyldec-l-yn-l-yl; n-dodec- 1-yn-l-yl; 3 -methylundec- 1-yn-l-yl; n-tridec- 1-yn-l-yl; 2-methyldodec-l-yn-l-yl; n-tetradec-1-yn-l-yl; 3-methyltridec-l-yn-l-yl; n-pentadec- 1-yn-l-yl; and 2-methyltetradec- 1-yn-l-yl. C4-C48 alkynyl represents an optionally substituted straight-chain or branched alkinyl radical having one to five triple bonds and 4 to 48 carbon atoms. The following may be mentioned by way of example and by way of preference: n-but-1-yn-l-yl; 2-butynyl; n-pent- 1-yn-l-yl; 3 -methylbut-l-yn-l-yl; n-hex-l-yn-l-yl; 3-methylpent- 1-yn-l-yl; n-hept-l-yn-l-yl; 2-methylhex-l-yn-l-yl; n-oct- 1-yn-l-yl; 3-methylhept- 1-yn-l-yl; n-non- 1-yn-l-yl; 2-m ethyl oct- 1-yn-l-yl; n-dec- 1-yn-l-yl; 3-methylnon- 1-yn-l-yl; n-undec- 1-yn-l-yl; 2-methyldec-l-yn-l-yl; n-dodec- 1-yn-l-yl; 3 -methylundec- 1-yn-l-yl; n-tridec- 1-yn-l-yl; 2-methyldodec-l-yn-l-yl; n-tetradec-1-yn-l-yl; 3-methyltridec-l-yn-l-yl; n-pentadec- 1-yn-l-yl; and 2-methyltetradec- 1-yn-l-yl.

[0052] As used herein, the term “aryl” generally refers to an all-carbon monocyclic or fused-ring polycyclic groups of 6 to 12 (Ce-12 aryl) or 6 to 10 carbon atoms (Ce-io aryl) having a completely conjugated pi-electron system. Examples include, but are not limited to, phenyl, naphthalenyl, fluorenyl, phenanthrenyl, tetrahydronaphthyl, indanyl, biphenyl, and anthracenyl. The aryl group may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl,Attorney Docket No.: P24-229-SEC-WO01thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NRXRY, wherein Rxand RYare independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and, combined, a five- or six-membered heteroalicyclic ring. Illustrative substituted alkyl group include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, aminomethyl, aminoethyl, hydoxymethyl, methoxymethyl, 2-fluoroethyl, and 2-methoxyethyl, etc.

[0053] As used herein, the term “halogen” or “halide” generally refers to fluorine, chlorine, bromine, and iodine. The term “haloalkyl” as used herein generally refers to an alkyl group that is substituted with one or more independently chosen halogens (e.g., “Ci-C6haloalkyl” groups have from 1 to 6 carbon atoms and at least one halogen). Examples of haloalkyl groups include, but are not limited to, mono-, di- or tri-fluoromethyl; mono-, di- or tri -chloromethyl; mono-, di-, tri-, tetra- or pentafluoroethyl; mono-, di-, tri-, tetra- or penta-chloroethyl; 2,2,2-trifluoroethyl; 1,2-difluoroethyl; 3-bromo-2-fluoropropyl; 1,2-dibromoethyl; and 1,2,2,2-tetrafluoro-l-trifluoromethyl-ethyl.

[0054] As used herein, the term “heteroalkyl” generally refers to an optionally substituted straight-chain or branched alkyl radical as defined above, in which one or more skeletal carbon atoms of the alkyl (preferably one to five carbon atoms) are selected from a heteroatom or heteroatom functionality (e.g., an atom other than carbon), e.g., oxygen (-O-), nitrogen (e.g., -NH-, -N=, -N(alkyl)-), sulfur (-S-S-, -S-, -S(=O)- or -S(=O)2), or combinations thereof. In some instances, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In one aspect, a heteroalkyl is a Ci-Ce heteroalkyl wherein the heteroalkyl is comprised of 1 to 6 carbon atoms and one or more atoms other than carbon, e.g., oxygen, nitrogen, sulfur, or combinations thereof. In some instances, a carbon atom or heteroatom is optionally oxidized (e g., -C(O)OCH2-, -CH2OCH2-, -CH2S(O)2NHCH2-, -NHC(O)NHCH2-, -CH2NHC(O)CH2-). Further examples of such heteroalkyl are, for example, -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH2OCH3, or -CH(CH3)OCH3, -CH2NHCH3, -CH2CH2NHCH3, -CH2CH2NHCH2CH2NHCH3, or -CH(CH3)NCH3. Unless stated otherwise specifically in the specification, a heteroalkyl is optionallyAttorney Docket No.: P24-229-SEC-WO01substituted for example, with oxo, halogen, amino, hydroxy, alkyl, alkenyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, a heteroalkyl is optionally substituted with halogen, methyl, ethyl, -CF3, -OH, -OMe, or -NH2. In some embodiments, a heteroalkyl is optionally substituted with methyl or NH2.

[0055] As used herein, the term “heteroalkenyl” generally refers to an optionally substituted straight-chain or branched alkenyl radical as defined above, wherein at least one carbon atom, preferably one to five carbon atoms, are independently replaced by a heteroatom or heteroatom functionality, selected from -NH-, -N=, -O-, -S-S-, -S-, -S(=O)- or -S(=O)2.

[0056] As used herein, the term “heteroalkynyl” generally refers to an optionally substituted straight-chain or branched alkynyl radical as defined above, wherein at least one carbon atom, preferably one to five carbon atoms, are independently replaced by a heteroatom or heteroatom functionality, selected from -NH-, -N=, -O-, -S-S-, -S-, -S(=O)- or -S(=O)2.

[0057] The terms “heterocyclic” or “heterocycle” or “heterocyclyl” or “cycloheteroalkyl” or “heterocycloalkyl” as used herein generally refer to a ring structure (monocycle or poly cycle) containing 3-12 ring atoms (3-12 membered heterocycle), 3-8 ring atoms (3-8 membered heterocycle or 3-8 membered cycloheteroalkyl), 3-6 ring atoms (3-6 membered heterocycle or 3-6 membered cycloheteroalkyl), or 5-6 ring atoms (5-6 membered heterocycle or 5-6 membered cycloheteroalkyl), in which at least one ring atom is carbon, and at least one ring atom is a heteroatom selected from N, O, and S, or a heteroatom group selected from C(=O), S(=O), and S(=O)2. A heterocyclic group may be aromatic or non-aromatic. Piperidine and oxetane are non-limiting examples of non-aromatic heterocycles. Thiazole and pyridine are non-limiting examples of aromatic heterocycles. Other examples of heterocycle include: aziridinyl, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, morpholinyl, piperazinyl,4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxothiomorpholinyl, butyrolactam, valerolactam, caprolactam, butyrolactone, valerolactone and caprolactone.Specifically, at least one ring contains at least one heteroatom or heteroatomAttorney Docket No.: P24-229-SEC-WO01functionality, preferably one to five heteroatoms or heteroatom functionalities, independently selected from -NH-, -N=, -O-, -S-S-, -S-, -S(=O)- or -S(=O)2- and is attached via a ring carbon atom or a heteroatom; whereby the polycyclic heterocycloalkyl group can be fused ring structures; spirocycles; bridged rings or a heterocycloalkyl group fused with one or more aromatic rings. The following may be mentioned by way of example and by way of preference: pyrrolidinyl; tetrahydrofuranyl; oxetan-3-ylidene; oxolan-3-ylidene; oxan-4-ylidene; oxepan-4-ylidene; morpholinyl; piperidinyl; furanyl; thienyl; thiazolyl; indolinyl; quinolinyl; tetrahydroquinolinyl; isoquinolinyl; benzothiazolyl; benzofuranyl; bicyclo[2.2.1]heptyl; 2-oxo-2,3-dihydro-lH-indol-3-ylidene; 4,5,6,7-tetrahydro-l-benzothiophen-4-ylidene; lH,4H,5H,6H-cyclopenta[b]pyrrol-4-ylidene; decahydroquinolin-4-ylidene; octahydro-2H-l -benzopyran-4-ylidene; hexahydro-lH-cyclopenta[c]furan-5-ylidene; 2,2-dioxo-21ambda6-thiaspiro[3.3]heptan-6-ylidene; 2-thiaspiro[3.3]heptan-6-ylidene; 2-oxaspiro[3.3]heptan-6-ylidene; 2-azabicyclo[2.2.2]octan-5-ylidene and 2-oxabicyclo[2.2.2]octan-5-ylidene.

[0058] Similarly, the term “cycloheteroalkenyl” refers to a monocycle or polycycle ring structure comprising carbon atom(s) and heteroatom(s) / heteroatom group(s), wherein the cycloheteroalkenyl comprises at least one C=C double bond, at least one ring atom that is carbon, and at least one ring atom that is a heteroatom selected from N, O, and S or a heteroatom group selected from C(=O), S(=O), and S(=O)2. Unless stated otherwise specifically in the specification, a heterocycle or heterocycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe.

[0059] The term “heteroaryl” as used herein generally refers to an optionally substituted aromatic group in which at least one aromatic ring comprises at least one heteroatom selected from N, O and S. Preferably, the optionally substituted aromatic heterocycle (or heteroaromatic radical) can be either monocyclic or polycyclic, having 3 to 8 atoms per ring, wherein at least one ring contains at least one heteroatom orAttorney Docket No.: P24-229-SEC-WO01heteroatom functionality, preferably one to five heteroatoms or heteroatom functionalities, independently selected from the group consisting of -NH-, -N=, -O-, -S-S-, -S-, -S(=O)- or -S(=O)2-, and is attached to the rest of the molecule via a ring carbon atom or a heteroatom, wherein each of the one or more rings can have 4 to 8 ring atoms. Heteroaryls include, for example, 5-12 membered heteroaryls, 5-10 membered heteroaryls, 5-7 membered monocyclic structures or 7-12 membered bicyclic structures. The number of heteroatoms in a heteroaryl can be 1, 2, 3, 4, or more. Examples include, but are not limited to, thienyl, pyridyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridine-2(lH)-keto, pyridine-4(lH)-keto, pyrrolyl, pyrazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-oxadiazolyl, imidazolyl, furanyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, naphthyl, benzothienyl, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolinyl, isoquinolinyl, and quinazolinyl. The heteroaryl group may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NRXRY, with Rxand RYas defined above.

[0060] As used herein, the term “amino” generally refers to primary amino group — l(-NH2), secondary amino group (-NH-), and tertiary amino group ( \).

[0061] As used herein, the term “alkylamino” generally refers to a secondary or tertiary amine that has the general structure -NH-R1or -N(R1)(R2), respectively, wherein R1and R2are selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Such alkylamino groups include, but are not limited to, mono- and di-(Ci-6 alkyl)amino groups, in which each C1-6 alkyl may be the same or different. In this case, the definition of “alkyl” as used in the term “alkylamino” differs from the definition of “alkyl” used for all other alkyl-containing groups, in the inclusion of cycloalkyl and (cycloalkyl)alkyl groups.

[0062] The terms “substituent” and “substituted,” as used herein, generally denote that a molecular moiety is covalently bonded to an atom within a molecule of interest. For example, a ring substituent may be a moiety such as a halogen, alkyl group,Attorney Docket No.: P24-229-SEC-WO01haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a ring member. Substituents of aromatic groups are generally covalently bonded to a ring carbon atom. A straight chain substituent may be a moiety such as a halogen, alkyl group, haloalkyl group or other group that is covalently bonded to an atom (preferably a carbon or nitrogen atom) that is a member of a straight chain.

[0063] As used herein, the term “optionally substituted” generally refers to that the referenced chemical moiety may be unsubstituted or substituted with one or more chemical groups. The term “substituted with one or more chemical group” generally refers to that one or more hydrogen atoms of a molecule are replaced by a different chemical group. In one embodiment of the invention, substituted means substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, hydroxy, oxo, thioxo, alkoxy, halogen, -CN, -NO2, -N3, -ORa, -NRaRb, -N+RaRbRc, -NRaC(=O)Rb, -SRa, -S-SRa, -C(=O)Ra, -CH(ORa)(ORb), -C(=O)ORa, -C(=O)SRa-, -C(=O)Ra, -OC(=O)Ra, -SC(=O)Ra, -C(=O)NRaRb, -OC(=O)NRaRb, -NRaC(=O)ORb, -NRaC(=NRb)NRcRd, -NRaC(=NRb)Rc, -C(=NRa)NRbRc, -NRaC(=O)NRbRc, -S(=O)Ra, -SO2Ra, -NRaSORb, -NRaSO2Rb, -S(=O)NRaRb, -C(=S)NRaRb, -NRaC(=S)Rb, -SO2NRaRb, and -OSO3Ra, -OPO3RaRb, -N=NRb, -OSiRaRbRc; wherein each of Ra, Rb, Rcand Rdis independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and alkoxy, and wherein each alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, Ra, Rb, Rcand Rdare optionally substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, hydroxy, oxo, thioxo, alkoxy, halogen, -CN, -NO2, -N3, -ORa’, -NRaRb, -N+RaRbRc, -NRaC(=O)Rb’, -SRa’, -S-SRa, -C(=O)Ra, -CH(ORa)(ORb), -C(=O)ORa’, -C(=O)SRa’, -C(=O)Ra’, -OC(=O)Ra’, -SC(=O)Ra, -C(=O)NRaRb, -OC(=O)NRaRb, -NHC(=0)NRaRb, - NRaC(=0)NHRb, -NRaC(=NRb)NRCRd, -NRa’C(=NRb’)Rc’, -C(=NRa)NRbRc, -NRaC(=O)NRbRc, -S(=O)Ra, -SO^’, -NRaSORb, -NRaSO2Rb, -S(=O)NRaRb’, -C(=S)NRaRb, -NRaC(=S)Rb, -SO2NRaRb, and -OSO3Ra’, -OPO3RaRb’, -N=NRb’, -OSiRaRbRc; wherein each of Ra, Rb, Rcand Rdis independently hydrogen, alkyl,Attorney Docket No.: P24-229-SEC-WO01alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and alkoxy.

[0064] As used herein, the term “linker” generally refers to a molecular group that connects, or is capable of connecting, a first group to at least one other group. In some cases, the linker uses covalent bonds to join the two other molecules. In general, linkers can be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker). In certain embodiments, the linker connects two or more groups comprising, or consisting of, at least one moiety, wherein each at least one moiety is independently selected from the group consisting of a bond, unsubstituted alkylene, substituted alkylene, -(alkylene-O)nn-, optionally substituted arylene, -O-, -C(O)-, -C(S)~, -N(RW)-, -S(0)o-2-, methine -(-CH)-, an amino acid, or a peptide; and combinations thereof; wherein: each nn is independently an integer from 1 to 100; and each Rwis independently H, Ci-Cs alkyl or a bond. Unless expressly indicated otherwise, no orientation of the linker is implied by the direction in which the formula of the linker group is written. By way of example, the formula -C(O)CH2CH2- represents both -C(O)CH2CH2“ and -CH2CH2C(O)-. In another example, the formula -C(O)CH2CH2- represents both *-C(O)CH2CH2- and -C(O)CH2CH2-*, wherein * denotes a point of connection, for example, connection to a PEG group. As used herein, the term “PEG linker” generally refers to a linker that connects with a PEG group. A PEG linker itself does not comprise the PEG moiety (CH2 O -CH2)I-IOO ---, but comprises other linker moieties disclosed above. In certain embodiments, when a sel ected moiety occurs two or more times in the same linker, the two or m ore occurrences are not adjacent. In certain embodiments, a linker is not a bond. The term “non-cleavable” general ly refers to the abili ty of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves a disulfide bond, etc.Recombinant Adeno-Associated Virus (AAV) Production

[0065] The term “cell” as used herein includes all types of eukaryotic. In certain embodiments the term refers to eukaryotic cells, especially mammalian cells. In certain embodiments, the term “cell” is meant to refer to human embryonic kidney (HEK) 293 cells or 293 cells, or a variant thereof, such as, e.g., a 293 variant that canAttorney Docket No.: P24-229-SEC-WO01grow in suspension. In certain embodiments it can mean variants of 293 cells that can grow, proliferate and be transfected in suspension culture, in particular those variants that can be cultured at high density (e.g., >about 2* 106cells / ml, greater >about 3 * 106cells / ml, or even optionally >about 4* 106cells / ml). An example of a variant are 293F cells, such as EXPI293F™ cells, Viral Production Cells 1.0 or Viral Production Cells 2.0.

[0066] The AAV production system can comprise a transfection reagent or a composition that facilitates entry of a macromolecule into a cell. In certain embodiments, the transfection reagent may comprises a cationic lipid, an amphipathic compound, a cationic polymer (e.g., a cationic polyacrylamide copolymer), a cationic cyclic amine, or combinations thereof. Examples of these compounds are disclosed in U. S. Pat. No. 8,921,448, U. S. Pat. No. 9,290,779, U. S. Pat. No. 9,677,077, U. S. Pat. No. 9,856,496, and U. S. Pat. No. 10,619,162, the detailed disclosure and figures of each is incorporated herein by reference in its entirety.

[0067] In certain embodiments a reagent for the introduction of macromolecules into cells can comprise one or more lipids which can be cationic lipids and / or neutral lipids. Suitable lipids include, but are not limited to, N-[l-(2,3-dioleyloxy) propyl]-N, N, N-trimethylamonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1.2-bis(oleoyloxy)-3-(4'-trimethylammonio) propane (DOTAP), dihydroxyldimyristyl spermine tetrahydrochloride (DHDMS), hydroxyl-dimyristylspermine tetrahydrochloride (HDMS), l,2-dioleoyl-3-(4'-trimethylammonio) butanoyl-sn-glycerol (DOTB), l,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4'-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), l,2-dioleoyl-3-dimethyl-hydroxy ethyl ammonium bromide (DORI), 1.2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DOME), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE), O, O'-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N, N, N-trimethylammonium chloride, spermine conjugated to one or more lipids (for example, 5-carboxyspermylglycine di octadecyl ami de (DOGS), HN^N^^Metramethyl-N, NI, NII, NIII-tet-rapalmityl spermine (TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde (DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS (Tris(hydroxymethyl)-Attorney Docket No.: P24-229-SEC-WO01aminomethane, tromethamine) conjugated fatty acids (TFAs) and / or peptides such as trilysyl-alanyl-TRIS mono-, di-, and tri-palmitate, 3B-[N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DCChol), N-(a-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dimethyl dioctadecylammonium bromide (DDAB), 2,3 -di oleyloxy -N-[2(spermine-carboxamido)ethyl]-N, N-dimethyl-l-propanamin-iniumtrinuoroacetate (DOSPA) and combinations thereof.

[0068] In certain embodiments the transfection reagent may further comprise at least one additional helper lipid. Helper lipids are known in the art and include, but are not limited to, neutral lipids. In certain embodiments, the neutral lipid is selected from the group consisting of DOPE, DOPC and cholesterol. In certain embodiments, the transfection reagent comprises at least one cationic lipid and at least one neutral lipid.

[0069] Those skilled in the art may appreciate that certain combinations of the above mentioned lipids have been shown to be particularly suited for the introduction of nucleic acids into cells including, for example, a 3:1 (w / w) combination of DOSPA and DOPE is available from Thermo Fisher Scientific under the trade name LIPOFECTAMINE™, a 1: 1 (w / w) combination of DOTMA and DOPE is available from Thermo Fisher Scientific under the trade name LIPOFECTIN®, a 1: 1 (M / M) combination of DIVIRIE and cholesterol is available from Thermo Fisher Scientific under the trade name DMRIE-C reagent; and a 1:1.5 (M / M) combination of TM-TPS and DOPE is available from Thermo Fisher Scientific. In certain embodiments the transfection reagent is a cationic lipid transfection reagent. In certain embodiments the transfection reagent is a polymer-based transfection reagent. Other commercially available cationic lipid transfection reagents include, without limitation, TRANSFAST™ (available from Promega Corporation); LYOVEC™ (available from InvivoGen); DOTAP liposomal transfection reagent (available from Roche);TRANSIT® transfection reagents (available from Minis); and Insect GENEJUICE® Transfection Reagent (EMD Millipore). Additional transfection reagents that may be used herein include, without limitation, LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000, available from Thermo Fisher Scientific; VIAFECT™ Transfection Reagent, FUGENE® 6 Transfection Reagent, and FUGENE® HDAttorney Docket No.: P24-229-SEC-WO01Transfection Reagent, each of which is available from Promega Corporation; and TRANSFECTIN™ Lipid Reagent, available from BioRad Laboratories, Inc.

[0070] In certain embodiments, the transfection reagent comprises a cationic polymer. Cationic polymers represent the second major class of transfection reagents. Suitable cationic polymers include, but are not limited to, a linear or branched polyethyleneimine (PEI) and variants thereof, PEI dendrimers, a polypropyleneimine (PPI), poly(amidoamine) (PAA) and dendrimers (PAMAM), cationic cyclodextrin, polyalkylamine, a polyhydroxyalkylamine, poly(butyleneimine) (PBI), spermine, a N-substituted polyallylamine, N-substituted chitosan, a N-substituted polyomithine, aN-substituted polylysine (PLL), a N-substituted polyvinylamine, poly(P-amino ester), hyperbranched poly(amino ester) (h-PAE), networked poly(amino ester) (n-PAE), poly(4-hydroxy-l -proline ester) (PHP-ester) and a poly-P-aminoacid. In certain embodiments, the transfection reagent is FectoVIR®-AAV Transfection Reagent (VWR) or PEIpro® DNA Transfection Reagent for Virus Production (VWR). In certain embodiments, the transfection reagent comprises a combination of cationic amine-containing polymer and an amphipathic compounds disclosed herein.

[0071] In certain embodiments, the transfection reagent is combined with the AAV transfer vector to form a DNA / transfection reagent complex prior to addition to the cells. In certain embodiments, the transfection reagent is combined with the AAV transfer vector, the pRep / Cap plasmid and the pHelper plasmid to form a DNA / transfection reagent complex prior to addition to the cells. In some cases, two of the three principal components — AAV vector, AAV helper, and adenoviral helper — are combined to be on one plasmid, thereby reducing the number of constructs to be transfected to two plasmids. Examples of such dual plasmid configuration as a packaging system for recombinant AAV include (1) a single construct containing transgene and replcap, while adenoviral helper functions are provided separately (pOXB), or (2) a single plasmid carrying transgene and adenoviral helper functions, while a replcap construct is supplied in trans (pLV).Lentiviral Expression Constructs

[0072] Lentiviruses are complex retroviruses that contain additional genes with regulatory or structural function other than the common retroviral genes gag, pol andAttorney Docket No.: P24-229-SEC-WO01env. The complexity may enable the lentivirus to modulate the life cycle of itself, for example, in the course of latent infection.

[0073] Human immunodeficiency virus (HIV) is an example of lentivirus. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as, for example, lymphocytes and macrophages. In vitro, HIV can infect primary cultures of monocyte- derived macrophages (MDM) and HeLa-Cd4 or T lymphoid cells arrested in the cell cycle by treatment with aphidicolin or g irradiation.

[0074] Infection of cells may be dependent on the active nuclear import of HIV preintegration complexes through the nuclear pores of the target cells, which occurs by the interaction of multiple, partly redundant, molecular determinants in the complex with the nuclear import machinery of the target cell. Identified determinants can include a functional nuclear localization signal (NLS) in the gag matrix (MA) protein, the karyophilic virion-associated protein, vpr, and a C-terminal phosphotyrosine residue in the gag MA protein.

[0075] The lentiviral genome has the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), a protease and an integrase; and the env gene encodes viral envelope glycoproteins. The 5’ and 3’ LTRs serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx (in HIV-1, HIV-2 and / or SIV).

[0076] Sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site) can be found adjacent to the 5’ LTR. If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect may prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.

[0077] In certain embodiments, the recombinant lentivirus is capable of infecting a non-dividing cell by transfecting a suitable host cell with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat. InAttorney Docket No.: P24-229-SEC-WO01certain embodiments, vectors lacking a functional tat gene are desirable. Thus, for example, a first vector can provide a nucleic acid encoding a viral gag and a viral pol and another vector can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene, identified as a transfer vector, into that packaging cell may yield a producer cell, which releases infectious viral particles carrying the foreign gene of interest.

[0078] The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.

[0079] According to the above-indicated configuration of vectors and foreign genes, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene can be derived from any virus, including retroviruses. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.

[0080] It may be desirable to target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell- type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.Retroviral vectors can be made target- specific by inserting, for example, a glycolipid or a protein. Targeting often is accomplished by using an antigen-binding portion of an antibody or a recombinant antibody -type molecule, such as a single chain antibody, to target the retroviral vector. Specific methods to achieve delivery of a retroviral vector to a specific target can be chosen by a technician.

[0081] Examples of retroviral-derived env genes include, but are not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). Other env genes such as Vesicular stomatitis virus (VSV) protein G (VSV-G), or that of hepatitis viruses and of influenza also can be used. For example, a lentivirus can be modified by replacing its native envelope protein with the glycoprotein from the vesicular stomatitis virus (VSV-G), essentially giving theAttorney Docket No.: P24-229-SEC-WO01lentivirus the ability to infect a wider range of cell types due to VSV-G’s broad tropism. This may enhance the efficiency of lentiviral vectors.

[0082] The vector providing the viral env nucleic acid sequence can be associated operably with regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter- enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences.

[0083] In certain embodiments, the transfection reagent is combined with a lentivirus plasmid or lentivirus transfection vector. Lentiviruses have the ability to infect and express their genes in both mitotic and post-mitotic cells. Methods of construction and use of lentivirus-based expression constructs are described in U. S. Pat. Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031, each of which is incorporated by reference in its entirety.Aggregation of Transfection Complexes

[0084] The transfection complexes may have different chemical and physical properties to affect their biological behaviors. These complex properties, primarily size and charge, are influenced by the conditions under which the complexes are formed. Certain characterization techniques such as dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta-potential (^-potential) measurement can help measure transfection complex size and surface charge. Certain characteristics of the transfection complexes need to be present for the transfection complex to be efficacious. For example, the transfection complex may need to stay within a certain size range and may need to have a net positive charge. Correlating these behaviors with improved functional delivery of the nucleic acids to host cells may find a variety of applications.

[0085] The composition of the transfection reagent may affect the size of the resulting complex post addition of nucleic acid and the rate at which the transfection complex grows. Typical transfection complexes begin growing and aggregating when poly cations meet polyanions in a buffered salt solution. For example, as shown in FIG. 1, a transfection reagent of cationic polymers and cationic lipids can bind toAttorney Docket No.: P24-229-SEC-WO01negatively charged nucleic acids, such as DNA molecules, to form a 3-component transfection complex at a first time point. This transfection complex can transfect a host cell. When two or more such transfection complexes collide, the electrostatic interactions of the 3 components can merge these collided transfection complexes into a bigger transfection complex at a second time point. When such collision continues, the transfection complex formed becomes bigger and bigger, eventually precipitate from the solution without entering host cells at a third time point.

[0086] Different transfection reagent formulations may have a different optimal complex formation time (window). In general, the ideal window is from 15-45 minutes. This relatively short complex formation time window may become problematic in large-scale transient transfection processes when adding a large volume of the transfection complex to the reaction vessel takes longer time (e.g., more than 30 minutes). To reduce the time transferring the transfection complex, a higher concentration of the transfection complex in the stock solution would be beneficial. But a higher concentration of the transfection complex may lead to faster aggregation of the transfection complex leading to precipitation of the aggregates, thereby a less productive process. Thus, there is a need to find better formulations for transfection complexes that increase the complex formation time window without increasing the complex formation volume at the same time.Transfection Complexes Stabilization

[0087] Adding a stabilization reagent, such as pegylated lipids, into the transfection composition, may slow down the rate at which the size of the transfection complexes grows as shown in FIG.2. A pegylated lipid is a lipid with one or more hydrophobic chains linked with one or more polyethylene glycol (PEG) chains. A pegylated lipid can be added as the fourth component to form the transfection complex. The hydrophilic PEG chains on the pegylated lipids may extend toward the aqueous environment surrounding the 4-component transfection complexes. This configuration may slow down the growth of average size of transfection complexes by steric effect, therefore preventing complex aggregation. As shown in FIG. 2, the presence of the pegylated lipid delays the size growth of the 4-component transfection complexes at the second and the third time points relative to the 3-component transfection complex in FIG. 1. As a result, the 4-component transfection complexesAttorney Docket No.: P24-229-SEC-WO01may have a longer optimal complex formation time (window) than that of the 3-component transfection complexesComplex Stabilization Agent

[0088] The present disclosure relates to compositions comprising a cationic polymer, an amphipathic compound, and a complex stabilization agent which is a pegylated lipid. The compositions can be used in the delivery of nucleic acid to cells in biological systems. The disclosure also relates to methods of making such compositions and their utilities in gene therapy as transfection agents. In some embodiments, the stabilization agent is a pegylated lipid.

[0089] Disclosed herein is a pegylated lipid according to Formula (I), that when combined with a cationic polymer and an amphipathic compound can stabilize a transfection complex containing a nucleic acid:X-L1Formula (I)or a salt or stereoisomer thereof, wherein:A is O, CH-L2-Y or N-L2-Y;B is H, -OH, -NH2, -OCH3, or -CH3;each LI and L2 is independently a linker;L3 is a PEG linker;each X and Y is independently alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein said alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; and q is an integer from 3 to 125.

[0090] In some embodiments, the pegylated lipid is according to Formula (la):X-LKBFormula (la)or a salt or stereoisomer thereof, wherein:Attorney Docket No.: P24-229-SEC-WO01(6) LI is a bond, L2 is a bond, and L3 isLAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0091] In some embodiments, the said pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:Attorney Docket No.: P24-229-SEC-WO01O?Ll is a bond, L2 is a bond, and L3 is5, O oLAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0092] In some embodiments, the pegylated lipid is according to Formula (lb):X-Lt H 13.C1!_2Formula (lb)or a salt or stereoisomer thereof, wherein:Attorney Docket No.: P24-229-SEC-WO01LAis independently -CH?-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)?-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0093] In some embodiments, the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:0LI is L2 isA>, L3 is a bond,-A. y--LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0094] In some embodiments, the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:z°-lA— rzo-rLB_ LI is, L2 is, L3 is abond, ’,Attorney Docket No.: P24-229-SEC-WO01^CT^N-L6—!H:LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0095] In some embodiments, the pegylated lipid is according to Formulas (Ila) or (Hb):oFormula (Ila)Formula (lib)or a salt or stereoisomer thereof, wherein:D is H, -OH, -NH?, -OCH?, or -CH?;Lcis a PEG linker;X is alkyl, alkenyl, or alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; and q is an integer from 3 to 125.

[0096] Regarding Formula (Hb): In some embodiments, Lcis -CH?-, -(CH?)?-, or -(CH?)?-.

[0097] In some embodiments, the pegylated lipid is according to Formulas (Illa) or (IHb),O oHO-rfEzAAttorney Docket No.: P24-229-SEC-WO01Formula (Illa)Formula (Illb)or a salt or stereoisomer thereof, wherein:D is H, -OH, -NH2, -OCH3, or -CH3;E is H or C1-C4 alkyl;Lcis a PEG linker;each X and Y is independently alkyl, alkenyl, or alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; andq is an integer from 3 to 125.

[0098] Regarding Formulas (Illa) and Formula (Illb): In some embodiments, Lcis -CH2-, — (CH2)2—, or -(CH2)3-. In some embodiments, each X and Y is independently alkenyl. In some embodiments, each X and Y independently comprises 8 to 20 carbons. In some embodiments, each X and Y independently comprises two non-conjugated alkenes. Regarding Formulas (I), (la), (lb), (Ila), (lib), (Illa), and (Illb): In some embodiments, each X and Y is independently C8-C2o alkyl, C8-C2o alkenyl, or C8-C2o alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl. Regarding Formulas (I), (la), (lb), (Ila) and (lib): In some embodiments, each X and Y is independently C8-C2o alkyl. In some embodiments, (1) X is C13 alkyl, and Y is C13 alkyl; or (2) X is C14 alkyl, and Y is C14 alkyl; or (3) X is C15 alkyl, and Y is C15 alkyl; or (4) X is Ci6 alkyl, and Y is Ci6 alkyl; or (5) X is C17 alkyl, and Y is C17 alkyl; or (6) X is Cis alkyl, and Y is Cis alkyl; or (7) X is C19 alkyl, and Y is C19 alkyl; or (8) X is C2o alkyl, and Y is C2o alkyl; or (9) X is Cs alkyl, and Y is Cs alkyl; or (10) X is C9 alkyl, and Y is C9 alkyl; or (11) X is C10 alkyl, and Y is C10 alkyl; or (12) X is C11 alkyl, and Y is C11 alkyl; or (13) X is C12 alkyl, and Y is C12 alkyl.Attorney Docket No.: P24-229-SEC-WO01

[0099] In some embodiments, the pegylated lipid is according to Formula (IV),Formula (IV)or a salt or stereoisomer thereof, wherein:B is H, -OH, -NH2, -OCH3, or -CH3;LI is a bond or a linker;X is a cholesterol or a derivative thereof, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein said alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; andq is an integer from 3 to 125.

[0100] In some embodiments, for all the pegylated lipids according to Formulas (I), (la), (lb), and (IV) disclosed herein, B is H. In some embodiments, for all the pegylated lipids according to Formulas (Ila), (lib), (Illa) and (Illb) disclosed herein, D is H.

[0101] The pegylated lipids herein include, but are not limited to, polyethylene glycol (PEG)-modified lipids, i.e., a lipid modified with polyethylene glycol. Nonlimiting examples of pegylated lipids include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a pegylated lipid can be 1.2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), 1.2-dilauroyl-sn-glycero-3 -phosphoethanolamine (DLPE) conjugated polyethylene glycol (DLPE-PEG), l,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE) conjugated polyethylene glycol (DMPE-PEG), dipalmitoylphosphatidylcholine (DPPC) conjugated polyethylene glycol (DPPC-PEG), PEG-1, 2-dimyristyloxlpropy 1-3-amine (PEG-c-DMA), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, or PEG-diacylglycamide (PEGDAG). Additional exemplarily pegylated lipids include DSPE-PEG2000, DSPE-PEG1500, DSPE-PEG1000, DSPE-PEG500, l,2-distearyloxypropyl-3-amine-Attorney Docket No.: P24-229-SEC-WO01PEG2000, hexadecylcarbamoylmethyl hexadecanoate-PEG2000, cholesteryl hemisuccinate-PEG2000, cholesteryl-PEG2000, 2-[(polyethylene glycol)-2000]-N, N-ditetradecyl acetamide (PEG2000-DMA), l,2-dimyristoyl-rac-glycero3-methoxypolyethylene glycol-2000 (PEG2000-DMG), PEG2000-C-DOMG, (2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide (PEG2000-DMA (also called ALC-0159)), and PEG2000-C-DMG. Additional stabilizing reagent can be cholesterol-hyperbranched polyglycerol, poly(2-methyl-2-oxazoline) (PMOZ)-DSPE, poly(2-ethyl-2-oxazoline) (PEOZ)-DSPE, poly(hydroxyethyl-l- asparagine)-succinyldioctadecylamine, and DSPE-poly(2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)- N, N-dimethyl-2-oxoethanamonium).

[0102] In some embodiments, the lipid moiety of the pegylated lipids (e.g., the X moiety or Y moiety in Formulas (I), (la), (lb), (Ila), (lib), (Illa), (Illb), or (IV)), for example, includes those having lengths of from about Cs to about C22, from about Cs to about C20, from about C10 to about C20, from about C12 to about C20, about C14 to about Cis, or from about C14 to about Ci6. In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of Cs, C9, C10, C11, C12, C13, C14, C15, Ci6, C17, Cis, C19, C20, C21, or C22. In some embodiments, the lipid moiety of the pegylated lipids is a cholesterol or a derivative thereof. In some embodiments, the PEG moiety of the PEG lipids, for example, has a size of about 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 Daltons. In some embodiments, pegylated lipids are described in US 8,158,601, US 8,492,359, WO2017 / 099823, WO2012 / 099755, and WO 2015 / 130584, each of which is incorporated herein by reference in its entirety.Cationic Polymer

[0103] Cationic polymer is another component of the 3-component transfection complex. For all the disclosed pegylated lipids in transfection complexes, the cationic polymer can comprise Formula (V);WR21R22Formula (V)Attorney Docket No.: P24-229-SEC-WO01wherein:each R1and R2is independently hydrogen or methyl;W is -NH- or -O-;R21comprises a structure of:RL / ^RRl ooR23wherein:each RLand RRis independently -(CH2)r-;each r is independently 1, 2, 3, 4, 5, or 6;R23is H or CH3;R22is independently alkyl, alkenyl, or heteroalkyl, wherein said alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0104] Regarding Formula (V): In some embodiments, R22is alkyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl. In some embodiments, R22is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, R22comprises 3 to 9 carbon atoms. In some embodiments, R22comprises 10 to 16 carbon atoms. In some embodiments, each r is independently 1, 2, or 3, and wherein R23is H. In some embodiments, the cationic polymer comprises a 5- to 7-membered cyclic amine. In some embodiments, the cationic polymer comprises 2 to 80 monomeric units.

[0105] For all the disclosed pegylated lipids in transfection complexes, the cationic polymer can comprise Formula (VI);R1R2W^OHN^OF^4R3Formula (VI)Attorney Docket No.: P24-229-SEC-WO01wherein:each R1and R2is independently hydrogen or methyl;W is -NH- or -O-;each R3and R4is independently alkyl, alkenyl, or heteroalkyl, wherein the alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, Ci- C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0106] Regarding Formula (VI): In some embodiments, R3is alkyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl. In some embodiments, R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, R3comprises 3 to 9 carbon atoms. In some embodiments, R3comprises 10 to 16 carbon atoms. In some embodiments, R3is alkenyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl. In some embodiments, R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, R3comprises 3 to 9 carbon atoms. In some embodiments, R3comprises 10 to 16 carbon atoms. In some embodiments, R3comprises 2 to 4 alkene bonds. In some embodiments, at least two of the 2 to 4 alkene bonds are conjugated. In some embodiments, at least two of the 2 to 4 alkene bonds are not conjugated. In some embodiments, R3is a heteroalkyl group, wherein the heteroalkyl group is optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl. In some embodiments, the heteroalkyl is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, the heteroalkyl group comprises 2 to 4 nitrogen atoms. In some embodiments, the heteroalkyl group comprises 3 to 6 nitrogen atoms. In some embodiments, the heteroalkyl group comprises 4 to 8 nitrogen atoms. In some embodiments, R3is a heteroalkyl group comprising one or more nitrogen atoms and one or two oxygen, wherein the heteroalkyl group is optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4Attorney Docket No.: P24-229-SEC-WO01alkyl, and C1-C4 haloalkyl. In some embodiments, the heteroalkyl group is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, the heteroalkyl group comprises 1 to 3 nitrogen atoms. In some embodiments, the heteroalkyl group comprises 3 to 5 nitrogen atoms. In some embodiments, the heteroalkyl group comprises 5 to 8 nitrogen atoms. In some embodiments, R3comprises one primary amine and one or more secondary or tertiary amine. In some embodiments, R3comprises 1 secondary amine. In some embodiments, R3comprises 2 secondary amine. In some embodiments, R3comprises 3 secondary amine. In some embodiments, R3comprises 4 secondary amine. In some embodiments, R3comprises 5 secondary amine. In some embodiments, R3further comprises another primary amine. In some embodiments, R3further comprises a tertiary amine. In some embodiments, R3further comprises two tertiary amine. In some embodiments, R4is C2-C6 alkyl. In some embodiments, R4is C3-C8 alkyl. In some embodiments, R4is C4-C10 alkyl. In some embodiments, R4is C3-C6 alkenyl. In some embodiments, C3-C8 alkenyl. In some embodiments, R4is C4-C10 alkenyl. In some embodiments, R4is substituted with 1-3 groups independently selected from F, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3. In some embodiments, R1is hydrogen and R2is methyl, or wherein R1is methyl and R2is hydrogen. In some embodiments, R1and R2are hydrogen. In some embodiments, R1and R2are methyl.

[0107] For all the disclosed pegylated lipids and cationic polymer, and all possible combinations of the disclosed pegylated lipids and cationic polymer in transfection complexes, the amphipathic compound is according to Formula (VII);R13 / \ R14R1< " L4-N\ _ J\l-L5" XR12Formula (VII)wherein each R11and R12is independently alkyl or alkenyl, wherein each the alkyl and alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;Attorney Docket No.: P24-229-SEC-WO01each R13and R14is independently hydrogen, alkyl, alkenyl, alkoxy, alkyl-C(O)-alkylene, alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-C(O)-, heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alky 1-heterocy cl e-alkeny 1 ene-C (O)-,alkenyl-heterocycle-alkenylene-C(O)-, heteroaryl-C(O)-,heteroaryl-alkylene-C(O)-, heteroaryl-alkenylene-C(O)-,alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-, alkyl-heteroaryl-alkenylene-C(O)-, oralkenyl-heteroaryl-alkenylene-C(O)-, wherein each the alkyl, alkenyl, alkylene, alkenylene, heterocycle, an heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;each L4 and L5 is independently C2-C8 alkylene, optionally substituted with one or two groups independently selected from the group consisting of halide, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andp is 0 or 1.

[0108] In some embodiments, each L4 and L5 is independently ethylene, n-propylene, 1-methyl-ethylene, 2-methyl-ethylene, 1-methyl-propylene, 2-m ethylpropylene, 3-methyl-propylene, n-butylene, 1-methyl-butylene, 2-methyl-butylene, 3-methyl-butylene, 4-methyl-butylene, 1 -ethyl -butylene, 2-ethyl-butylene, 3 -ethylbutylene, or 4-ethyl-butylene. In some embodiments, L4 and L5 are different. In some embodiments, L4 and L5 are the same. In some embodiments, L4 and L5 is substituted with one or two groups of F or CF3. In some embodiments, each R11and R12is independently C10 to C25 alkyl. In some embodiments, each R11and R12is independently C10 to C25 alkenyl. In some embodiments, each R11and R12is independently substituted with F, methyl, ethyl, or CF3. In some embodiments, each R11and R12independently comprises 1 to 4 alkene bonds. In some embodiments, each R11and R12independently comprises 2 to 4 alkene bonds. In some embodiments, for each R11and R12, at least two of the 2 to 4 alkene bonds are conjugated. In some embodiments, for each R11and R12, at least two of the 2 to 4 alkene bonds are notAttorney Docket No.: P24-229-SEC-WO01conjugated. In some embodiments, at least one of the 1 to 4 alkene bonds is cis. In some embodiments, at least one of the 1 to 4 alkene bonds is trans. In some embodiments, two of the 2 to 4 alkene bonds are cis. In some embodiments, wherein two of the 2 to 4 alkene bonds are trans. In some embodiments, each R11and R12comprises 10 to 15 carbon atoms. In some embodiments, each R11and R12comprises 16 to 20 carbon atoms. In some embodiments, each R11and R12comprises 21 to 25 carbon atoms. In some embodiments, each R11and R12independently comprises:Qor. In some embodiments, R11and R12are the same. In some embodiments, R11and R12are different. In some embodiments, both R13and R14are hydrogen. In some embodiments, R13is hydrogen and R14is C2 to C10 alkyl, C2 to C10 alkenyl, or C2 to C10 alkoxy, alkyl-C(O)-alkylene, alkenyl-C(O)-alkylene,alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkenylene-C(O)-, alkenyl-heterocycle-alkenylene-C(O)-, alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-,alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-. In some embodiments, wherein each R13and R14is independently C2 to C10 alkyl. In some embodiments, each R13and R14is independently C2 to C10 alkenyl. In some embodiments, each R13and R14is independently C2 to C10 alkoxy. In some embodiments, each R13and R14is independently alkyl-C(O)-alkylene,alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, or alkenyl-C(O)-. In some embodiments, each R13and R14is independently heterocycle-C(O)-, heterocycle-alkylene-C(O)-,heterocycle-alkenylene-C(O)-, alkyl-heterocycle-alkylene-C(O)-,Attorney Docket No.: P24-229-SEC-WO01alkenyl-heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkenylene-C(O)-, or alkenyl-heterocycle-alkenylene-C(O)-. In some embodiments, each R13and R14is independently heteroaryl-C(O)-, heteroaryl-alkylene-C(O)-,heteroaryl-alkenylene-C(O)-, alkyl-heteroaryl-alkylene-C(O)-,alkenyl-heteroaryl-alkylene-C(O)-, alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, the heterocycle is azetidine, 1,3-diazetidine, pyrrolidine, piperidine, piperazine, or morpholine. In some embodiments, each R13and R14is independently heterocycle-Ci-C4 alkylene-C(O)-, and wherein the heterocycle is optionally substituted with fluorine, methyl, ethyl, propyl, isopropyl, or CF3. In some embodiments, the heteroaryl is pyrrole, furan, imidazole, pyrazole, oxazole, isoxazole, pyridine, or pyrazine. In some embodiments, R13and R14is independently heteroaryl- C1-C4 alkylene-C(O)-, and wherein the heteroaryl is optionally substituted with fluorine, methyl, ethyl, propyl, isopropyl, or CF3. In some embodiments, R13and R14are different. In some embodiments, R13and R14are the same. In some embodiments, the cationic polymer is a block copolymer, an alternating copolymer, a random or statistical copolymer, or a gradient copolymer. In some embodiments, the cationic polymer has a linear structure, a graft structure, a branched structure, a crosslinked or network structure, a star structure, a comb structure, a ladder structure, or a dendritic structure. In some embodiments, number average molecular weight (Mn) of the cationic polymer is from about 2,000 to about 100,000. In some embodiments, Mnis from about 2,000 to about 5,000. In some embodiments, Mnis from about 3,000 to about 6,000. In some embodiments, Mnis from about 5,000 to about 10,000. In some embodiments, Mnis from about 10,000 to about 15,000. In some embodiments, Mnis from about 15,000 to about 20,000. In some embodiments, Mnis from about 20,000 to about 25,000. In some embodiments, Mnis from about 25,000 to about 30,000. In some embodiments, Mnis from about 30,000 to about 35,000. In some embodiments, Mnis from about 35,000 to about 40,000. In some embodiments, Mnis from about 40,000 to about 45,000. In some embodiments, Mnis from about 45,000 to about 50,000. In some embodiments, Mnis from about 50,000 to about 55,000. In some embodiments, Mnis from about 55,000 to about 60,000. In some embodiments, Mnis from about 60,000 to about 65,000. InAttorney Docket No.: P24-229-SEC-WO01some embodiments, Mnis from about 65,000 to about 70,000. In some embodiments, Mnis from about 70,000 to about 80,000. In some embodiments, Mnis from about 80,000 to about 90,000. In some embodiments, Mnis from about 90,000 to about 100,000. In some embodiments, Mw / Mnis from about 1.00 to about 1.10, and wherein Mwis weight average molecular weight. In some embodiments, Mw / Mnis from about 1.10 to about 1.20. In some embodiments, Mw / Mnis from about 1.20 to about 1.30. In some embodiments, Mw / Mnis from about 1.30 to about 1.40. In some embodiments, Mw / Mn is from about 1.40 to about 1.50. In some embodiments, wherein a molar ratio of monomeric units comprising R3to monomeric units comprising R4in the cationic polymer is from about 0.30 to about 0.53. In some embodiments, a ratio of monomeric units comprising R3to monomeric units comprising R4in the cationic polymer is from about 0.34 to about 0.49. In some embodiments, a ratio of monomeric units comprising R3to monomeric units comprising R4in the cationic polymer is from about 0.36 to about 0.47. In some embodiments, W is -NH-. In some embodiments, W is -O-. In some embodiments, the composition further comprises an alcohol and a buffer. In some embodiments, the alcohol is ethanol. In some embodiments, the buffer is glycine-HCl. In some embodiments, the composition further comprises the nucleic acid. In some embodiments, the composition further comprises DNA, RNA, or any combination thereof.

[0109] In certain embodiments, disclosed herein is a transfection reagent comprising a composition of any embodiments disclosed herein and a nucleic acid. In certain embodiments, the nucleic acid is one or more adeno-associated virus (AAV) plasmids. In certain embodiments, the nucleic acid is one or more lentivirus (LV) plasmids.

[0110] In certain embodiments, disclosed herein is a method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of the embodiments disclosed herein with the nucleic acid to produce a mixed solution, and contacting the cell with the mixed solution, thereby transfecting the nucleic acid into the cell. In some embodiments, the nucleic acid is one or more plasmids used to manufacture adeno-associated virus (AAV). In some embodiments, the nucleic acid is one or more plasmids used to manufacture lentivirus (LV). In some embodiments, an average genome titer is no less than 1.5><10A10 genome copies per milliliter (GC / mL)Attorney Docket No.: P24-229-SEC-WO01at 30 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.5><10A10 GC / mL at 60 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.5><10A10 GC / mL at 90 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.5xl0A10 GC / mL at 120 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.5xl0A10 GC / mL at 3 hours after the mixing. In some embodiments, the average genome titer is no less than 1.5xl0A10 GC / mL at 4 hours after the mixing. In some embodiments, the average genome titer is no less than 1.5xl0A10 GC / mL at 5 hours after the mixing. In some embodiments, the average genome titer is no less than 1.5xl0A10 GC / mL at 6 hours after the mixing. In some embodiments, the average genome titer is no less than 1.5 x 10Al 0 GC / mL at 7 hours after the mixing. In some embodiments, the average genome titer is no less than 1.5xl0A10 GC / mL at 8 hours after the mixing.

[0111] In some embodiments, an average genome titer is no less than 1.0×1011genome copies per milliliter (GC / mL) at 30 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 60 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 90 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 120 minutes after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 3 hours after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 4 hours after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 5 hours after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 6 hours after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 7 hours after the mixing. In some embodiments, the average genome titer is no less than 1.0×1011GC / mL at 8 hours after the mixing.

[0112] When compared with an average genome titer at 10 minutes after complex initiation: in some embodiments, an average genome titer at 3 hours after complex initiation decreases no more than 50%. In some embodiments, the average genome titer at 3 hours after complex initiation decreases no more than 40%. In some embodiments, the average genome titer at 3 hours after complex initiation decreasesAttorney Docket No.: P24-229-SEC-WO01no more than 35%. In some embodiments, the average genome titer at 3 hours after complex initiation decreases no more than 30%. In some embodiments, the average genome titer at 3 hours after complex initiation decreases no more than 25%. In some embodiments, the average genome titer at 3 hours after complex initiation decreases no more than 20%. In some embodiments, the average genome titer at 3 hours after complex initiation decreases no more than 15%. In some embodiments, the average genome titer decreases no more than 50% at 4 hours after complex initiation. In some embodiments, the average genome titer decreases no more than 50% at 5 hours after complex initiation. In some embodiments, the average genome titer decreases no more than 50% at 6 hours after complex initiation. In some embodiments, the average genome titer decreases no more than 50% at 7 hours after complex initiation. In some embodiments, the average genome titer decreases no more than 50% at 8 hours after complex initiation.

[0113] In certain embodiments, disclosed herein is a method of forming and stabilizing a transfection complex, comprising mixing a composition of any one of those disclosed herein with the nucleic acid, thereby producing a plurality of transfection complexes comprising the pegylated lipid, the cationic polymer, the amphipathic compound, and the nucleic acid. In some embodiments, the nucleic acid is one or more plasmids used to manufacture adeno-associated virus (AAV). In some embodiments, the nucleic acid is one or more plasmids used to manufacture lentivirus (LV). In some embodiments, an average radius of the plurality of transfection complexes is no more than 1000 nm at 60 minutes after the mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 120 minutes after the mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 180 minutes after the mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 240 minutes after the mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 300 minutes after the mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 360 minutes after the mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 420 minutes after theAttorney Docket No.: P24-229-SEC-WO01mixing. In some embodiments, the average radius of the plurality of transfection complexes is no more than 1000 nm at 480 minutes after the mixing.

[0114] In certain embodiments, disclosed herein is a preparing a transfection reagent mixture, comprising: (a) adding a pegylated lipid to a mixture of a cationic polymer and an amphipathic compound, thereby obtaining another mixture; and (b) mixing the another mixture, thereby obtaining the transfection reagent mixture. In some embodiments, each of the pegylated lipid, the cationic polymer, and the amphipathic compound is according to any one of those disclosed herein.

[0115] The monomeric units of the polymer described here may comprises one or more amines present in their side chains. These amines can be primary, secondary or tertiary, or a combination thereof,. In certain embodiments the precursors of the acrylamide monomers may contain primary and / or secondary amine side groups protected with tert-butoxycarbonyl (BOC) protecting group. The BOC protected acrylamide monomers can be used to form homopolymers or various types of copolymers with acrylamide and / or acrylate co-monomers. The BOC groups are subsequently removed under acidic conditions post-polymerization to form the required polycations. The acidic conditions can be trifluoroacetic acid in water or dilute hydrochloric acid.

[0116] The development, synthesis, and characterization of cationic amine-containing copolymers are described. Various amine-containing copolymers containing tertiary, secondary, and / or primary amines are synthesized using free radical polymerization. Specifically, reversible-addition fragmentation chain transfer (RAFT) polymerization is used to synthesize polyacrylamides with well-defined structures, compositions, and molecular weights (Mw / Mn< 1.5). Architectures include, but are not limited to, random / statistical, gradient, block, linear, branched, cross-linked / network, star, and dendritic structures.

[0117] The present disclosure provides a composition comprising a polymer, an amphipathic compound, and a pegylated lipid to assist a nucleic acid transfer into cells via a complex comprising the nucleic acid and the composition.

[0118] In certain embodiments, compositions comprising nucleic acids, cationic amine-containing polymers and copolymers, amphipathic compounds, and pegylatedAttorney Docket No.: P24-229-SEC-WO01lipids, and processes using such compositions to deliver a nucleic acid to a cell in vivo or in vitro for the purposes of altering expression of a gene in the cell are described.

[0119] In certain embodiments, compositions and compounds are described that facilitate delivery of nucleic acid to a cell in vitro and in vivo. The nucleic acid may comprise a double-stranded or single-stranded structure having a nucleotide sequence substantially identical to part of an expressed target nucleic acid within the cell.Further, the use of a composition comprising cationic amine-containing polymer and copolymers, amphipathic compounds and pegylated lipids significantly increases nucleic acid transfer efficiency. The nucleic acid then alters expression of a selected endogenous nucleic acid.

[0120] In certain embodiments, the composition comprising cationic amine-containing polymers and copolymers, amphipathic compounds and pegylated lipids is used to assist transfection of DNA, RNA, mRNA or RNAi into a cell. The nucleic acid then alters the cell's natural process.

[0121] RNA interference (RNAi) is a phenomenon wherein double-stranded RNA, when present in a cell, inhibits expression of a gene that has an identical or nearly identical sequence. Inhibition is caused by degradation of the messenger RNA (mRNA) transcribed from the target gene. The double-stranded RNA responsible for inducing RNAi is termed interfering RNA. dsRNA introduced into the cytoplasm of a cell is first processed into RNA fragments 21-25 nucleotides long. It has been shown in in vitro studies that these dsRNAs, termed small interfering RNAs (siRNA) are generated at least in part by the RNAse Ill-like enzyme Dicer. Each siRNA is unwound into two single-stranded (ss) ssRNAs, the passenger strand and the guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex.

[0122] RNAi has become a valuable research tool, both in cell culture and in living organisms, because synthetic dsRNA introduced into cells can selectively and robustly induce suppression of specific genes of interest. RNAi may be used for large-scale screens that systematically shut down each gene in the cell, which can help identify theAttorney Docket No.: P24-229-SEC-WO01components necessary for a particular cellular process or an event such as cell division. The pathway is also used as a practical tool in biotechnology and medicine. The compositions comprising cationic amine-containing polymers and copolymers, amphipathic compounds and pegylated lipids described in this specification provide a mechanism to transfect siRNA and other nucleic acids into cells.

[0123] The development, synthesis, and characterization of cationic amine-containing polymers and copolymers are described. Various amine-containing polymers and copolymers were synthesized using free radical polymerization.Specifically, reversible-addition fragmentation chain transfer (RAFT) polymerization was used to synthesize polyacrylamides with well-defined structures, compositions, and molecular weights (Mw / Mn <1.5). Architectures include, but are not limited to, random / statistical, gradient, block, linear, branched, cross-linked / network, star, and dendritic structures. RAFT and other controlled free radical polymerization techniques such as atom transfer radical polymerization (ATRP) are effective ways to synthesize well-defined and novel polymers. The controlled synthesis of RAFT polymers can be achieved using conventional radical initiators such as azobisisobutyronitrile (AIBN), and the reversible chain transfer of dithiocarbonyl compounds.

[0124] Polymers: A polymer is a molecule built up by repetitive bonding together of smaller units called monomers. In this application the term polymer includes both oligomers which have two to about 80 monomers and polymers having more than 80 monomers. The polymer can be linear, branched network, star, comb, or ladder types of polymer. The polymer can be a homopolymer in which a single monomer is used or can be copolymer in which two or more monomers are used. Types of copolymers include alternating, random, block and graft. The main chain of a polymer is composed of the atoms whose bonds are required for propagation of polymer length. The side chain of a polymer is composed of the atoms whose bonds are not required for propagation of polymer length. To those skilled in the art of polymerization, there are several categories of polymerization processes that can be utilized in the described process.

[0125] Types of Monomers: A wide variety of monomers can be used in the polymerization processes. These include positive charged organic monomers such asAttorney Docket No.: P24-229-SEC-WO01amines, imidine, guanidine, imine, hydroxylamine, hydrazine, heterocycles (like azetidine, 1,3 -diazetidine, pyrrolidine, piperidine, piperazine, imidazole, oxazole, isoxazole, pyrazine pyridine, morpholine, pyrimidine, or pyrene). The amines can be pH-sensitive in that the pKa of the amine is within the physiologic range of 4 to 8. Specific amines include spermine, spermidine, N, N’-bis(2-aminoethyl)-l,3-propanediamine (AEPD), and 3,3’-diamino-N, N-25 dimethyldipropylammonium bromide. Monomers can also be hydrophobic, hydrophilic or amphipathic. Monomers can also be intercalating agents such as acridine, thiazole orange, or ethidium bromide. The polymers may have other groups that increase their utility. These groups can be incorporated into monomers prior to polymer formation or attached to the polymer after its formation. These groups include: targeting groups that are used for targeting the polymer-nucleic acid complexes to specific cells or tissues

[0126] Steric Stabilizer: A steric stabilizer is a long chain hydrophilic group that prevents aggregation of final polymer by sterically hindering particle to particle electrostatic interactions. Examples include: alkyl groups, PEG chains, polysaccharides, alkyl amines. Electrostatic interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges.

[0127] Buffers: Buffers are made from a weak acid or weak base and their salts. Buffer solutions resist changes in pH when additional acid or base is added to the solution.

[0128] Biochemical reactions: Biological, chemical, or biochemical reactions involve the formation or cleavage of ionic and / or covalent bonds.

[0129] Reactive: A compound is reactive if it is capable of forming either an ionic or a covalent bond with another compound. The portions of reactive compounds that are capable of forming covalent bonds are referred to as reactive functional groups.

[0130] Steroid: A steroid derivative means a sterol, a sterol in which the hydroxyl moiety has been modified (for example, acylated), or a steroid hormone, or an analog thereof. The modification can include spacer groups, linkers, or reactive groups.

[0131] Sferics: Steric hindrance, or sferics, is the prevention or retardation of a chemical reaction because of neighboring groups on the same molecule.Attorney Docket No.: P24-229-SEC-WO01Embodiments

[0132] A composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formula (I),X-L1Formula (I)or a salt or stereoisomer thereof, wherein:A is O, CH-L2-Y or N-L2-Y;B is H, -OH, -NH2, -OCH3, or -CH3;each LI and L2 is independently a linker;L3 is a PEG linker;each X and Y is independently alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein the alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; and q is an integer from 3 to 125.

[0133] Embodiment 2. The composition of Embodiment 1, wherein the pegylated lipid is according to Formula (la):X-Lt, 13 J OlL2JqFormula (la)or a salt or stereoisomer thereof, wherein:OAttorney Docket No.: P24-229-SEC-WO01LAis independently -CH?-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently a bond, -CH?-, -(CH?)?-, or -(CH?)?-; and * denotes a connection leading to A.

[0134] Embodiment s. The composition of Embodiment 2, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:O? LI is a bond, L2 is a bond, and L3 is5, O oLAis independently -CH2-, -(CH?)?-, or -(CH?)?-;Attorney Docket No.: P24-229-SEC-WO01LBis independently -CH2-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0135] Embodiment 4. The composition of any of Embodiment 1 to 3, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:LI is a bond, L2 is a bond, and L3 is a bond.

[0136] Embodiment s. The composition of any of Embodiment 1 to 4, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:LI is a bond, L2 is a bond, and L3 is a bond;each X and Y is independently alkyl, alkenyl, heteroalkyl or heteroalkenyl, wherein the alkyl, alkenyl, heteroalkyl and heteroalkenyl are optionally substituted; andq is an integer from 3 to 70.

[0137] Embodiment 6. The composition of any of Embodiment 1 to 5, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl or alkenyl, wherein the alkyl and alkenyl are optionally substituted.

[0138] Embodiment 7. The composition of any of Embodiment 1 to 6, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl or alkenyl.

[0139] Embodiment 8. The composition of any of Embodiment 1 to 7, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:q is an integer from 30 to 80, 30 to 70, 40 to 50, 42 to 48 or 45.

[0140] Embodiment 9. The composition of any of Embodiment 1 to 8, wherein the pegylated lipid is according to Formula (la), or a salt or stereoisomer thereof, wherein:B is -OCH3.Attorney Docket No.: P24-229-SEC-WO01

[0141] Embodiment 10. The composition of any of Embodiment 1 to 9, wherein the pegylated lipid is according to compound of formula

[0142] Embodiment 11. The composition of Embodiment 1, wherein the pegylated lipid is according to Formula (lb):X-Lf H 1 3., OB1!_2Formula (lb)or a salt or stereoisomer thereof, wherein:O O o— LA— r (1) LI is?, L2 is, L3 is a bond, O HN6HAttorney Docket No.: P24-229-SEC-WO01LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0143] Embodiment 12. The composition of Embodiment 11, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:O O11 II 7. V^O-LA-i*.LI is ’, L2 is *, L3 is abond,,n, orH^cr^N-L6— |H <LAis independently -CH?-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)?-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and* denotes a connection leading to A.

[0144] Embodiment 13. The composition of Embodiment 11 or 12, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:O O• %<^O-LA— f.,LI is5, L2 isA>, L3 is abond;LAis independently -CH?-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)?-; and* denotes a connection leading to A.

[0145] Embodiment 14. The composition of any of Embodiment 11 to 13, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:Attorney Docket No.: P24-229-SEC-WO01 o o• V^o-LA— f. Y^O— f.,LI is5, L2 is 7 *, L3 is abond;LAis -CH2-; and* denotes a connection leading to A.

[0146] Embodiment 15. The composition of any of Embodiment 11 to 14, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl, alkenyl, heteroalkyl or heteroalkenyl, wherein the alkyl, alkenyl, heteroalkyl and heteroalkenyl are optionally substituted; andq is an integer from 3 to 70.

[0147] Embodiment 16. The composition of any of Embodiment 11 to 15, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl or alkenyl, wherein the alkyl and alkenyl are optionally substituted.

[0148] Embodiment 17. The composition of any of Embodiment 11 to 16, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl or alkenyl.

[0149] Embodiment 18. The composition of any of Embodiment 11 to 17, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:q is an integer from 30 to 80, 30 to 70, 40 to 50, 42 to 48 or 45.

[0150] Embodiment 19. The composition of any of Embodiment 11 to 18, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:B is -OCH3.

[0151] Embodiment 20. The composition of any of Embodiment 11 to 19, wherein the pegylated lipid is a compound of formulaAttorney Docket No.: P24-229-SEC-WO016

[0152] Embodiment 21. The composition of Embodiment 11 or 12, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; and* denotes a connection leading to A.

[0153] Embodiment 22. The composition of any of Embodiments 11, 12 or 21, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:LAis -CH?-.

[0154] Embodiment 23. The composition of any of Embodiment 11, 12, 21 or 22, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl, alkenyl, heteroalkyl or heteroalkenyl, wherein the alkyl, alkenyl, heteroalkyl and heteroalkenyl are optionally substituted; andq is an integer from 3 to 70.

[0155] Embodiment 24. The composition of any of Embodiment 11, 12, 21 to 23, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl or alkenyl, wherein the alkyl and alkenyl are optionally substituted.Attorney Docket No.: P24-229-SEC-WO01

[0156] Embodiment 25. The composition of any of Embodiment 11, 12, 21 to 24, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:each X and Y is independently alkyl or alkenyl.

[0157] Embodiment 26. The composition of any of Embodiment 11, 12, 21 to 25, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:q is an integer from 30 to 80, 30 to 70, 40 to 50, 42 to 48 or 45.

[0158] Embodiment 27. The composition of any of Embodiment 11, 12, 21 to 26, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:B is -OCH3.

[0159] Embodiment 28. The composition of any of Embodiment 11, 12, 21 to 27, wherein the pegylated lipid is a compound of formulao Si o:: Co

[0160] Embodiment 29. The composition of Embodiment 11, wherein the pegylated lipid is according to Formula (lb), or a salt or stereoisomer thereof, wherein:°-LA-r o^- 7 LI is, L2 is, L3 is abond, ’,^cr^N-L6— I H 5. LAis independently -CH2-, -(CH?)?-, -(CH?)s-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)s-; andAttorney Docket No.: P24-229-SEC-WO01* denotes a connection leading to A.

[0161] Embodiment 30. A composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formulas (Ila) or (lib),OFormula (Ila)Formula (lib)or a salt or stereoisomer thereof, wherein:D is H, -OH, -NH2, -OCH3, or -CH3;Lcis a PEG linker;X is alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; andq is an integer from 3 to 125.

[0162] Embodiment 31. The composition of Embodiment 30, wherein Lcis -CH2-, -(CH2)2-, or -(CH2)3-.

[0163] Embodiment 32. A composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formulas (Illa) or (Illb),Attorney Docket No.: P24-229-SEC-WO01Formula (Illb)or a salt or stereoisomer thereof, wherein:D is H, -OH, -NH2, -OCH3, or -CH3;E is H or C1-C4 alkyl;Lcis a PEG linker;each X and Y is independently alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; andq is an integer from 3 to 125.

[0164] Embodiment 33. The composition of Embodiment 32, wherein Lcis -CH2-, -(CH2)2-, or -(CH2)3-.

[0165] Embodiment 34. The composition of Embodiment 33, wherein each X and Y is independently alkenyl.

[0166] Embodiment 35. The composition of Embodiment 34, wherein each X and Y independently comprises 8 to 20 carbons.

[0167] Embodiment 36. The composition of Embodiment 35, wherein each X and Y independently comprises two non-conjugated alkenes.

[0168] Embodiment 37. The composition of any one of Embodiments 1-36, wherein in Formulas (I), (la), (lb), (Ila), (lib), (Illa), and (Illb):each X and Y is independently C8-C2o alkyl, C8-C2o alkenyl, or Cs-C2o alkynyl, wherein the alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl.

[0169] Embodiment 38. The composition of Embodiment 37, wherein in Formulas (I), (la), (lb), (Ila) and (lib):each X and Y is independently Cs-C2o alkyl.

[0170] Embodiment 39. The composition of Embodiment 38, wherein:Attorney Docket No.: P24-229-SEC-WO01(1) X is C13 alkyl, and Y is C13 alkyl; or(2) X is C14 alkyl, and Y is C14 alkyl; or(3) X is C15 alkyl, and Y is C15 alkyl; or(4) X is Ci6 alkyl, and Y is Ci6 alkyl; or(5) X is C17 alkyl, and Y is C17 alkyl; or(6) X is Cis alkyl, and Y is Cis alkyl; or(7) X is C19 alkyl, and Y is C19 alkyl; or(8) X is C20 alkyl, and Y is C20 alkyl; or(9) X is Cs alkyl, and Y is C8 alkyl; or(10) X is C9 alkyl, and Y is C9 alkyl; or(11) X is C10 alkyl, and Y is C10 alkyl; or(12) X is Cn alkyl, and Y is C11 alkyl; or(13) X is C12 alkyl, and Y is C12 alkyl.

[0171] Embodiment 40. A composition for delivery of a nucleic acid into a cell, comprising:(a) a cationic polymer;(b) an amphipathic compound; and(c) a pegylated lipid according to Formula (IV),X-L1^Formula (IV)or a salt or stereoisomer thereof, wherein:B is H, -OH, -NH2, -OCH3, or -CH3;LI is a bond or a linker;X is a cholesterol or a derivative thereof, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein said alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; andq is an integer from 3 to 125.

[0172] Embodiment 41. The composition of any one of Embodiments 1-40, wherein the cationic polymer comprises Formula (V);Attorney Docket No.: P24-229-SEC-WO01R1R2W'^OHN'^OR21R22Formula (V)wherein:each R1and R2is independently hydrogen or methyl;W is -NH- or -O-;R21comprises a structure of:RL / ^RRl ooR23wherein:each RLand RRis independently -(CH2)r-;each r is independently 1, 2, 3, 4, 5, or 6;R23is H or CH3;R22is independently alkyl, alkenyl, or heteroalkyl, wherein the alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0173] Embodiment 42. The composition of Embodiment 41, wherein R22is alkyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl.

[0174] Embodiment 43. The composition of Embodiment 41, wherein R22is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0175] Embodiment 44. The composition of Embodiment 41, wherein R22comprises 3 to 9 carbon atoms.

[0176] Embodiment 45. The composition of Embodiment 41, wherein R22comprises 10 to 16 carbon atoms.Attorney Docket No.: P24-229-SEC-WO01

[0177] Embodiment 46. The composition of any one of Embodiments 41-46, wherein each r is independently 1, 2, or 3, and wherein R23is H.

[0178] Embodiment 47. The composition of any one of Embodiments 41-47, wherein the cationic polymer comprises a 5- to 7-membered cyclic amine.

[0179] Embodiment 48. The composition of any one of Embodiments 41-48, wherein the cationic polymer comprises 2 to 80 monomeric units.

[0180] Embodiment 49. The composition of any one of Embodiments 1-40, wherein the cationic polymer comprises Formula (VI);R1R2W'^OHN'^OF^4R3Formula (VI)wherein:each R1and R2is independently hydrogen or methyl;W is -NH- or -O-;each R3and R4is independently alkyl, alkenyl, or heteroalkyl, wherein the alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, Ci- C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0181] Embodiment 50. The composition of Embodiment 49, wherein R3 is alkyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl.

[0182] Embodiment 51. The composition of Embodiment 50, wherein R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0183] Embodiment 52. The composition of Embodiment 50, wherein R3comprises 3 to 9 carbon atoms.

[0184] Embodiment 53. The composition of Embodiment 50, wherein R3comprises 10 to 16 carbon atoms.Attorney Docket No.: P24-229-SEC-WO01

[0185] Embodiment 54. The composition of Embodiment 49, wherein R3is alkenyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl.

[0186] Embodiment 55. The composition of Embodiment 54, wherein R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0187] Embodiment 56. The composition of Embodiment 54, wherein R3comprises 3 to 9 carbon atoms.

[0188] Embodiment 57. The composition of Embodiment 54, wherein R3comprises 10 to 16 carbon atoms.

[0189] Embodiment 58. The composition of Embodiment 54, wherein R3comprises 2 to 4 alkene bonds.

[0190] Embodiment 59. The composition of Embodiment 58, wherein at least two of the 2 to 4 alkene bonds are conjugated.

[0191] Embodiment 60. The composition of Embodiment 58, wherein at least two of the 2 to 4 alkene bonds are not conjugated.

[0192] Embodiment 61. The composition of Embodiment 49, wherein R3 is a heteroalkyl group, wherein the heteroalkyl group is optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl.

[0193] Embodiment 62. The composition of Embodiment 61, wherein the heteroalkyl is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0194] Embodiment 63. The composition of Embodiment 61, wherein the heteroalkyl group comprises 2 to 4 nitrogen atoms.

[0195] Embodiment 64. The composition of Embodiment 61, wherein the heteroalkyl group comprises 3 to 6 nitrogen atoms.

[0196] Embodiment 65. The composition of Embodiment 61, wherein the heteroalkyl group comprises 4 to 8 nitrogen atoms.

[0197] Embodiment 66. The composition of Embodiment 49, wherein R3is a heteroalkyl group comprising one or more nitrogen atoms and one or two oxygen, wherein the heteroalkyl group is optionally substituted with 1-3 groups independentlyAttorney Docket No.: P24-229-SEC-WO01selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl.

[0198] Embodiment 67. The composition of Embodiment 66, wherein the heteroalkyl group is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0199] Embodiment 68. The composition of Embodiment 66, wherein the heteroalkyl group comprises 1 to 3 nitrogen atoms.

[0200] Embodiment 69. The composition of Embodiment 66, wherein the heteroalkyl group comprises 3 to 5 nitrogen atoms.

[0201] Embodiment 70. The composition of Embodiment 66, wherein the heteroalkyl group comprises 5 to 8 nitrogen atoms.

[0202] Embodiment 71. The composition of any one of Embodiments 1-70, wherein R3 comprises one primary amine and one or more secondary or tertiary amine.

[0203] Embodiment 72. The composition of Embodiment 71, wherein R3 comprises 1 secondary amine.

[0204] Embodiment 73. The composition of Embodiment 71, wherein R3comprises 2 secondary amine.

[0205] Embodiment 74. The composition of Embodiment 71, wherein R3comprises 3 secondary amine.

[0206] Embodiment 75. The composition of Embodiment 71, wherein R3comprises 4 secondary amine.

[0207] Embodiment 76. The composition of Embodiment 71, wherein R3comprises 5 secondary amine.

[0208] Embodiment 77. The composition of any one of Embodiments 71-76, wherein R3 further comprises another primary amine.

[0209] Embodiment 78. The composition of any one of Embodiments 71-77, wherein R3 further comprises a tertiary amine.

[0210] Embodiment 79. The composition of any one of Embodiments 71-78, wherein R3 further comprises two tertiary amine.

[0211] Embodiment 80. The composition of any one of Embodiments 50-70, wherein R4is C2-C6 alkyl.Attorney Docket No.: P24-229-SEC-WO01

[0212] Embodiment 81. The composition of any one of Embodiments 50-70, wherein R4is C3-C8 alkyl.

[0213] Embodiment 82. The composition of any one of Embodiments 50-70, wherein R4is C4-C10 alkyl.

[0214] Embodiment 83. The composition of any one of Embodiments 50-70, wherein R4is C3-C6 alkenyl.

[0215] Embodiment 84. The composition of any one of Embodiments 50-70, wherein R4is C3-C8 alkenyl.

[0216] Embodiment 85. The composition of any one of Embodiments 50-70, wherein R4is C4-C10 alkenyl.

[0217] Embodiment 86. The composition of any one of Embodiments 71-79, wherein R4is C2-C6 alkyl.

[0218] Embodiment 87. The composition of any one of Embodiments71-79, wherein R4is C3-C8 alkyl.

[0219] Embodiment 88. The composition of any one of Embodiments 71-79, wherein R4is C4-C10 alkyl.

[0220] Embodiment 89. The composition of any one of Embodiments 71-79, wherein R4is C3-C6 alkenyl.

[0221] Embodiment 90. The composition of any one of Embodiments 71-79, wherein R4is C3-C8 alkenyl.

[0222] Embodiment 91. The composition of any one of Embodiments 71-79, wherein R4is C4-C10 alkenyl.

[0223] Embodiment 92. The composition of any one of Embodiments 80-91, wherein R4is substituted with 1-3 groups independently selected from F, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0224] Embodiment 93. The composition of any one of Embodiments 49, 50, 52-54, 56-61, 63-66, or 68-92, wherein R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0225] Embodiment 94. The composition of any one of Embodiments 49-93, wherein R1is hydrogen and R2is methyl, or wherein R1is methyl and R2is hydrogen.

[0226] Embodiment 95. The composition of any one of Embodiments 49-93, wherein R1and R2are hydrogen.Attorney Docket No.: P24-229-SEC-WO01

[0227] Embodiment 96. The composition of any one of Embodiments 49-93, wherein R1and R2are methyl.

[0228] Embodiment 97. The composition of any one of Embodiments 1-96, wherein the amphipathic compound is according to Formula (VII);R13 / \ R14R1< " L4-N\ _ J\l-L5" XR12Formula (VII)wherein each R11and R12is independently alkyl or alkenyl, wherein each the alkyl and alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;each R13and R14is independently hydrogen, alkyl, alkenyl, alkoxy, alkyl-C(O)-alkylene, alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-C(O)-, heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alky 1-heterocy cl e-alkeny 1 ene-C (O)-,alkenyl-heterocycle-alkenylene-C(O)-, heteroaryl-C(O)-,heteroaryl-alkylene-C(O)-, heteroaryl-alkenylene-C(O)-,alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-, alkyl-heteroaryl-alkenylene-C(O)-, oralkenyl-heteroaryl-alkenylene-C(O)-, wherein each the alkyl, alkenyl, alkylene, alkenylene, heterocycle, an heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;each L4 and L5 is independently C2-C8 alkylene, optionally substituted with one or two groups independently selected from the group consisting of halide, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andp is 0 or 1.

[0229] Embodiment 98. The composition of Embodiment 97, wherein each L4 andL5 is independently ethylene, n-propylene, 1-methyl-ethylene, 2-methyl-ethylene,Attorney Docket No.: P24-229-SEC-WO011-methyl-propylene, 2-methyl-propylene, 3-methyl-propylene, n-butylene, 1 -methylbutylene, 2-methyl-butylene, 3-methyl-butylene, 4-methyl-butylene, 1 -ethyl -butylene, 2-ethyl-butylene, 3-ethyl-butylene, or 4-ethyl-butylene.

[0230] Embodiment 99. The composition of Embodiment 98, wherein L4 and L5 are different.

[0231] Embodiment 100. The composition of Embodiment 98, wherein L4 and L5 are the same.

[0232] Embodiment 101. The composition of any one of Embodiments 97-100, wherein L4 and L5 is substituted with one or two groups of F or CF3.

[0233] Embodiment 102. The composition of any one of Embodiments 97-101, wherein each R11and R12is independently C10 to C25 alkyl.

[0234] Embodiment 103. The composition of any one of Embodiments 97-101, wherein each R11and R12is independently C10 to C25 alkenyl.

[0235] Embodiment 104. The composition of any one of Embodiment 102 or 103, wherein each Rll and R12 is independently substituted with F, methyl, ethyl, or CF3.

[0236] Embodiment 105. The composition of Embodiments 103 or 104, wherein each R11and R12independently comprises 1 to 4 alkene bonds.

[0237] Embodiment 106. The composition of Embodiment 105, wherein each R11and R12independently comprises 2 to 4 alkene bonds.

[0238] Embodiment 107. The composition of Embodiment 106, wherein, for each R11and R12, at least two of the 2 to 4 alkene bonds are conjugated.

[0239] Embodiment 108. The composition of Embodiment 106, wherein for each R11and R12, at least two of the 2 to 4 alkene bonds are not conjugated.

[0240] Embodiment 109. The composition of any one of Embodiments 105-108 wherein at least one of the 1 to 4 alkene bonds is cis.

[0241] Embodiment 110. The composition of any one of Embodiments 105-109, wherein at least one of the 1 to 4 alkene bonds is trans.

[0242] Embodiment 111. The composition of any one of Embodiments 106-110, wherein two of the 2 to 4 alkene bonds are cis.

[0243] Embodiment 112. The composition of any one of Embodiments 106-111, wherein two of the 2 to 4 alkene bonds are trans.Attorney Docket No.: P24-229-SEC-WO01

[0244] Embodiment 113. The composition of any one of Embodiments 102-112, wherein each R11and R12comprises 10 to 15 carbon atoms.

[0245] Embodiment 114. The composition of any one of Embodiments 102-112, wherein each R11and R12comprises 16 to 20 carbon atoms.

[0246] Embodiment 115. The composition of any one of Embodiments 102-112, wherein each R11and R12comprises 21 to 25 carbon atoms.

[0247] Embodiment 116. The composition of any one of Embodiments 103-115,

[0248] Embodiment 117. The composition of any one of Embodiments 102-116, wherein R11and R12,are the same.

[0249] Embodiment 118. The composition of any one of Embodiments 102-116, wherein R11and R12are different.

[0250] Embodiment 119. The composition of any one of Embodiments 97-118, wherein both R13and R14are hydrogen.

[0251] Embodiment 120. The composition of any one of Embodiments 97-118, wherein R13is hydrogen and R14is C2 to C10 alkyl, C2 to C10 alkenyl, or C2 to C10 alkoxy, alkyl-C(O)-alkylene, alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, alkenyl-C(O)-,heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkenylene-C(O)-, alkenyl-heterocycle-alkenylene-C(O)-, alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-,alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-.

[0252] Embodiment 121. The composition of any one of Embodiments 97-118, wherein each R13and R14is independently C2 to C10 alkyl.

[0253] Embodiment 122. The composition of any one of Embodiments 97-118, wherein each R13and R14is independently C2 to C10 alkenyl.Attorney Docket No.: P24-229-SEC-WO01

[0254] Embodiment 123. The composition of any one of Embodiments 97-118, wherein each R13and R14is independently C2 to C10 alkoxy.

[0255] Embodiment 124. The composition of any one of Embodiments 97-118, wherein each R13and R14is independently alkyl-C(O)-alkylene,alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, or alkenyl-C(O)-.

[0256] Embodiment 125. The composition of any one of Embodiments 97-118, wherein each R13and R14is independently heterocycle-C(O)-,heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkenylene-C(O)-, or alkenyl-heterocycle-alkenylene-C(O)-.

[0257] Embodiment 126. The composition of any one of Embodiments 97-118, wherein each R13and R14is independently heteroaryl-C(O)-,heteroaryl-alkylene-C(O)-, heteroaryl-alkenylene-C(O)-,alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-,alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-.

[0258] Embodiment 127. The composition of any one of Embodiments 97-126, wherein p is 0.

[0259] Embodiment 128. The composition of any one of Embodiments 97-126, wherein p is 1.

[0260] Embodiment 129. The composition of any one of Embodiments 97-118, 120, 125, 127, or 128, wherein the heterocycle is azetidine, 1,3-diazetidine, pyrrolidine, piperidine, piperazine, or morpholine.

[0261] Embodiment 130. The composition of Embodiment 129, wherein each R13and R14is independently heterocycle-Ci-C4 alkylene-C(O)-, and wherein the heterocycle is optionally substituted with fluorine, methyl, ethyl, propyl, isopropyl, or CF3.

[0262] Embodiment 131. The composition of any one of Embodiments 97-118, 120, or 126-128, wherein the heteroaryl is pyrrole, furan, imidazole, pyrazole, oxazole, isoxazole, pyridine, or pyrazine.Attorney Docket No.: P24-229-SEC-WO01

[0263] Embodiment 132. The composition of Embodiment 129, wherein R13and R14is independently heteroaryl- C1-C4 alkylene-C(O)-, and wherein the heteroaryl is optionally substituted with fluorine, methyl, ethyl, propyl, isopropyl, or CF3.

[0264] Embodiment 133. The composition of any one of Embodiments 97-118 or 121-132, wherein R13and R14are different.

[0265] Embodiment 134. The composition of any one of Embodiments 97-118 or 121-132, wherein R13and R14are the same.

[0266] Embodiment 135. The composition of any one of Embodiments 97-134, wherein the cationic polymer is a block copolymer, an alternating copolymer, a random or statistical copolymer, or a gradient copolymer.

[0267] Embodiment 136. The composition of any one of Embodiments 97-134, wherein the cationic polymer has a linear structure, a graft structure, a branched structure, a crosslinked or network structure, a star structure, a comb structure, a ladder structure, or a dendritic structure.

[0268] Embodiment 137. The composition of any one of Embodiments 97-136, wherein number average molecular weight (Mn) of the cationic polymer is from about 2,000 to about 100,000.

[0269] Embodiment 138. The composition of Embodiment 137, wherein Mnis from about 2,000 to about 5,000.

[0270] Embodiment 139. The composition of Embodiment 137, wherein Mnis from about 3,000 to about 6,000.

[0271] Embodiment 140. The composition of Embodiment 137, wherein Mnis from about 5,000 to about 10,000.

[0272] Embodiment 141. The composition of Embodiment 137, wherein Mnis from about 10,000 to about 15,000.

[0273] Embodiment 142. The composition of Embodiment 137, wherein Mnis from about 15,000 to about 20,000.

[0274] Embodiment 143. The composition of Embodiment 137, wherein Mnis from about 20,000 to about 25,000.

[0275] Embodiment 144. The composition of Embodiment 137, wherein Mnis from about 25,000 to about 30,000.Attorney Docket No.: P24-229-SEC-WO01

[0276] Embodiment 145. The composition of Embodiment 137, wherein Mnis from about 30,000 to about 35,000.

[0277] Embodiment 146. The composition of Embodiment 137, wherein Mnis from about 35,000 to about 40,000.

[0278] Embodiment 147. The composition of Embodiment 137, wherein Mnis from about 40,000 to about 45,000.

[0279] Embodiment 148. The composition of Embodiment 137, wherein Mnis from about 45,000 to about 50,000.

[0280] Embodiment 149. The composition of Embodiment 137, wherein Mnis from about 50,000 to about 55,000.

[0281] Embodiment 150. The composition of Embodiment 137, wherein Mnis from about 55,000 to about 60,000.

[0282] Embodiment 151. The composition of Embodiment 137, wherein Mnis from about 60,000 to about 65,000.

[0283] Embodiment 152. The composition of Embodiment 137, wherein Mnis from about 65,000 to about 70,000.

[0284] Embodiment 153. The composition of Embodiment 137, wherein Mnis from about 70,000 to about 80,000.

[0285] Embodiment 154. The composition of Embodiment 137, wherein Mnis from about 80,000 to about 90,000.

[0286] Embodiment 155. The composition of Embodiment 137, wherein Mnis from about 90,000 to about 100,000.

[0287] Embodiment 156. The composition of any one of Embodiments 97-155, wherein Mw / Mnis from about 1.00 to about 1.10, and wherein Mwis weight average molecular weight.

[0288] Embodiment 157. The composition of any one of Embodiments 97-155, wherein Mw / Mnis from about 1.10 to about 1.20, and wherein Mwis weight average molecular weight.

[0289] Embodiment 158. The composition of any one of Embodiments 97-155, wherein Mw / Mnis from about 1.20 to about 1.30, and wherein Mwis weight average molecular weight.Attorney Docket No.: P24-229-SEC-WO01

[0290] Embodiment 159. The composition of any one of Embodiments 97-155, wherein Mw / Mnis from about 1.30 to about 1.40, and wherein Mwis weight average molecular weight.

[0291] Embodiment 160. The composition of any one of Embodiments 97-155, wherein Mw / Mnis from about 1.40 to about 1.50, and wherein Mwis weight average molecular weight.

[0292] Embodiment 161. The composition of any one of Embodiments 97-160, wherein a molar ratio of monomeric units comprising R3to monomeric units comprising R4in the cationic polymer is from about 0.30 to about 0.53.

[0293] Embodiment 162. The composition of any one of Embodiments 97-160, wherein a ratio of monomeric units comprising R3to monomeric units comprising R4in the cationic polymer is from about 0.34 to about 0.49.

[0294] Embodiment 163. The composition of any one of Embodiments 97-160, wherein a ratio of monomeric units comprising R3to monomeric units comprising R4in the cationic polymer is from about 0.36 to about 0.47.

[0295] Embodiment 164. The composition of any one of Embodiments 97-163, wherein W is -NH-.

[0296] Embodiment 165. The composition of any one of Embodiments 97-163, wherein W is -O-.

[0297] Embodiment 166. The composition of any one of Embodiments 1-165, comprising 7ra / / .slT-VirusGEN®, AAViator™, 7ra / / .slT-Lenti®, 7 / z / / / .sIT-X2®, Traw IT-PRO® or 7 / Y / / 7. S1T-2020® and a pegylated lipid according to any of formula (I), (Ila), (lib), (Illa), (Illb) or (IV).

[0298] Embodiment 167. The composition of any one of Embodiments 97-166, further comprising an alcohol and a buffer.

[0299] Embodiment 168. The composition of Embodiment 167, wherein the alcohol is ethanol.

[0300] Embodiment 169. The composition of Embodiment 167 or Embodiment 168, wherein the buffer is glycine-HCl.

[0301] Embodiment 169 A. The composition of any one of Embodiments 1-31 and 37-169, B is H.Attorney Docket No.: P24-229-SEC-WO01

[0302] Embodiment 169B. The composition of any one of Embodiments 32-169, D is H.

[0303] Embodiment 170. The composition of any one of Embodiments 97-169, 169A or 169B, further comprising the nucleic acid.

[0304] Embodiment 171. The composition of any one of Embodiments 97-169, 169A or 169B, further comprising DNA, RNA, or any combination thereof.

[0305] Embodiment 172. A transfection reagent comprising a composition of any one of Embodiments 1-169, 169Aor 169B, and a nucleic acid.

[0306] Embodiment 173. The transfection reagent of Embodiment 172, wherein the nucleic acid is one or more adeno-associated virus (AAV) plasmids.

[0307] Embodiment 174. The transfection reagent of Embodiment 172, wherein the nucleic acid is one or more lentivirus (LV) plasmids.

[0308] Embodiment 175. A method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of Embodiments 1-171 with the nucleic acid to produce a mixed solution, and contacting the cell with the mixed solution, thereby transfecting the nucleic acid into the cell.

[0309] Embodiment 176. The method of Embodiment 175, wherein the nucleic acid is one or more plasmids used to manufacture adeno-associated virus (AAV).

[0310] Embodiment 177. The method of Embodiment 175, wherein the nucleic acid is one or more plasmids used to manufacture lentivirus (LV).

[0311] Embodiment 178. The method of any one of Embodiments 175-177, wherein an average genome titer is no less than 1.5><10A10 genome copies per milliliter (GC / mL) at 30 minutes after the mixing.

[0312] Embodiment 179. The method of Embodiment 178, wherein the average genome titer is no less than 1.5><10A10 GC / mL at 60 minutes after the mixing.

[0313] Embodiment 180. The method of Embodiment 178, wherein the average genome titer is no less than 1.5xl0A10 GC / mL at 90 minutes after the mixing.

[0314] Embodiment 181. The method of Embodiment 178, wherein the average genome titer is no less than 1.5xl0A10 GC / mL at 120 minutes after the mixing.

[0315] Embodiment 182. The method of Embodiment 178, wherein the average genome titer is no less than 1.5xl0A10 GC / mL at 8 hours after the mixing.Attorney Docket No.: P24-229-SEC-WO01

[0316] Embodiment 183. The method of any one of Embodiments 175-177, wherein an average genome titer is no less than 1.0><10All genome copies per milliliter (GC / mL) at 30 minutes after the mixing.

[0317] Embodiment 184. The method of Embodiment 183, wherein the average genome titer is no less than 1.0×1011genome copies per milliliter (GC / mL) at 60 minutes after the mixing.

[0318] Embodiment 185. The method of Embodiment 183, wherein the average genome titer is no less than 1.0×1011genome copies per milliliter (GC / mL) at 90 minutes after the mixing.

[0319] Embodiment 186. The method of Embodiment 183, wherein the average genome titer is no less than 1.0><10All genome copies per milliliter (GC / mL) at 120 minutes after the mixing.

[0320] Embodiment 187. The method of Embodiment 183, wherein the average genome titer is no less than 1.0×1011genome copies per milliliter (GC / mL) at 8 hours after the mixing.

[0321] Embodiment 188. The method of any one of Embodiments 175-179, wherein an average genome titer decreases no more than 50% at 3 hours after complex initiation.

[0322] Embodiment 189. The method of Embodiment 188, wherein the average genome titer decreases no more than 40% at 3 hours after complex initiation.

[0323] Embodiment 190. The method of Embodiment 188, wherein the average genome titer decreases no more than 50% at 4 hours after complex initiation.

[0324] Embodiment 191. A method of forming and stabilizing a transfection complex, comprising mixing a composition of any one of Embodiments 1-157 with the nucleic acid, thereby producing a plurality of transfection complexes comprising the pegylated lipid, the cationic polymer, the amphipathic compound, and the nucleic acid.

[0325] Embodiment 192. The method of Embodiment 191, wherein the nucleic acid is one or more plasmids used to manufacture adeno-associated virus (AAV).

[0326] Embodiment 193. The method of Embodiment 191, wherein the nucleic acid is one or more plasmids used to manufacture lentivirus (LV).Attorney Docket No.: P24-229-SEC-WO01

[0327] Embodiment 194. The method of any one of Embodiments 191-193, wherein an average radius of the plurality of transfection complexes is no more than 1000 nm at 60 minutes after the mixing.

[0328] Embodiment 195. The method of Embodiment 194, wherein the average radius of the plurality of transfection complexes is no more than 1000 nm at 120 minutes after the mixing.

[0329] Embodiment 196. The method of Embodiment 194, wherein the average radius of the plurality of transfection complexes is no more than 1000 nm at 180 minutes after the mixing.

[0330] Embodiment 197. The method of Embodiment 194, wherein the average radius of the plurality of transfection complexes is no more than 1000 nm at 240 minutes after the mixing.

[0331] Embodiment 198. A method of preparing a transfection reagent mixture, comprising:(a) adding a pegylated lipid to a mixture of a cationic polymer and an amphipathic compound, thereby obtaining another mixture; and (b) mixing the another mixture, thereby obtaining the transfection reagent mixture.

[0332] Embodiment 199. The method of Embodiment 198, wherein each of the pegylated lipid, the cationic polymer, and the amphipathic compound is according to any one of Embodiments 1-198.Examples

[0333] Example synthesis of polymers. Synthesis of the polymers can follow the procedures disclosed in U. S. Pat. Nos. 8,921,448 and 9,677,077, each of which is incorporated by reference in its entirety. For example, polymers with a acrylamide or (meth)acrylamide backbone having a amine-containing side group containing one or more primary, secondary or tertiary amines can be synthesized according to known procedures. The copolymers can be a combination of two or more different cationic repeat unit structures according to Formula (I). Copolymers can be a combination of acrylamide or (meth)acrylamide amine-containing cationic units and alkyl acrylamideAttorney Docket No.: P24-229-SEC-WO01or alkyl (meth)acrylamide units. Other combinations of the acrylamide or (meth)acrylamide with different side chains are possible.

[0334] The tert-butyloxycarbonyl (BOC) protected amine-containing acrylamide monomers can be synthesized and polymerized. The monomers can be synthesized by reacting acryloyl chloride or methacryloyl chloride with amines (primary or secondary) in the presence of a base (e.g., diisopropylethylamine) and solvent (usually dichloromethane). Structure and purity of the monomers can be determined byXH NMR. These monomers then undergo polymerization and copolymerization. Once the (co)polymers are purified by precipitation (e.g., into hexane), they are analyzed by gel permeation chromatography (organic solvent phase) andXH NMR. If additional amine groups in the side chain of the monomer are present (and protected by BOC), the (co)polymers can be deprotected under acidic conditions to remove the BOC protecting groups; structure and purity can be confirmed byXH NMR or other analytical methods.

[0335] For example, BOC protected amine-containing monomers can be synthesized based on the reaction of either acryloyl chloride or methacryloyl chloride with a primary amine-containing or alcohol-containing moiety in the presence of a base. See Scheme 1. Both R21and R22comprise additional primary or secondary amines that can be protected by BOC if the particular amine is not used in the acylation reaction with acryloyl chloride or methacryloyl chloride.

[0336] Scheme 1: an example general synthetic route leading to monomers of the co-polymer according to Formula (V)HW-R21diisopropylamineH2N— R22dichloromethane

[0337] Example polymer synthesis: The monomers described above were polymerized using RAFT in order to synthesize polymers of well-defined molecularAttorney Docket No.: P24-229-SEC-WO01weights, compositions, and architectures. The polymerization reaction is illustrated in Scheme 2.

[0338] Scheme 2: an example general synthetic route to make cationic polymer according to Formula (V) via a polymerization reactionR1_,R2CTA, initiatorO+W HN BuAc, heat W" " A) HhK "^O^R21^R22R21R22Formula (V)For Formula (V), each R1and R2is independently hydrogen or methyl; W is -NH- or -O-;R21comprises a structure of:RL / ^RRR23wherein:each RLand RRis independently -(CH2)r-;each r is independently 1, 2, 3, 4, 5, or 6;R23is H or CH3;R22is independently alkyl, alkenyl, or heteroalkyl, wherein the alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0339] Scheme 2 shows a RAFT polymerization of BOC protected (meth)acrylamide monomers in the presence of a chain transfer agent (CTA), free radical initiator (initiator), solvent (butyl acetate, BuAc), and heat (60-100 °C).

[0340] The following general procedure is an example. Monomers (0.8 mmol total), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPCPA, 0.0042 mmol), AIBN (0.00064 mmol), and butyl acetate (1.10 mL) are added to a 20 mL glass vial with stirrer bar. The vial is sealed with a rubber cap and the solution bubbled with nitrogen using a long syringe with a second syringe as the outlet for about 1 h. TheAttorney Docket No.: P24-229-SEC-WO01syringes are removed, and the vial is heated to 80 °C for about 15 h using an oil bath. The solution is allowed to cool to room temperature, and solids are precipitated into hexane. The product is re-dissolved in dichloromethane and precipitated into hexane dried under reduced pressure for several hours.

[0341] The BOC protected polymers are deprotected post-polymerization to yield primary and secondary amines in the polymer side groups. Below is a general deprotection procedure as an example.

[0342] BOC-protected polymer (0.150 g) is dissolved in a 2 N HC1 solution of acetic acidic (4 mL) and is stirred for 1 h. Water (15 mL) is added to the solution, which is then dialyzed against salt water and then deionized water over a period of about 48 h. The dialyzed solution is then frozen and lyophilized to dryness to provide the desired polymer.

[0343] Cationic polymer according to Formula (VI) can synthesized using similar synthetic routes as shown in Scheme 3 and Scheme 4 below.

[0344] Scheme 3: an example general synthetic route leading to monomers of the co-polymer according to Formula (VI)diisopropylamineH2N-R3dichloromethane

[0345] BOC protected amine-containing monomers can be synthesized based on the reaction of either acryloyl chloride or methacryloyl chloride with a primary amine-containing or alcohol-containing moiety in the presence of a base. See Scheme 3. Both R4and R4comprise additional primary or secondary amines that can be protected by BOC if the particular amine is not used in the acylation reaction with acryloyl chloride or methacryloyl chloride.

[0346] Example polymer synthesis: The monomers described above were then polymerized using RAFT in order to synthesize polymers of well-defined molecularAttorney Docket No.: P24-229-SEC-WO01weights, compositions, and architectures. The polymerization reaction is illustrated in Scheme 4.

[0347] Scheme 4: an example general synthetic route to make cationic polymer according to Formula (VI) via a polymerization reactionCTA, initiatorBuAc, heatFormula (VI)For Formula (V), each R1and R2is independently hydrogen or methyl; W is -NH- or -O-;each R3and R4is independently alkyl, alkenyl, or heteroalkyl, wherein said alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, Ci- C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; andeach m and n is independently an integer from 1 to 10.

[0348] Scheme 4 shows a RAFT polymerization of BOC protected (meth)acrylamide monomers in the presence of a chain transfer agent (CTA), free radical initiator (initiator), solvent (butyl acetate, BuAc), and heat (60-100 °C).

[0349] Example amphipathic compound synthesis: Synthesis of amphipathic compounds can follow the procedures disclosed in U. S. Pat. No. 11,739, which is incorporated by reference in its entirety. A general synthetic route to amphipathic compounds is shown in Scheme 5. A bisamine is acylated with an appropriate acyl chloride under basic conditions to provide a bisamide, which is subsequently reduced by LiAlFU to provide the bis-secondary amine (amphipathic compound 1). A second acylation affords another bisamide (amphipathic compound 2), which can be reduced again by LiAlFU to provide a bis-tertiary amine (amphipathic compound 3).

[0350] Scheme 5: General synthetic scheme leading to an amphipathic compound.Attorney Docket No.: P24-229-SEC-WO01acylation LiAIH4acylationamphipathic amphipathiccompound 2 compound 1p is 0 or 1amphipathiccompound 3

[0351] To a cooled solution of bisamine (1 eq) and EtiN (2.1 eq) in CHCh is added, dropwise, a solution of acyl chloride (2.05-2.1 eq) in CHCI3. The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with CHCh to double the volume, washed three times with saturated Na2CCh, washed once with saturated NaCl, and dried with MgSCh or Na2SO4. The solvent is removed using a rotary evaporator. The bisamide is purified by recrystallization or by column chromatography.

[0352] To a stirred suspension of lithium aluminum hydride (LAH, 3 eq) in tetrahydrofuran (THF), under nitrogen, is added, dropwise, a solution of bisamide (1 eq) in THF. When the addition is complete, the reaction mixture is refluxed under nitrogen overnight. Then the reaction mixture is cooled (cold water bath), and excess hydride is decomposed following standard procedure(s). The mixture is filtered, and the precipitate is washed with THF. The filtrate is diluted with CHCh to at least double the volume, washed twice with water, washed once with saturated NaCl, and dried with MgSCh or Na2SC>4. The solvents are removed using a rotary evaporator.The amine is purified by column chromatography.Attorney Docket No.: P24-229-SEC-WO01

[0353] The second round of acylation and reduction follow similar procedures to attach another side chain to the non-cyclic amine.

[0354] Exemplar Materials and Methods

[0355] Dynamic Light Scattering (DLS) Protocol for Measurement of Transfection Complex Size

[0356] 7ra / / .sIT-VirusGEN® (500 pL, Minis Bio LLC, Cat. No. MIR 6700) was mixed with stabilizing lipid solution (4 pL). To form the transfection complex, a 1 mg / mL DNA solution (2 pL) in water was added to a final volume of 192 pL phosphate-buffered saline (PBS; Corning, Cat. No. 21-040-CV). Subsequently, 6 pL of the 7ra / / .sIT-VirusGEN® + stabilizing lipid solution was added to the PBS solution and mixed at room temperature. Immediately after complexes are mixed, a 50 pL aliquot was transferred to a MicroCuvette (Wyatt Disposable MicroCuvette for the NanoStar (Part no. 162960 rev C)) and inserted into the DLS instrument (Wyatt DynaPro NanoStar). Using Wyatt’s Dynamics (v7) software, data was collected within 1-2 minutes of addition of the transfection reagent to nucleic acid. A typical DLS experiment was performed at 25°C and the event schedule parameters were as follows: auto-attenuation enabled, acquisition time (s) = 10, collect acquisitions = 4. DLS readings were taken over several minutes to hours either continuously or at various timepoints. The transfection complex radius and normalized intensity was plotted against time to assess transfection complex growth.

[0357] Cell Culture for 293 Suspension Cells

[0358] Viral Production Cells 2.0 (293-VP 2.0; Thermo Fisher Scientific, Cat. No. A49784) were maintained in Viral Production Media (VPM; Thermo Fisher Scientific, Cat. No. A4817901) supplemented with 4 mM GlutaMAX™ (Thermo Fisher Scientific, Cat. No. 35050061). Cells were maintained in 8% CO2 at 37°C on a 2.5 cm orbital shaker at 125 rpm.

[0359] Adeno-associated virus (AAV) Production and Analysis Protocol

[0360] The day before transfection, 293 suspension cells were passaged to ensure the cells were actively dividing and would reach a density of 4.0 - 5.0 x 106cells / mL the day of transfection. The day of transfection, cells were seeded in 6-well non-tissue culture treated plates (Coming, Cat. No. 351146) at a density of 3.0 x 106cells / mL. A 1 mg / mL DNA solution composed of a 1:1:1 plasmid mass ratio of a transfer vectorAttorney Docket No.: P24-229-SEC-WO01pALD-ITR-WPRE-GFP (Aldevron), an AAV8 or AAV9 RepCap vector (Genemedi), and a helper vector (pALD-Helper; Aldevron) was mixed in water. For each milliliter of culture, 3 pL of 7ra / / .sIT-VirusGEN® (Minis Bio LLC, Cat. No. MIR 6700), with or without supplementation with a pegylated lipid, and 1.5 pg of DNA were added to a final volume of 50 pL phosphate-buffered saline (PBS; Corning, Cat. No. 21-040-CV) for a final transfection reagent to DNA ratio of 2: 1 (vol:wt). Complexes start forming immediately following the addition of the transfection reagent (which is added last to the complex). At various time points after the initiation of complex formation, 50 pL of transfection complex was added per milliliter of culture for a final complexation volume of 5%. Alternately, separate transfection complexes were formed at different times with the addition of the complexes at the same time point. Cells were transfected for 48-72 hours before harvesting virus.

[0361] When the transfection complex is 7ra / / .sIT-VirusGEN® (Minis Bio LLC, Cat. No. MIR 6700) only and does not contain the stabilizing agent (pegylated lipid) for AAV9 transfection, the average AAV9 genome titer (GC / mL) and average radius of transfection complex particles (nm) at various time points from 30 minutes to 120 minutes are shown in FIG. 3. As shown in FIG. 3, the particle sizes grows from less than 1000 nm at 30 minutes to more than 1,600 nm at 120 minutes. In this case, the total volume of the transfection complex added to the reaction vessel is about 5% of the culture volume. As the average size of the transfection complexes grows, the average AAV9 genome titer decreases from about 1.5* 10Al 1 GC / mL at 30 minutes to about 6*10A10 GC / mL at 120 minutes.

[0362] When a more concentrated transfection complex is used for the addition (total volume of the transfection complex is about 2% of the culture volume) under otherwise similar transfection conditions, the results are shown in FIG. 4. In this case, the particle sizes grows from about 1500 nm at 30 minutes to about 2,000 nm at 60 minutes. Then the transfection complexes begin to precipitate out at 90 minutes and 120 minutes. The average AAV9 genome titer decreases from about 1.0* 10Al 1 GC / mL at 30 minutes to about 5* 10A10 GC / mL at 60 minutes.

[0363] Similar experiments are conducted by adding the stabilizing agent (pegylated lipids) to the transfection reagents such that the pegylated lipids become part of the transfection complexes. When the total volume of the resulting stabilizedAttorney Docket No.: P24-229-SEC-WO01transfection complex is about 5% of the culture volume, the experimental results are shown in FIG. 5. As shown in FIG. 5, the particle sizes grows much slower: from about 700 nm at 30 minutes to about 900 nm at 120 minutes, the average AAV9 genome titer grows from about 2* 10Al 1 GC / mL at 30 minutes to about 2.5 * 10Al 1 GC / mL at 120 minutes.

[0364] When a more concentrated stabilized transfection complex is used for the addition (total volume of the transfection complex is about 2% of the culture volume) under otherwise similar transfection conditions, the results are shown in FIG. 6. In this case, the particle sizes remains around 750 nm from 30 minutes to 120 minutes. The average AAV9 genome titer decreases from about 2.5 * 10Al 1 GC / mL at 30 minutes to about 2.2* 10Al 1 GC / mL at 120 minutes.

[0365] AAV Harvest (48-72 hours post-transfection)

[0366] To harvest AAV, cells were incubated with 0. IX volume of a 10X Cell Lysis Buffer, composed of 500 mM Tris pH 8, 10% Tween®20, 20 mM MgCh, and 1000 U / mL Recombinant Dr. Nuclease (Syd Labs, Cat. No. BP4200) at 37 °C for 1.5 hours while shaking. Cells were then incubated with 0.1X volume of 5 M NaCl at 37 °C for 30 minutes while shaking. The lysate was centrifuged at 4,100 x g for 10 minutes to remove cell debris, and the supernatant transferred to a clean tube and stored at -80 °C.

[0367] AAV Genome and Capsid Quantitation

[0368] Genome quantitation was performed using digital PCR (dPCR) on a QIAcuity Digital PCR System (Qiagen, Cat. No. 911001). AAV lysates were diluted in a water-based buffer consisting of IX GeneAmp PCR Buffer (Thermo Fisher Scientific, Cat. No. 4379878) and 0.05% Poloxamer 188 (Minis Bio LLC, Cat. No. MIR 6230). Each reaction contained IX QIAcuity Probe PCR Mastermix (Qiagen, Cat. No. 250102), 0.8 pM CMV Forward Primer (IDT; 5’-TTCCTACTTGGCAGTACATCTACG -3’) and CMV Reverse Primer (IDT; 5’-GTCAATGGGGTGGAGACTTGG -3’), 0.4 pM CMV Probe (IDT; 5’- 156-FAM / TGAGTCAAA / ZEN / CCGCTATCCACGCCCA / 3IABkFQ / -3’), and 4.5 pL diluted AAV lysate in a final volume of 15 pL. Samples were loaded into a QIAcuity Nanoplate (Qiagen, Cat. No. 250021) and cycled using the following parameters: 95 °C for 10 minutes, 40 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds.Atorney Docket No.: P24-229-SEC-WO01

[0369] Capsids were quantified using an ELISA kit for the appropriate serotypes (Progen, Cat. No. PRATV, PRAAV5, PRAAV8, PRAAV9) or a Lumit-based assay (Promega, Cat. No. VB2020) developed for Minis Bio LLC by Promega Corporation.

[0370] Lentivirus (LV) Production and Analysis Protocol

[0371] Viral Production Cells 1.0 (293-VP 2.0; Thermo Fisher Scientific, Cat. No. A49784) were maintained in LV-MAX Production Medium (Thermo Fisher Scientific Cat. No. A3583402). Cells were maintained in 8% CO2 at 37 °C on a 2.5 cm orbital shaker at 125 rpm.

[0372] Viral Production Cells 1.0 (2 mL) were seeded into untreated 6-well plates at 4.0 x 106cells / mL the same day as transfection. Transfection complexes were formed in PBS at 3: 1 reagent: DNA (vokmass) with 10 ug / mL of plasmid DNA using TransIT-VirusGEN with or without supplementation with pegylated lipid. 200 pl of transfection complex (containing 2 pg of a 1: 1:4:6 pALD-VSV-G-A, pALD-Rev-A, pALD-GagPol-A, pALD-LentiEGFP-A (Aldevron)) was added per well after a 30 min or 3 hr complex formation. Lentivirus was harvested 48 hourspost transfection by centrifuging the cells at 350 x g for 5 minutes and collecting the clarified supernatants and storing at -80 °C. LV was titered via a functional transduction assay: briefly, various dilutions of LV were added to cultures of 293T / 17 cells in the presence of polybrene. Cells were assessed for GFP expression using a Guava EasyCyte HT Flow Cytometer 72 hrs post transduction.

[0373] FIG. 7 shows that the stabilizing agent (pegylated lipids) has a concentration-related effect on the transfection of LV based on the functional LV titer. In this case, the concentrations of the pegylated lipids added are 0, 3 ng / pL, 5 ng / pL, or 7 ng / pL, and the functional LV titers are measured at 30 minutes and 3 hr after complex formation. In the absence of the pegylated lipids, the functional LV titers dropped from about 2.6* 10A8 TU / mL at 30 minutes to about 6* 10A7 TU / mL at 3 hrs. In contrast, when the pegylated lipids are added at 3 ng / pL concentration, the functional LV titers increase from about 1.2*10A8 TU / mL at 30 minutes to about 2.2*10A8 TU / mL at 3 hrs. However, when the concentration of the pegylated lipids increase to 5 ng / pL, the functional LV titers decrease when compared to those when the pegylated concentration is 3 ng / pL. Nevertheless, the functional LV titers stillAttorney Docket No.: P24-229-SEC-WO01increase from about 5.5><10A7 TU / mL at 30 minutes to about 8.5*10A7 TU / mL at 3 hrs.

[0374] A Variety of Pegylated Lipids Tested

[0375] A variety of pegylated lipids with different structures are evaluated as the stabilizing agent for transfection complexes. Two milliliters of Viral Production 2.0 cells (Thermo Fisher) grown in Viral Production Medium (Thermo Fisher) were seeded at 3 million cells / mL into untreated 6-well plates immediately prior to transfection. VirusGEN® or Stabilized VirusGEN transfection reagent was complexed with pDNA at a ratio of 1.5: 1 (vokmass), 2 pg / ml plasmid DNA (pALD-ITR-WPRE-GFP, Genemedi AAV8 Rep / Cap, pALD Helper; 1:1:1 mass ratios). AAV was harvested from cells and supernatant at 96 hours post-transfection using chemical lysis. Genome copies were determined by dPCR (Qiagen QIAcuity) using primers and a probe targeting the CMV promoter. Serotype-specific Lumit Kits (Promega) were used to determine the capsid titer. Error bars represent the standard deviation from duplicate wells.

[0376] The pegylated lipids tested were DMG-PEG 2000 (tested two batches: Rep 1 and Rep 2), DSPE PEG 2000, and ALC-0159. AAV8 genome titer (GC / mL) were measured at 30 minutes and 120 minutes. The results are shown in FIG. 8. In all cases, adding the pegylated lipids resulted in an increase of the AAV8 genome titer at all time points. In addition, at 120 minutes time point, the control (without the pegylated lipids) provides an AAV8 genome titer at about 8* 10Al 0 GC / mL, while the stabilized transfection complexes provide AAV8 genome titer from about 1.6><10All GC / mL to about 2.8* 10All GC / mL.

[0377] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the invention are not limited toAttorney Docket No.: P24-229-SEC-WO01the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0378] A Variety of Transfection Reagents Tested

[0379] The stabilizing effect with a variety of transfection reagents is evaluated. When the transfection complex is a 7 / z / / / .sIT transfection reagent (for instance, 7ra / / .sIT-VirusGEN® (Minis Bio LLC, Cat. No. MIR 6700) with AevIT™ AAV Enhancer (MIR 8000), 7 / Y / / 7.sIT-Lenti® (Minis Bio LLC, Cat. No. MIR 6600), 7ra / 7.sIT-X2® (Mims Bio LLC, Cat. No. MIR 6000), 7 / z / / / .sIT-PRO® (Mims Bio LLC, Cat. No. MIR 5700), or TransYT-2020® (Mims Bio LLC, Cat. No. MIR 5400)) with and without the stabilizing agent (pegylated lipid) for AAV9 transfection, the average AAV9 genome titer (GC / mL) of transfection complexes at various time points from 30 minutes to 180 minutes are shown in FIG. 9. The total volume of the transfection complex added to the reaction vessel is about 5% of the culture volume. Genome titers at 180 min were improved in the presence of the stabilizing agent compared to the same conditions at 180 min without stabilizing agent. In some cases the titers matched or improved on the optimal 30 min timepoints without stabilizer.

[0380] Fig. 9. Experimental Details: 2 mL of Viral Production 2.0 cells (Thermo Fisher Scientific) grown in Cellvento 4 HEK (Sigma Aldrich) supplemented with 6 mM L-Glutamine were seeded in untreated 6-well plates at 3 million cells / ml.Recombinant AAV9 / RPE65 was generated via transient transfection of a mixture of 3 plasmids (pMIR 736-RPE65 (Aldevron), pALD-HELP (Aldevron) and pAAV-RepCap (AAV9 Genemedi) at a 1:1:1 plasmid mass ratio) using TransIT-VirusGEN® (1.7:1 reagent: DNA ratio (vokmass); complexed in PBS + 1 pl / ml RevIT; 2 pg / ml pDNAdose), TransIT-Lenti® (2.2:1 reagent: DNA ratio (vokmass); complexed in Opti-MEM (ThermoFisher) 2 pg / ml pDNAdose), TransIT-X2 System® (2.2:1 reagent: DNA ratio (vokmass); complexed in Opti-MEM (Source) 2 pg / ml pDNAAttorney Docket No.: P24-229-SEC-WO01dose), TransIT-PRO® (1.1:1 reagent: DNA ratio (vol:mass); complexed in CHOgro (MirusBio) 2.1 pg / ml pDNAdose) and TransIT-2020® (2.2:1 reagent: DNA ratio (vol:mass); complexed in Opti-MEM (ThermoFisher) 2 pg / ml pDNAdose).Transfection reagents were tested with or without supplementation with VirusGEN Transfection Complex Stabilizer. 5% culture volume complexes were allowed to form for 30 minutes or 180 minutes before adding to cells. AAV was harvested at 72 hours post-transfection using chemical lysis. Genome titers were determined via dPCR using primers and a probe targeting the BREL promoter region. Total assembled capsids were determined using the Pan-AAV9-Lumit Assay (Promega) and the percentage of full capsids were determined by dividing the number of genome copies by total assembled capsids for each condition. The error bars represent the standard deviation of duplicate wells.

Claims

1. Attorney Docket No.: P24-229-SEC-WO012.We Claim:3.Claims:

1. A composition for delivery of a nucleic acid into a cell, comprising:5.(a) a cationic polymer;6.(b) an amphipathic compound; and7.(c) a pegylated lipid according to Formula (I),9. 10.X-LW311.Formula (I)12.or a salt or stereoisomer thereof, wherein:13.A is O, CH-L2-Y or N-L2-Y;14.B is H, -OH, -NH2, -OCH3, or -CH3;15.each LI and L2 is independently a linker;16.L3 is a PEG linker;17.each X and Y is independently alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl, wherein said alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted; and q is an integer from 3 to 125.

2. The composition of claim 1, wherein said pegylated lipid is according to Formula (la):

20. 22.Formula (la)23.or a salt or stereoisomer thereof, wherein:

25.

26. Attorney Docket No.: P24-229-SEC-WO0128.

29. LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently a bond, -CH?-, -(CH?)?-, or -(CH?)?-; and * denotes a connection leading to A.

3. The composition of claim 1, wherein said pegylated lipid is according to Formula (lb):31.X-L1 H L3, O32.B33.l!_235. 37.Formula (lb)38.or a salt or stereoisomer thereof, wherein: Attorney Docket No.: P24-229-SEC-WO0140.

41. LAis independently -CH2-, -(CH?)?-, -(CH?)?-, -(CH?)4-, or -(CH?)s-; LBis independently -CH?-, -(CH?)?-, or -(CH?)?-; and42.* denotes a connection leading to A.

4. A composition for delivery of a nucleic acid into a cell, comprising:44.(a) a cationic polymer;45.(b) an amphipathic compound; and46.(c) a pegylated lipid according to Formulas (Ila) or (lib),47.Formula (Ila)49.

50. Attorney Docket No.: P24-229-SEC-WO0152. 54.Formula (lib)55.or a salt or stereoisomer thereof, wherein:56.D is H, -OH, -NH2, -OCH3, or -CH3;57.Lcis a PEG linker;58.X is alkyl, alkenyl, or alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; and59.q is an integer from 3 to 125.

5. A composition for delivery of a nucleic acid into a cell, comprising:61.(a) a cationic polymer;62.(b) an amphipathic compound; and63.(c) a pegylated lipid according to Formulas (Illa) or (IHb),65. 67.or a salt or stereoisomer thereof, wherein:68.D is H, -OH, -NH2, -OCH3, or -CH3;69.E is H or C1-C4 alkyl;70.Lcis a PEG linker;71.each X and Y is independently alkyl, alkenyl, or alkynyl, wherein said alkyl, alkenyl, and alkynyl are optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; and Attorney Docket No.: P24-229-SEC-WO0172.q is an integer from 3 to 125.

6. The composition of any one of claims 1-5, wherein said cationic polymer comprises Formula (V);74.W^OHN-^O76.

77. R21R2278.Formula (V)79.wherein:80.each R1and R2is independently hydrogen or methyl;81.W is -NH- or -O-;82.R21comprises a structure of:83.RLRR85.

86. R2387.wherein:88.each RLand RRis independently -(CH2)r-;89.each r is independently 1, 2, 3, 4, 5, or 6;90.R23is H or CH3;91.R22is independently alkyl, alkenyl, or heteroalkyl, wherein said alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; and92.each m and n is independently an integer from 1 to 10.

7. The composition of any one of claims 1-5, wherein said cationic polymer comprises Formula (VI);94.R1R296. 98.Formula (VI)99.wherein: Attorney Docket No.: P24-229-SEC-WO01100.each R1and R2is independently hydrogen or methyl;101.W is -NH- or -O-;102.each R3and R4is independently alkyl, alkenyl, or heteroalkyl, wherein said alkyl, alkenyl, and heteroalkyl are optionally substituted with one or more groups independently selected from the group consisting of halide, amino, Ci- C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; and103.each m and n is independently an integer from 1 to 10.

8. The composition of any one of claims 1-7, wherein said amphipathic compound is according to Formula (VII);105.R13 / \ R14106.R1< " L4- _ N-L5" XR12108. 110.Formula (VII)111.wherein each R11and R12is independently alkyl or alkenyl, wherein each said alkyl and alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl;112.each R13and R14is independently hydrogen, alkyl, alkenyl, alkoxy, alkyl-C(O)-alkylene, alkenyl-C(O)-alkylene, alkyl-C(O)-alkenylene, alkenyl-C(O)-alkenylene, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-C(O)-, heterocycle-alkylene-C(O)-, heterocycle- alkenylene-C(O)-,113.alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alky 1-heterocy cl e-alkeny 1 ene-C (O)-,114.alkenyl-heterocycle-alkenylene-C(O)-, heteroaryl-C(O)-,115.heteroaryl-alkylene-C(O)-, heteroaryl-alkenylene-C(O)-,116.alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)-, alkyl-heteroaryl-alkenylene-C(O)-, or117.alkenyl-heteroaryl-alkenylene-C(O)-, wherein each said alkyl, alkenyl, alkylene, alkenylene, heterocycle, an heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halide, C1-C4 alkyl, and C1-C4 haloalkyl; Attorney Docket No.: P24-229-SEC-WO01118.each L4 and L5 is independently C2-C8 alkylene, optionally substituted with one or two groups independently selected from the group consisting of halide, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; and119.p is 0 or 1.

9. The composition of any one of claims 1-8, further comprising said nucleic acid.

10. A transfection reagent comprising a composition of any one of claims 1-8, and a nucleic acid.

11. A method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of claims 1-8 with said nucleic acid to produce a mixed solution, and contacting said cell with said mixed solution, thereby transfecting said nucleic acid into said cell.

12. The method of claim 11, wherein an average genome titer is no less than 1.5><10A10 genome copies per milliliter (GC / mL) at 30 minutes after said mixing.

13. The method of claims 11 or 12, wherein an average genome titer decreases no more than 50% at 3 hours after complex initiation.

14. A method of forming and stabilizing a transfection complex, comprising mixing a composition of any one of claims 1-8 with said nucleic acid, thereby producing a plurality of transfection complexes comprising said pegylated lipid, said cationic polymer, said amphipathic compound, and said nucleic acid.

15. The method of claim 14, wherein an average radius of said plurality of transfection complexes is no more than 1000 nm at 60 minutes after said mixing.

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