Transfection reagent comprising cationic polymer and amphipathic compound

Abstract

Cationic amine-containing polymers and copolymers as well as novel amphipathic compound have been designed and synthesized for efficient delivery of nucleic acids to cells in biological systems, specifically for in vitro cell transfection research.

Description

Attorney Docket No. P24-228-SEC-WO01TRANSFECTION REAGENT COMPRISING CATIONIC POLYMER AND AMPHIPATHIC COMPOUNDCross-Reference to Related Applications

[0001] The present application claims the benefit of priority of U.S. provisional patent application no. 63 / 704,015, filed on October 6, 2024, the content of which is hereby incorporated in its entirety.Field of the Invention

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

[0003] The control of living processes is mediated through nucleic acids. Nucleic acids encode proteins which, as enzymes, hormones and other regulatory factors, carry out the processes which enable living organisms to function. Nucleic acids also encode for regulatory sequences which control the expression of proteins.

[0004] Because of its central role in living organisms, nucleic acids make an ideal therapeutic target. It is thought that many diseases could be controlled by the manipulation of nucleic acids in living organisms.

[0005] 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 fragile molecules which are highly negatively charged (one negative charge per phosphate group) and which are readily cleaved by nucleases present both in extracellular fluids and intracellular compartments. As a highly charged molecule it will not cross the lipid membranes surrounding the cell, nor can it readily escape from endosomal compartments involved in the uptake of macromolecules into cells. Even RNAi molecules, although smaller in molecular weight, show significant problems of stability and uptake.

[0006] Cationic lipid formulations suffer from a number of shortcomings. The lipids used in these formulations are often toxic to cells, and their use as delivery vehicles for nucleic acid to cells can be limited by the toxicity of this component. Cationic lipid formulations are also unstable and have a relatively short shelf life. TheAttorney Docket No. P24-228-SEC-WO01 short shelf life is at least partly due to the tendency of these formulations to aggregate. Furthermore, lipid formulations are generally expensive.

[0007] The use of cationic polymers overcomes some, but not all, of the problems associated with amphipathic compound formulations. Polycationic polymers are, however, generally cytotoxic although some cationic polymers with lower toxicity have been reported. Cationic polymers are generally cheap to produce, and do not have the shelf life problems associated with amphipathic compounds.

[0008] Cationic polymers are very efficient at condensing nucleic acids into a small volume and at protecting nucleic acids from degradation by serum nucleases. Interaction is through an equilibrium reaction in which adjustment of the environmental conditions, (salt concentration, pH, molecular weight of each of the polymers) will affect the composition and form of the complexes.

[0009] In the formation of the toroids, the processes of condensation of nucleic acids and aggregation of particles are competing, so that these systems tend to be unstable with time and form larger aggregates. This is influenced by the charge ratio of the complexes, and can be reduced by using an excess of one of the components. Generally such complexes are, therefore, made with an excess of polymer, although similar complexes with an excess of nucleic acids also have some favorable properties.

[0010] The lack of efficiency of cationic polymer-nucleic acid delivery systems may relate to the efficiency with which they can be taken up into cells, and with which they can escape from the endosomal compartment of the cell, into the cytoplasm. For this reason there has been much research into incorporating ligands and other biologically-active molecules which recognize cell surface receptors involved in endocytosis, and into the use of molecules, such as amphipathic peptides, which can disrupt endosomal membranes.

[0011] Cationic polyamines such as polyethylenimine (PEI), poly(L-lysine), polyamidoamines, chitosan, poly(amino ester)s and polyacrylates have been widely investigated as nucleic acid delivery vehicles.

[0012] It is an object of the invention to overcome at least some of the above problems.Attorney Docket No. P24-228-SEC-WO01Summary

[0013] In one aspect, provided herein is a composition for delivery of a nucleic acid into a cell, comprising:(i) a polymer comprising Formula (I);Formula (I) wherein each R1and R2is independently hydrogen or methyl; each R3and R4is independently alkyl, alkenyl, or heteroalkyl, wherein the alkyl, alkenyl, and heteroalkyl are optionally substituted with atoms one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; each m and n is independently an integer from 1 to 10; and(ii) an amphipathic compound of Formula (II)Formula (II) wherein each R11and R12is independently alkyl or alkenyl, wherein each of the alkyl and alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, Ci- 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)- alkyl-heterocycle-alkenylene-C(O)- alkenyl-heterocycle-alkenylene-C(O)-, heteroaryl -C(O)- heteroary 1 -alkyl ene-C(O)-, heteroary 1 -alkenyl ene-C (O)-, alkyl-heteroaryl-alkylene-C(O)-Attorney Docket No. P24-228-SEC-WO01 alkenyl-heteroaryl-alkylene-C(O)- alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-, wherein each of 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 LI and L2 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; and p is 0 or 1.

[0014] In another aspect, provided herein is a transfection reagent comprising a composition of any one of those disclosed here, and a nucleic acid.

[0015] In still another aspect, provide 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.

[0016] 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

[0017] 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.Attorney Docket No. P24-228-SEC-WO01Brief Description of the Drawings

[0018] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention 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:

[0019] FIG. 1 shows examples of cationic polyacrylamide structures tested for transfection efficiency with and without an example amphipathic compound MCI 180.

[0020] FIG. 2 shows an example amphipathic compound MCI 180.

[0021] FIG. 3 shows transfection efficiency of various cationic polymers, cationic polymer-amphipathic compound mixes, and amphipathic compound complexes with DNA in HEK 293 VP 2.0 cells, as measured by luciferase.

[0022] FIG. 4 shows the transfection efficiency of various polymer:lipid;pLuc DNA ratios in HEK293 VP 2.0 cells, as measured by luciferase.DETAILED DESCRIPTION

[0023] The present disclosure pertains to the use of compositions comprising synthetic cationic copolymers and amphipathic compounds as nucleic acid transfection agents.

[0024] 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.

[0025] 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.Attorney Docket No. P24-228-SEC-WO01

[0026] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0027] 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.

[0028] 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.

[0029] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.

[0030] As used herein, the term “Ci-Ce alkyl” generally refers to a straight or branched hydrocarbon chain 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.

[0031] 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.Attorney Docket No. P24-228-SEC-WO01

[0032] 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.

[0033] As used herein, the term “alkylene” generally refers to an optionally substituted divalent straight or branched hydrocarbon chain having from 1 to 6 carbon atoms, and the straight or branched hydrocarbon chain is attached to the rest of the molecule by two single bonds. Examples include, but are not limited to methylene (methanediyl), ethylene (ethanediyl) or trimethylene (propane- 1,3 -diyl).

[0034] 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-m ethylbutoxy, 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.

[0035] 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.

[0036] As used herein, the term “alkenyl” generally refers to straight or branched chain alkene groups, which comprise at least one unsaturated carbon-carbon double bond and is attached to the rest of the molecule by a single bond. 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. The term “alkynyl” as used herein generally refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond and is attached to the rest of the molecule by a single 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.

[0037] As used herein, “alkenylene” means a straight or branched divalent unsaturated aliphatic hydrocarbon group (an alkenediyl) having from 2 to 6 carbonAttorney Docket No. P24-228-SEC-WO01 atoms and containing at least one carbon-carbon double bond, the group being attached to the remainder of the molecule by two single bonds. Examples include, but are not limited to ethenediyl (ethene- 1 ,2-diyl), propenediyl (propene- 1 ,2-diyl) or butenediyl (butene- 1,2-diyl).

[0038] 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-Ce haloalkyl” 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 .

[0039] As used herein, the term “heteroalkyl” generally refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, 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 optionally substituted 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.

[0040] The term “cycloalkyl” as used herein generally refer 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 aAttorney Docket No. P24-228-SEC-WO01 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- IH-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[2.1.1]hexan-2-ylidene; bicyclo[2.2.1]heptan-2-ylidene; cholesteryl; cholesteryl-derivatives and adamantyl.

[0041] 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. 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 statedAttorney Docket No. P24-228-SEC-WO01 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.

[0042] The term “heteroaryl” as used herein generally refers to an aromatic group in which at least one aromatic ring comprises at least one heteroatom selected from N, O and S. 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.

[0043] As used herein, the term “amino” generally refers to primary amino group —l / (-NH2), secondary amino group (-NH-), and tertiary amino group ( \).

[0044] 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.Attorney Docket No. P24-228-SEC-WO01

[0045] 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, haloalkyl 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.

[0046] The term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, volume, time, viral titer, and concentration. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about,” the claims include equivalents to the quantities.Recombinant Adeno- Associated Virus (AAV) Production

[0047] 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 can grow 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 or Viral Production Cells 2.0.

[0048] 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 cationicAttorney Docket No. P24-228-SEC-WO01 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.

[0049] 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 -di oleyloxy) propyl]- N,N,N-trimethylamonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE),1.2-bis(oleoyloxy)-3-(4'-trimethylammonio) propane (DOTAP), dihydroxyldimyristyl spermine tetrahydrochloride (DHDMS), hydroxyl -dimyristyl spermine 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-hydroxyethyl 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), N, N^N^N111-tetramethyl - N,NINH,NIII-tet-rapalmityl spermine (TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde (DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS (Tris(hydroxymethyl)- aminomethane, 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- 1 -propanamin-iniumtrinuoroacetate (DOSPA) and combinations thereof.

[0050] 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,Attorney Docket No. P24-228-SEC-WO01 the transfection reagent comprises at least one cationic lipid and at least one neutral lipid.

[0051] 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 Life Technologies Corporation, Carlsbad, Calif, under the trade name LIPOFECTAM1NE™, 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 Life Technologies Corporation, Carlsbad, Calif, under the trade name DMRIE-C reagent; and a 1 : 1.5 (M / M) combination of TM-TPS and DOPE is available from Life Tech. 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® HD Transfection Reagent, each of which is available from Promega Corporation; and TRANSFECTIN™ Lipid Reagent, available from BioRad Laboratories, Inc.

[0052] 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 polyornithine, a N- 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 certainAttorney Docket No. P24-228-SEC-WO01 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.

[0053] 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.

[0054] The present disclosure relates to compositions comprising a cationic polymer and an amphipathic compound. 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.

[0055] Disclosed herein is a composition for delivery of a nucleic acid into a cell, comprising:(i) a polymer comprising Formula (I);Formula (I) wherein each R1and R2is independently hydrogen or methyl; 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; each m and n is independently an integer from 1 to 10; and(ii) an amphipathic compound of Formula (II);Formula (II)Attorney Docket No. P24-228-SEC-WO01 wherein each R11and R12is independently alkyl or alkenyl, wherein each of 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 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)- heterocy cl e-alky 1 ene-C (O)-, heterocy cl e- alkenyl ene-C (O)-, alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkenylene-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)-, or alkenyl-heteroaryl-alkenylene-C(O)-, wherein each of 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 LI and L2 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; and p is 0 or 1.

[0056] Regarding Formula (I) in the composition: in certain embodiments, both R1and R2are hydrogen. In certain embodiments, R3is alkyl. In certain embodiments, R3is alkenyl. In certain embodiments, R3comprises 2 to 4 alkene bonds, either conjugated or not conjugated. In certain embodiments, R3is heteroalkyl comprising 1 to 8 heteroatoms. In certain embodiments, R3comprises one primary amine. In certain embodiments, R3comprises one or more secondary amines. In certain embodiments, R3comprises two or more secondary amines. In certain embodiments, R3 comprises one or more tertiary amine. In certain embodiments, R3does not comprise cyclic amine. In certain embodiments, R3comprises one or more branching atoms selected from carbon and nitrogen. In certain embodiments, R3does not have a branching atom. In certain embodiments, R3further comprises one or more oxygen atoms. In certain embodiments, R3further comprises one or more substitutions independently selected from F, methyl, and CF3. In certain embodiments, R3does not have a substitution. In certain embodiments, R3comprises 3 to 9 carbon atoms. In certainAttorney Docket No. P24-228-SEC-WO01 embodiments, R3comprises 10 to 16 carbon atoms. In certain embodiments, R3comprises more than 16 carbon atoms. In certain embodiments, R4is C2-C6 alkyl. In certain embodiments, R4is C3-C8 alkyl. In certain embodiments, R4is C4-C10 alkyl. In certain embodiments, R4is C2-C6 alkenyl. In certain embodiments, R4is C3-C8 alkenyl. In certain embodiments, R4is C4-C10 alkenyl. In certain embodiments, R4is substituted with 1-3 groups independently selected from F, methyl, ethyl, n-propyl, isopropyl, and CF3. In certain embodiments, R4does not have a substitution.

[0057] Regarding Formula (II) in the composition and for all the embodiments of Formula (I) disclosed here: in certain embodiments, LI and L2 are the same. In certain embodiments, LI and L2 are different. In certain embodiments, R11and R12are the same. In certain embodiments, R13and R14are the same. In certain embodiments, R13and R14are different. In certain embodiments, p is 0. In certain embodiments, p is 1. In certain embodiments, each LI and L2 is independently ethylene, n-propylene, 1 -methyl -ethylene, 2-methyl-ethylene, 1-methyl-propylene, 2- methyl-propylene, 3-methyl-propylene, n-butylene, 1 -methyl -butylene, 2-methyl- butylene, 3 -methyl -butylene, 4-methyl-butylene, 1 -ethyl -butylene, 2-ethyl-butylene, 3-ethyl-butylene, or 4 -ethyl -butylene. In certain embodiments, each LI and L2 independently comprises no more than 8 carbon atoms. In certain embodiments, each LI and L2 independently comprises no more than 6 carbon atoms. In certain embodiments, one of LI or L2 comprises a branching carbon atom. In certain embodiments, each LI and L2 comprises a branching carbon atom. In certain embodiments, neither LI nor L2 comprises a branching carbon atom. In certain embodiments, LI and L2 is substituted with one or two groups of F or CF3. In certain embodiments, one of LI or L2 does not have a substitution. In certain embodiments, neither LI nor L2 has a substitution. In some embodiments, each R11and R12is independently C10 to C25 alkyl. In certain embodiments, each R11and R12is independently C10 to C25 alkenyl. In certain embodiments, each R11and R12is independently substituted with 1-3 groups independently selected from F, methyl, ethyl, or CF3. In certain embodiments, each R11and R12independently comprises 1 to 4 alkene bonds. In certain embodiments, each R11and R12independently comprises 2 to 4 alkene bonds. In certain embodiments, any two alkene bonds of the 2 to 4 alkene bonds are not conjugated. In certain embodiments, at least two alkene bonds of the 2 to 4 alkene bonds are conjugated. In certain embodiments, each R11and R12comprises 10 to 15 carbon atoms. In certain embodiments, each R11and R12comprises 16 to 20Attorney Docket No. P24-228-SEC-WO01 carbon atoms. In certain embodiments, each R11and R12comprises 21 to 25 carbon atoms. In some embodiments, each R11and R12independently comprises:certain embodiments, both R13and R14are hydrogen. In certain 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)- heterocy cl e-alky 1 ene-C (O)-, heterocy cl e- alkenyl ene-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 certain embodiments, each R13and R14is independently C2 to C10 alkyl. In certain embodiments, each R13and R14is independently C2 to C10 alkenyl. In certain embodiments, each R13and R14is independently C2 to C10 alkoxyl. In certain embodiments, each R13and R14is independently C2 to C10 alkyl. In certain embodiments, each R13and R14is independently C2 to C10 alkenyl. In certain embodiments, each R13and R14is independently C2 to C10 alkoxy. In certain embodiments, R13or R14comprises a carbonyl group bonded to the branching nitrogen atom. In certain embodiments, both R13and R14comprises a carbonyl group bonded to the branching nitrogen atom. In certain embodiments, each R13and R14independently comprises a heterocycle or heteroaryl group. In certain embodiments, the heterocycle or heteroaryl group is at a terminal position of R13or R14. In certain embodiments, the heterocycle or heteroaryl group is inserted at a middle position of R13or R14. In certain embodiments, the heterocycle in R13or R14is azetidine, 1,3- diazetidine, pyrrolidine, piperidine, piperazine, or morpholine. In certain embodiments, the heteroaryl in R13or R14is pyrrole, furan, imidazole, pyrazole, oxazole, isoxazole, pyridine, or pyrazine. In certain embodiments, the heterocycle orAttorney Docket No. P24-228-SEC-WO01 heteroaryl in R13or R14is substituted with fluorine, methyl, ethyl, propyl, isopropyl, or CF3.

[0058] Regarding the composition, and for all the embodiments of Formula (I) and all the embodiments of Formula (II): in certain embodiments, number average molecular weight (Mn) of said polymer is from about from about 30,000 to about 35,000, 35,000 to about 70,000, from about 35,000 to about 40,000, from about 40,000 to about 4,5000, from about 45,000 to about 50,000, from about 55,000 to about 60,000, from about 60,000 to about 6,5000, from about 65,000 to about 70,000, or from about 70,000 to about 75,000. In certain embodiments, Mwis weight average molecular weight of the polymer and Mnis number average molecular weight, and Mw / Mn is from about 1.00 to about 1.10, from about 1.10 to about 1.20, from about 1.20 to about 1.30, from about 1.30 to about 1.40, or from about 1.40 to about 1.50. In certain embodiments, Mw / Mnis no more than 1.50. In certain embodiments, a molar ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.30 to about 0.53, from about 0.34 to about 0.49, or about 0.36 to about 0.47. In certain embodiments, the composition further comprises an alcohol and a buffer. In certain embodiments, the alcohol is ethanol. In certain embodiments, the alcohol is 80% ethanol. In certain embodiments, the alcohol is isopropanol. In certain embodiments, the buffer is glycine-HCl.

[0059] In certain embodiments, disclosed herein a transfection reagent comprising a composition of any embodiments disclosed herein. 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.

[0060] 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 theAttorney Docket No. P24-228-SEC-WO01 required polycations. The acidic conditions can be trifluoroacetic acid in water or dilute hydrochloric acid.

[0061] 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.

[0062] The present disclosure provides a composition comprising a polymer and an amphipathic compound to assist a nucleic acid transfer into cells via a complex comprising the nucleic acid and the composition.

[0063] In certain embodiments, compositions comprising nucleic acids, cationic amine-containing polymers and copolymers, and amphipathic compounds, 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.

[0064] 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 and amphipathic compounds significantly increased nucleic acid transfer efficiency. The nucleic acid then alters expression of a selected endogenous nucleic acid.

[0065] In certain embodiments, the composition comprising cationic amine- containing polymers and copolymers and amphipathic compounds is used to assist transfection of DNA, RNA, mRNA or RNAi into a cell. The nucleic acid then alters the cell's natural process.

[0066] 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 ofAttorney Docket No. P24-228-SEC-WO01 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.

[0067] 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 the components 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 cationic polyacrylamides described in this specification provide a mechanism to transfect siRNA and other nucleic acids into cells.

[0068] 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.

[0069] 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 typesAttorney Docket No. P24-228-SEC-WO01 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.

[0070] Types of Monomers: A wide variety of monomers can be used in the polymerization processes. These include positive charged organic monomers such as amines, 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

[0071] 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.

[0072] 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.

[0073] Biochemical reactions: Biological, chemical, or biochemical reactions involve the formation or cleavage of ionic and / or covalent bonds.Attorney Docket No. P24-228-SEC-WO01

[0074] 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.

[0075] 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.

[0076] Sterics: Steric hindrance, or sterics, is the prevention or retardation of a chemical reaction because of neighboring groups on the same molecule.Embodiments

[0077] Embodiment 1. A composition for delivery of a nucleic acid into a cell, comprising:(i) a polymer comprising Formula (I);Formula (I) 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 atoms one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy; each m and n is independently an integer from 1 to 10; and(ii) an amphipathic compound of Formula (II)Formula (II) wherein each R11and R12is independently alkyl or alkenyl, wherein each of the alkyl and alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, Ci- C4 alkyl, and C1-C4 haloalkyl;Attorney Docket No. P24-228-SEC-WO01 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)- alkyl-heterocycle-alkenylene-C(O)- alkenyl-heterocycle-alkenylene-C(O)-, heteroaryl -C(O)- heteroary 1 -alkyl ene-C(O)-, heteroary 1 -alkenyl ene-C (O)-, alkyl-heteroaryl-alkylene-C(O)- alkenyl-heteroaryl-alkylene-C(O)-, alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-, wherein each of 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 LI and L2 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; and p is 0 or 1.

[0078] Embodiment 2. The composition of Embodiment 1, wherein R3is alkyl optionally substituted with 1-3 groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl.

[0079] Embodiment 3. The composition of Embodiment 2, wherein R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n- propyl, isopropyl, and CF3.

[0080] Embodiment 4. The composition of Embodiment 2, wherein R3comprises 3 to 9 carbon atoms.

[0081] Embodiment 5. The composition of Embodiment 2, wherein R3comprises 10 to 16 carbon atoms.Attorney Docket No. P24-228-SEC-WO01

[0082] Embodiment 6. The composition of Embodiment 1, 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.

[0083] Embodiment 7. The composition of Embodiment 2, wherein R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n- propyl, isopropyl, and CF3.

[0084] Embodiment 8. The composition of Embodiment 6, wherein R3comprises 3 to 9 carbon atoms.

[0085] Embodiment 9. The composition of Embodiment 6, wherein R3comprises 10 to 16 carbon atoms.

[0086] Embodiment 10. The composition of Embodiment 6, wherein R3comprises 2 to 4 alkene bonds.

[0087] Embodiment 11. The composition of Embodiment 10, wherein at least two of the 2 to 4 alkene bonds are conjugated.

[0088] Embodiment 12. The composition of Embodiment 10, wherein at least two of the 2 to 4 alkene bonds are not conjugated.

[0089] Embodiment 13. The composition of Embodiment 1, wherein 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.

[0090] Embodiment 14. The composition of Embodiment 13, wherein the heteroalkyl group is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0091] Embodiment 15. The composition of Embodiment 13, wherein the heteroalkyl group comprises 2 to 4 nitrogen atoms.

[0092] Embodiment 16. The composition of Embodiment 13, wherein the heteroalkyl group comprises 3 to 6 nitrogen atoms.

[0093] Embodiment 17. The composition of Embodiment 13, wherein the heteroalkyl group comprises 4 to 8 nitrogen atoms.

[0094] Embodiment 18. The composition of Embodiment 1, wherein 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.Attorney Docket No. P24-228-SEC-WO01

[0095] Embodiment 19. The composition of Embodiment 18, wherein the heteroalkyl group is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0096] Embodiment 20. The composition of Embodiment 18, wherein the heteroalkyl group comprises 1 to 3 nitrogen atoms.

[0097] Embodiment 21. The composition of Embodiment 18, wherein the heteroalkyl group comprises 3 to 5 nitrogen atoms.

[0098] Embodiment 22. The composition of Embodiment 18, wherein the heteroalkyl group comprises 5 to 8 nitrogen atoms.

[0099] Embodiment 23. The composition of any one of Embodiments 1-22, wherein R3comprises one primary amine and one or more secondary or tertiary amine.

[0100] Embodiment 24. The composition of Embodiment 23, wherein R3comprises 1 secondary amine.

[0101] Embodiment 25. The composition of Embodiment 23, wherein R3comprises 2 secondary amine.

[0102] Embodiment 26. The composition of Embodiment 23, wherein R3comprises 3 secondary amine.

[0103] Embodiment 27. The composition of Embodiment 23, wherein R3comprises 4 secondary amine.

[0104] Embodiment 28. The composition of Embodiment 23, wherein R3comprises 5 secondary amine.

[0105] Embodiment 29. The composition of any one of Embodiments 23-28, wherein R3further comprises another primary amine.

[0106] Embodiment 30. The composition of any one of Embodiments 23-29, wherein R3further comprises a tertiary amine.

[0107] Embodiment 31. The composition of any one of Embodiments 23-29, wherein R3further comprises two tertiary amine.

[0108] Embodiment 32. The composition of any one of Embodiments 2-22, wherein R4is C2-C6 alkyl.

[0109] Embodiment 33. The composition of any one of Embodiments 2-22, wherein R4is C3-C8 alkyl.

[0110] Embodiment 34. The composition of any one of Embodiments 2-22, wherein R4is C4-C10 alkyl.Attorney Docket No. P24-228-SEC-WO01[OHl] Embodiment 35. The composition of any one of Embodiments 2-22, wherein R4is C3-C6 alkenyl.

[0112] Embodiment 36. The composition of any one of Embodiments 2-22, wherein R4is C3-C8 alkenyl.

[0113] Embodiment 37. The composition of any one of Embodiments 2-22, wherein R4is C4-C10 alkenyl.

[0114] Embodiment 38. The composition of any one of Embodiments 23-31, wherein R4is C2-C6 alkyl.

[0115] Embodiment 39. The composition of any one of Embodiments 23-31, wherein R4is C3-C8 alkyl.

[0116] Embodiment 40. The composition of any one of Embodiments 23-31, wherein R4is C4-C10 alkyl.

[0117] Embodiment 41. The composition of any one of Embodiments 23-31, wherein R4is C3-C6 alkenyl.

[0118] Embodiment 42. The composition of any one of Embodiments 23-31, wherein R4is C3-C8 alkenyl.

[0119] Embodiment 43. The composition of any one of Embodiments 23-31, wherein R4is C4-C10 alkenyl.

[0120] Embodiment 44. The composition of any one of Embodiments 32-43, wherein R4is substituted with 1-3 groups independently selected from F, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0121] Embodiment 45. The composition of any one of Embodiments 1, 2, 4-6, 8-13, 15-18, or 20- Embodiment 44, wherein R3is substituted with 1-3 groups independently selected from F, amino, methyl, ethyl, n-propyl, isopropyl, and CF3.

[0122] Embodiment 46. The composition of any one of Embodiments 1-45, wherein R1and R2are hydrogen.

[0123] Embodiment 47. The composition of any one of Embodiments 1-45, wherein R1and R2are methyl.

[0124] Embodiment 48. The composition of any one of Embodiments 1-47, wherein each LI and L2 is independently ethylene, n-propylene, 1 -methyl -ethylene, 2-methyl-ethylene, 1-methyl-propylene, 2-methyl-propylene, 3-methyl-propylene, n- butylene, 1 -methyl -butylene, 2-methyl-butylene, 3-methyl-butylene, 4-methyl- butylene, 1 -ethyl -butylene, 2-ethyl-butylene, 3-ethyl-butylene, or 4-ethyl-butylene.Attorney Docket No. P24-228-SEC-WO01

[0125] Embodiment 49. The composition of Embodiment 48, wherein LI andL2 are different.

[0126] Embodiment 50. The composition of Embodiment 48, wherein LI andL2 are the same.

[0127] Embodiment 51. The composition of any one of Embodiments 1-50, wherein LI and L2 is substituted with one or two groups of F or CF3.

[0128] Embodiment 52. The composition of any one of Embodiments 1-51, wherein each R11and R12is independently C10 to C25 alkyl.

[0129] Embodiment 53. The composition of any one of Embodiments 1-51, wherein each R11and R12is independently C10 to C25 alkenyl.

[0130] Embodiment 54. The composition of any one of Embodiment 52 or 53, wherein each R11and R12is independently substituted with F, methyl, ethyl, or CF3.

[0131] Embodiment 55. The composition of Embodiments 53 or 54, wherein each R11and R12independently comprises 1 to 4 alkene bonds.

[0132] Embodiment 56. The composition of Embodiment 55, wherein each R11and R12independently comprises 2 to 4 alkene bonds.

[0133] Embodiment 57. The composition of Embodiment 56, wherein, for each R11and R12, at least two of the 2 to 4 alkene bonds are conjugated.

[0134] Embodiment 58. The composition of Embodiment 56, wherein for each R11and R12, at least two of the 2 to 4 alkene bonds are not conjugated.

[0135] Embodiment 59. The composition of any one of Embodiments 55-58 wherein at least one of the 1 to 4 alkene bonds is cis.

[0136] Embodiment 60. The composition of any one of Embodiments 55-59, wherein at least one of the 1 to 4 alkene bonds is trans.

[0137] Embodiment 61. The composition of any one of Embodiments 56-60, wherein two of the 2 to 4 alkene bonds are cis.

[0138] Embodiment 62. The composition of any one of Embodiments 56-61, wherein two of the 2 to 4 alkene bonds are trans.

[0139] Embodiment 63. The composition of any one of Embodiments 52-62, wherein each R11and R12comprise 10 to 15 carbon atoms.

[0140] Embodiment 64. The composition of any one of Embodiments 52-62, wherein each R11and Embodiment R12comprise 16 to 20 carbon atoms.

[0141] Embodiment 65. The composition of any one of Embodiments 52-62, wherein each R11and R12comprise 21 to 25 carbon atoms.Attorney Docket No. P24-228-SEC-WO01

[0142] Embodiment 66. The composition of any one of Embodiments 53-65, wherein each R11and R12independently comprises:

[0143] Embodiment 67. The composition of any one of Embodiments 52-66, wherein R11and R12are the same.

[0144] Embodiment 68. The composition of any one of Embodiments 52-66, wherein R11and R12are different.

[0145] Embodiment 69. The composition of any one of Embodiments 1-68, wherein both R13and R14are hydrogen.

[0146] Embodiment 70. The composition of any one of Embodiments 1-68, 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)- heterocy cl e-alky 1 ene-C (O)-, heterocy cl e- alkenyl ene-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)-.

[0147] Embodiment 71. The composition of any one of Embodiments 1-68, wherein each R13and R14is independently C2 to C10 alkyl.

[0148] Embodiment 72. The composition of any one of Embodiments 1-68, wherein each R13and R14is independently C2 to C10 alkenyl.

[0149] Embodiment 73. The composition of any one of Embodiments 1-68, wherein each R13and R14is independently C2 to C10 alkoxy.

[0150] Embodiment 74. The composition of any one of Embodiments 1-68, 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)-.Attorney Docket No. P24-228-SEC-WO01

[0151] Embodiment 75. The composition of any one of Embodiments 1-68, wherein each R13and R14is independently heterocycle-C(O)- heterocy cl e-alky 1 ene-C (O)-, heterocy cl e- alkenyl ene-C (O)-, alkyl-heterocycle-alkylene-C(O)-, alkenyl-heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkenylene-C(O)-, or alkenyl-heterocycle-alkenylene-C(O)-.

[0152] Embodiment 76. The composition of any one of Embodiments 1-68, wherein each R13and R14is independently heteroaryl-C(O)-, heteroaryl -alkyl ene-C(O)-, heteroary 1 -alkenyl ene-C (O)-, alkyl-heteroaryl-alkylene-C(O)-, alkenyl-heteroaryl-alkylene-C(O)- alkyl-heteroaryl-alkenylene-C(O)-, or alkenyl-heteroaryl-alkenylene-C(O)-.

[0153] Embodiment 77. The composition of any one of Embodiments 1-76, wherein p is 0.

[0154] Embodiment 78. The composition of any one of Embodiments 1-76, wherein p is 1.

[0155] Embodiment 79. The composition of any one of Embodiments 1-68, 70, 75, 77, or 78, wherein the heterocycle is azetidine, 1,3-diazetidine, pyrrolidine, piperidine, piperazine, or morpholine.

[0156] Embodiment 80. The composition of Embodiment 79, 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.

[0157] Embodiment 81. The composition of any one of Embodiments 1-68, 70, or 76-78, wherein the heteroaryl is pyrrole, furan, imidazole, pyrazole, oxazole, isoxazole, pyridine, or pyrazine.

[0158] Embodiment 82. The composition of Embodiment 79, wherein each R13and R14is independently heteroaryl- C1-C4 alkylene-C(O)-, and wherein the heteroaryl is optionally substituted with fluorine, methyl, ethyl, propyl, isopropyl, or CF3.

[0159] Embodiment 83. The composition of any one of Embodiments 1-68 or 71-82, wherein R13and R14are different.

[0160] Embodiment 84. The composition of any one of Embodiments 1-68 or 71-82, wherein R13and R14are the same.Attorney Docket No. P24-228-SEC-WO01

[0161] Embodiment 85. The composition of any one of Embodiments 1-84, wherein the polymer is a block copolymer, an alternating copolymer, a random or statistical copolymer, or a gradient copolymer.

[0162] Embodiment 86. The composition of any one of Embodiments 1-84, wherein the 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.

[0163] Embodiment 87. The composition of any one of Embodiments 1-86, wherein number average molecular weight (Mn) of the polymer is from about 35,000 to about 70,000.

[0164] Embodiment 88. The composition of Embodiment 87, wherein Mnis from about 35,000 to about 40,000.

[0165] Embodiment 89. The composition of Embodiment 87, wherein Mnis from about 40,000 to about 45,000.

[0166] Embodiment 90. The composition of Embodiment 87, wherein Mnis from about 45,000 to about 50,000.

[0167] Embodiment 91. The composition of Embodiment 87, wherein Mnis from about 50,000 to about 55,000.

[0168] Embodiment 92. The composition of Embodiment 87, wherein Mnis from about 55,000 to about 60,000.

[0169] Embodiment 93. The composition of Embodiment 87, wherein Mnis from about 60,000 to about 65,000.

[0170] Embodiment 94. The composition of Embodiment 87, wherein Mnis from about 65,000 to about 70,000.

[0171] Embodiment 95. The composition of any one of Embodiments 1-94, wherein Mw / Mn is from about 1.00 to about 1.10, and wherein Mw is weight average molecular weight.

[0172] Embodiment 96. The composition of any one of Embodiments 1-94, wherein Mw / Mn is from about 1.10 to about 1.20, and wherein Mw is weight average molecular weight.

[0173] Embodiment 97. The composition of any one of Embodiments 1-94, wherein Mw / Mn is from about 1.20 to about 1.30, and wherein Mw is weight average molecular weight.Attorney Docket No. P24-228-SEC-WO01

[0174] Embodiment 98. The composition of any one of Embodiments 1-94, wherein Mw / Mn is from about 1.30 to about 1.40, and wherein Mw is weight average molecular weight.

[0175] Embodiment 99. The composition of any one of Embodiments 1-94, wherein Mw / Mn is from about 1.40 to about 1.50, and wherein Mw is weight average molecular weight.

[0176] Embodiment 100. The composition of any one of Embodiments 1-99, wherein a molar ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.30 to about 0.53.

[0177] Embodiment 101. The composition of any one of Embodiments 1-99, wherein a ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.34 to about 0.49.

[0178] Embodiment 102. The composition of any one of Embodiments 1-99, wherein a ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.36 to about 0.47.

[0179] Embodiment 103. The composition of any one of Embodiments 1-102, wherein W is -NH-.

[0180] Embodiment 104. The composition of any one of Embodiments 1-102, wherein W is -O-.

[0181] Embodiment 105. The composition of any one of Embodiments 1-104, further comprising an alcohol and a buffer.

[0182] Embodiment 106. The composition of Embodiment 105, wherein the alcohol is ethanol.

[0183] Embodiment 107. The composition of Embodiment 105 or Embodiment 106, wherein the buffer is glycine-HCl.

[0184] Embodiment 108. The composition of any one of Embodiments 1-107, further comprising the nucleic acid.

[0185] Embodiment 109. The composition of any one of Embodiments 1-107, wherein R3 and R4 are different.

[0186] Embodiment 110. The composition of any one of Embodiments 1-107, wherein R3is an alkyl or heteroalkyl group, wherein said alkyl or heteroalkyl group is optionally substituted with 1-3 groups independently selected from the group consisting of amino and C1-C4 alkyl.Attorney Docket No. P24-228-SEC-WO01

[0187] Embodiment 111. The composition of any one of Embodiments 1-107, wherein R3is a heteroalkyl group, wherein said heteroalkyl group is optionally substituted with an amino group.

[0188] Embodiment 112. The composition of any one of Embodiments 1-107, wherein R3is an alkyl group, wherein said alkyl group is optionally substituted with an amino group.

[0189] Embodiment 113. The composition of any one of Embodiments 1-107, further comprising DNA, RNA, or any combination thereof.

[0190] Embodiment 114. A transfection reagent comprising a composition of any one of Embodiments 1-112, and a nucleic acid.

[0191] Embodiment 115. The transfection reagent of Embodiment 114, wherein the nucleic acid is one or more adeno-associated virus (AAV) plasmids.

[0192] Embodiment 116. A method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of Embodiments 1-112 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.

[0193] Embodiment 117. The method of Embodiment 116, wherein the nucleic acid is one or more adeno-associated virus (AAV) plasmids.

[0194] Embodiment 118. A composition for delivery of a nucleic acid into a cell, comprising:(i) a polymer comprising Formula (I);Formula (I) 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; each m and n is independently an integer from 1 to 10; and(ii) an amphipathic compound of Formula (II);Attorney Docket No. P24-228-SEC-WO01Formula (II) 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, oxo, 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)-, heteroalkyl-C(O)-, 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)-, 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)-, or alkenyl-heteroaryl-alkenylene-C(O)-, cycloalkyl-O-C(O)-, wherein each said alkyl, heteroalkyl, alkenyl, alkylene, alkenylene, cycloalkyl, heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl. each LI and L2 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; and p is 0 or 1.

[0195] Embodiment 119. The composition of Embodiment 118, wherein R3is an alkyl or heteroalkyl group, wherein said alkyl or heteroalkyl group is optionally substituted with 1-3 groups independently selected from the group consisting of amino and C1-C4 alkyl.

[0196] Embodiment 120. The composition of Embodiments 118 and 119, wherein R3is an alkyl group, wherein said alkyl group is optionally substituted with an amino group.Attorney Docket No. P24-228-SEC-WO01

[0197] Embodiment 121. The composition of any one of Embodiment 118 to 120, wherein R3is an Ci-Ce alkyl group, wherein said Ci-Ce alkyl group is optionally substituted with an amino group.

[0198]

[0199] Embodiment 122. The composition of Embodiments 118 and 119, wherein R3is an heteroalkyl group, wherein said heteroalkyl group is optionally substituted with an amino group.

[0200] Embodiment 123. The composition of Embodiments 118, 120 or 122, wherein R3is an Ci-Ce heteroalkyl group, wherein said Ci-Ce heteroalkyl group is optionally substituted with an amino group.

[0201] Embodiment 124. The composition of Embodiment 118, 120, 122 or 123 wherein the heteroalkyl group comprises of 1 or 2 nitrogen atoms.

[0202] Embodiment 125. The composition of any one of Embodiment 118 to 124, wherein R4is C2-C6 alkyl, wherein said C2-C6 alkyl is optionally substituted with an amino group.

[0203] Embodiment 126. The composition of any one of Embodiment 118 to 125, wherein R1and R2are hydrogen.

[0204] Embodiment 127. The composition of any one of Embodiment 118 to 126, wherein W is -NH-.

[0205] Embodiment 128. The composition of any one of Embodiment 118 to 127, wherein each LI and L2 is independently C2-C4 alkylene.

[0206] Embodiment 129. The composition of any one of Embodiment 118 to 125, wherein LI and L2 are the same.

[0207] Embodiment 130. The composition of any one of Embodiment 118 to 129, wherein each R11and R12is independently C10 to C25 alkenyl and comprises 1 to 4 alkene bonds wherein each said alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, oxo, C1-C4 alkyl, and C1-C4 haloalkyl, preferably oxo.

[0208] Embodiment 131. The composition of any one of Embodiment 118 to 130, wherein each R11and R12is independently Cis alkylene and comprises 2 alkene bonds.

[0209] Embodiment 132. The composition of any one of Embodiment 118 to 131, wherein R11and R12are the same.Attorney Docket No. P24-228-SEC-WO01

[0210] Embodiment 133. The composition of any one of Embodiment 118 to 132, wherein each R13and R14is independently hydrogen, C2 to C10 alkyl, C2 to C10 alkenyl, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-alkylene-C(O)- alkyl-heterocycle-alkylene-C(O)-, heteroaryl-alkylene-C(O)-, or alkyl-heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkenyl, alkylene, heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl.

[0211] Embodiment 134. The composition of any one of Embodiment 118 to 133, wherein each R13and R14is independently hydrogen, C2 to C10 alkyl, alkyl-C(O)- or heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkylene and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl.

[0212] Embodiment 135. The composition of any one of Embodiment 118 to 134, wherein each R11and R12is independently C10 to C25 alkenyl and comprises 1 to 4 alkene bonds, wherein each said alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, oxo, C1-C4 alkyl, and C1-C4 haloalkyl and R13and R14is independently hydrogen, C2 to C10 alkyl, C2 to C10 alkenyl, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkylene-C(O)-, heteroaryl-alkylene-C(O)-, or alkyl-heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkenyl, alkylene, heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl.

[0213] Embodiment 136. The composition of any one of Embodiment 118 to 135, wherein each R11and R12is independently Cis alkylene and comprises 2 alkene bonds and R13and R14is independently hydrogen, C2 to C10 alkyl, alkyl-C(O)- or heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkylene and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl.

[0214] Embodiment 137. The composition of any one of Embodiment 118 to 136, wherein each R11and R12is Cis alkylene and comprises 2 alkene bonds and R13and R14is hydrogen, C2 to C10 alkyl, alkyl-C(O)- or heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkylene and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl.Attorney Docket No. P24-228-SEC-WO01

[0215] Embodiment 138. The composition of any one of Embodiment 118 to 137, wherein p is 1.

[0216] Embodiment 139. The composition of any one of Embodiment 118 to 138, wherein p is 0.

[0217] Embodiment 140. The composition of any one of Embodiment 118 to 139, wherein R13and R14are the same.

[0218] Embodiment 141. The composition of any one of Embodiment 118 to 140, comprising:(i) a polymer comprising Formula (I);Formula (I) wherein each R1and R2are hydrogen;W is -NH-;R3is an Ci-Ce heteroalkyl group, wherein said Ci-Ce heteroalkyl group is optionally substituted with an amino group;R4is C2-C6 alkyl, wherein said C2-C6 alkyl is optionally substituted with an amino group; and(ii) an amphipathic compound of Formula (II);Formula (II) wherein each R11and R12is independently C10 to C25 alkenyl and comprises 1 to 4 alkene bonds, wherein each said alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, oxo, C1-C4 alkyl, and C1-C4 haloalkyl; each R13and R14is independently hydrogen, C2 to C10 alkyl, C2 to C10 alkenyl, alkyl-C(O)-, alkenyl -C(O)-, heterocycle-alkylene-C(O)- alkyl-heterocycle-alkylene-C(O)-, heteroaryl-alkylene-C(O)-, or alkyl-heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkenyl, alkylene,Attorney Docket No. P24-228-SEC-WO01 heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl; each LI and L2 is independently C2-C4 alkylene; and p is 0.

[0219] Embodiment 142. The composition of any one of Embodiment 118 to 141, comprising:(i) a polymer comprising Formula (I);Formula (I) wherein each R1and R2are hydrogen;W is -NH-;R3is an Ci-Ce heteroalkyl group, wherein said Ci-Ce heteroalkyl group is optionally substituted with an amino group;R4is C2-C6 alkyl, wherein said C2-C6 alkyl is optionally substituted with an amino group; and(ii) an amphipathic compound of Formula (II);Formula (II) wherein R11and R12is C10 to C25 alkenyl and comprises 1 to 4 alkene bonds, wherein each said alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, oxo, C1-C4 alkyl, and Ci- C4 haloalkyl;R13and R14is hydrogen, C2 to C10 alkyl, C2 to C10 alkenyl, alkyl-C(O)-, alkenyl-C(O)-, heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkylene-C(O)- heteroaryl-alkylene-C(O)-, or alkyl-heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkenyl, alkylene, heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl;LI and L2 is C2-C4 alkylene; andAttorney Docket No. P24-228-SEC-WO01 p is 0.

[0220] Embodiment 143. The composition of any one of Embodiment 118 to 142, comprising:(i) a polymer comprising Formula (I);Formula (I) wherein each R1and R2are hydrogen;W is -NH-;R3is an Ci-Ce heteroalkyl group, wherein said Ci-Ce heteroalkyl group is optionally substituted with an amino group;R4is C2-C6 alkyl, wherein said C2-C6 alkyl is optionally substituted with an amino group; and(ii) an amphipathic compound of Formula (II);Formula (II) wherein R11and R12is Cis alkylene and comprises 2 alkene bonds;R13and R14is hydrogen, C2 to C10 alkyl, alkyl-C(O)- or heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkylene and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl;LI and L2 is C2-C4 alkylene; and p is 0.

[0221] Embodiment 143. The composition of any one of Embodiments 118-142, wherein the polymer is a block copolymer, an alternating copolymer, a random or statistical copolymer, or a gradient copolymer.

[0222] Embodiment 144. The composition of any one of Embodiments 118-143, wherein the polymer is a block copolymer or a statistical copolymer.

[0223] Embodiment 145. The composition of any one of Embodiments 118-144, wherein the polymer has a linear structure, a graft structure, a branched structure, aAttorney Docket No. P24-228-SEC-WO01 crosslinked or network structure, a star structure, a comb structure, a ladder structure, or a dendritic structure.

[0224] Embodiment 146. The composition of any one of Embodiments 118-145, wherein number average molecular weight (Mn) of the polymer is from about 35,000 to about 70,000.

[0225] Embodiment 147. The composition of Embodiment 146, wherein Mnis from about 35,000 to about 40,000.

[0226] Embodiment 148. The composition of Embodiment 146, wherein Mnis from about 40,000 to about 45,000.

[0227] Embodiment 149. The composition of Embodiment 146, wherein Mnis from about 45,000 to about 50,000.

[0228] Embodiment 150. The composition of Embodiment 146, wherein Mnis from about 50,000 to about 55,000.

[0229] Embodiment 151. The composition of Embodiment 146, wherein Mnis from about 55,000 to about 60,000.

[0230] Embodiment 152. The composition of Embodiment 146, wherein Mnis from about 60,000 to about 65,000.

[0231] Embodiment 153. The composition of Embodiment 146, wherein Mnis from about 65,000 to about 70,000.

[0232] Embodiment 154. The composition of any one of Embodiments 118-153, wherein Mw / Mn is from about 1.00 to about 1.10, and wherein Mw is weight average molecular weight.

[0233] Embodiment 155. The composition of any one of Embodiments 118-153, wherein Mw / Mn is from about 1.10 to about 1.20, and wherein Mw is weight average molecular weight.

[0234] Embodiment 156. The composition of any one of Embodiments 118-153, wherein Mw / Mn is from about 1.20 to about 1.30, and wherein Mw is weight average molecular weight.

[0235] Embodiment 157. The composition of any one of Embodiments 118-153, wherein Mw / Mn is from about 1.30 to about 1.40, and wherein Mw is weight average molecular weight.

[0236] Embodiment 158. The composition of any one of Embodiments 118-153, wherein Mw / Mn is from about 1.40 to about 1.50, and wherein Mw is weight average molecular weight.Attorney Docket No. P24-228-SEC-WO01

[0237] Embodiment 159. The composition of any one of Embodiments 118-158, wherein a molar ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.30 to about 0.53.

[0238] Embodiment 160. The composition of any one of Embodiments 118-158, wherein a ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.34 to about 0.49.

[0239] Embodiment 161. The composition of any one of Embodiments 118-158, wherein a ratio of monomeric units comprising R3to monomeric units comprising R4in the polymer is from about 0.36 to about 0.47.

[0240] Embodiment 162. A transfection reagent comprising a composition of any one of Embodiment 118 to 161, and a nucleic acid.

[0241] Embodiment 163. A method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of Embodiment 118 to 161 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.Examples

[0242] 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 acrylamide or alkyl (meth)acrylamide units. Other combinations of the acrylamide or (meth)acrylamide with different side chains are possible.

[0243] 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 byJH NMR. These monomers then undergo polymerization and copolymerization. Once the (co)polymers are purified by precipitation (e.g., into hexane), they areAttorney Docket No. P24-228-SEC-WO01 analyzed by gel permeation chromatography (organic solvent phase) andJH 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 by H NMR or other analytical methods.

[0244] 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 moiety in the presence of a base. See Scheme 1.

[0245] Scheme 1 : an example general synthetic route leading to monomers of the co-polymerH2N— R3diisopropylamineorH2N— R4dichloromethanexis H or methyl R

[0246] Example polymer synthesis: The monomers described were polymerized using RAFT in order to synthesize polymers of well-defined molecular weights, compositions, and architectures. The following general procedure is an example.

[0247] 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. The syringes 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.

[0248] 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.Attorney Docket No. P24-228-SEC-WO01

[0249] 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.

[0250] 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 2. A bisamine is acylated with an appropriate acyl chloride under basic conditions to provide a bisamide, which is subsequently reduced by LiAlEU to provide the bis-secondary amine (amphipathic compound 1). A second acylation affords another bisamide (amphipathic compound 2), which can be reduced again by LiAUU to provide a bis-tertiary amine (amphipathic compound 3).

[0251] Scheme 2 : General synthetic scheme leading to an amphipathic compound.amphipathic compound 2 compound 1 p is 0 or 1amphipathic compound 3

[0252] To a cooled solution of bisamine (1 eq) and EtsN (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 onceAttorney Docket No. P24-228-SEC-WO01 with saturated NaCl, and dried with MgSC or Na2SO4. The solvent is removed using a rotary evaporator. The bisamide is purified by recrystallization or by column chromatography.

[0253] To a stirred suspension of lithium aluminum hydride (LAH, 3eq) 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 CHCI3 to at least double the volume, washed twice with water, washed once with saturated NaCl, and dried with MgSCU or Na2SO4. The solvents are removed using a rotary evaporator. The amine is purified by column chromatography.

[0254] The second round of acylation and reduction follow similar procedures to attach another side chain to the non-cyclic amine.

[0255] Example. Transfection efficiency of example compositions relative to a commercially available reagent (PEIpro).

[0256] In this case, transfection efficiency of various polymers, lipid-polymer mixes, and lipid complexes with DNA in HEK 293 VP 2.0 cells, as measured by luciferase expression. Chemical structures of the polymers and lipid used in this Example can be found in FIG. 1 and FIG. 2. The commercially available reagent PEIpro was used as a control.

[0257] Experimental Details: 100 pl Viral Production 2.0 cells (Thermo Fisher Scientific) grown in Viral Production Medium (Thermo Fisher Scientific) seeded into untreated 96-well plates at 2 million cells / mL the same day as the transfection.Transfection complexes were formed in Opti-MEM (100 pL) containing 1 mg DNA at 2: 1 polymerDNA (mass:mass), 2: 1 lipid:DNA (mass:mass) or 2:2: 1 polymer:lipid:DNA (mass:mass:mass). Ten pL of transfection complex containing 100 ng of pCI-Luc pDNA was added 15-20 min post-complex formation to each well. Cells were harvested for luciferase expression 24 h post-transfection by addition of an equal volume of lysis buffer (2% Triton X 100, 100 mM potassium phosphate, pH 7.5, 1 mM EDTA), incubated at 4° C for 30 min and diluted 10-fold in lysis buffer. Luciferase activity was measured on a Berthold luminometer by pipetting 10 pl of diluted cell lysate into a white luminometer plate and measuring luminescence following a single injection of luciferase reaction buffer (12.6 mM potassiumAttorney Docket No. P24-228-SEC-WO01 phosphate, pH 7.5, 1 mM EDTA, 13.6 mM MgSCL, 2.85 mM ATP, 3 mM Luciferin). A 10 s integrated read was performed. Error bars represent the standard deviation from technical duplicates. The results are shown in FIG. 3.

[0258] Experimental Details: 100 pl Viral Production 2.0 cells (Thermo Fisher Scientific) grown in Cellvento 4HEK (Sigma Aldrich) seeded into untreated 96-well plates at 2 million cells / mL the same day as the transfection. Transfection complexes were formed in DMEM (100 pL) containing 1.5 pg DNA at 1 : 1 polymer :DNA (mass:mass), 2: 1 lipid:DNA (mass:mass), 1 : 1 : 1 polymer:lipid:DNA (mass:mass:mass) or 2:2: 1 polymer:lipid:DNA (mass:mass:mass). 10 pL of transfection complex containing 150 ng of pCI-Luc pDNA was added 30 min post-complex formation to each well. Cells were harvested for luciferase expression 24 h post-transfection by addition of an equal volume of lysis buffer (2% Triton X 100, 100 mM potassium phosphate, pH 7.5, 1 mM EDTA), incubated at 4° C for 30 min and diluted 10-fold in lysis buffer. Luciferase activity was measured on a Veritas Luminometer by pipetting 10 pl of diluted cell lysate into a white luminometer plate and measuring luminescence following a single injection of luciferase reaction buffer (12.6 mM potassium phosphate, pH 7.5, 1 mM EDTA, 13.6 mM MgSO4, 2.85 mM ATP, 3 mM Luciferin). A 10 s integrated read was performed. Error bars represent the standard deviation from technical duplicates. The results are shown in FIG. 4.

[0259] As shown in FIG. 3 and FIG. 4, individually the transfection performance of the cationic amine-containing polymers or the amphipathic compound tested is worse than that of the commercially available reagent. However, the combination of the same cationic amine-containing polymers and the amphipathic compound in a composition drastically improved the transfection performance. Additionally, many properties of the polymer appear to influence the transfection performance. Such properties include but are not limited to, molecular weight of the polymer, length of the side chain, makeup of the copolymer from the types of the side chains to the ratio between different side chain. This demonstrates the transfection efficiency of the compositions disclosed herein when complexed with pDNA and transfected into cells in culture.

[0260] 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. WhileAttorney Docket No. P24-228-SEC-WO01 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 to the 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.

Claims

Attorney Docket No. P24-228-SEC-WO01We Claim:Claims:

1. A composition for delivery of a nucleic acid into a cell, comprising:(i) a polymer comprising Formula (I);Formula (I) 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; each m and n is independently an integer from 1 to 10; and(ii) an amphipathic compound of Formula (II);Formula (II) 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, oxo, 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)-, heteroalkyl-C(O)-, 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)-, 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)-, or alkenyl-heteroaryl-alkenylene-C(O)-, cycloalkyl-O-C(O)-, wherein each said alkyl, heteroalkyl, alkenyl, alkylene,Attorney Docket No. P24-228-SEC-WO01 alkenylene, cycloalkyl, heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of halide, amino, C1-C4 alkyl, and C1-C4 haloalkyl. each LI and L2 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; and p is 0 or 1.

2. The composition of claim 1, wherein R3is an alkyl or heteroalkyl group, wherein said alkyl or heteroalkyl group is optionally substituted with 1-3 groups independently selected from the group consisting of amino and C1-C4 alkyl.

3. The composition of claim 1 or 2, wherein R3is an heteroalkyl group, wherein said heteroalkyl group is optionally substituted with an amino group.

4. The composition of any one of claims 1-3, wherein R4is C2-C6 alkyl, wherein said C2-C6 alkyl is optionally substituted with an amino group.

5. The composition of any one of claims 1-4, wherein R1and R2are hydrogen.

6. The composition of any one of claims 1-5, wherein W is -NH-.

7. The composition of any one of claims 1-6, wherein each LI and L2 is independently C2-C4 alkylene.

8. The composition of any one of claim 1 1-7, wherein LI and L2 are the same.

9. The composition of any one of claims 1-8, wherein each R11and R12is independently C10 to C25 alkenyl and comprises 1 to 4 alkene bonds, wherein each said C10 to C25 alkenyl is optionally substituted with one or more groups independently selected from the group consisting of halide, oxo, C1-C4 alkyl, and Ci- C4 haloalkyl.

10. The composition of any one of claims 1-9, wherein R11and R12are the same.

11. The composition of any one of claims 1-10, wherein each R13and R14is independently hydrogen, C2 to C10 alkyl, C2 to C10 alkenyl, alkyl-C(O)- alkenyl-C(O)-, heterocycle-alkylene-C(O)-, alkyl-heterocycle-alkylene-C(O)- heteroaryl-alkylene-C(O)-, or alkyl-heteroaryl-alkylene-C(O)-, wherein each said alkyl, alkenyl, alkylene, heterocycle and heteroaryl is optionally substituted with one or more groups independently selected from the group consisting of amino and C1-C4 alkyl.

12. The composition of any one of claims 1-11, wherein p is 0.Attorney Docket No. P24-228-SEC-WO0113. The composition of any one of claims 1-12, wherein R13and R14are the same.

14. A transfection reagent comprising a composition of any one of claims 1-13, and a nucleic acid.

15. A method of transfecting a nucleic acid into a cell, comprising mixing a composition of any one of claims 1-13 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.

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