Statistical charge-altering releasable transporters for nucleic acid delivery

Abstract

Provided are statistical copolymers derived from lipid carbonate monomers and cationic aminoester monomers useful in the delivery of nucleic acids across cell membranes, both in vitro and in vivo. The compounds described herein are shown to be particularly effective in delivery of mRNA into cells with resultant high protein expression, both in vitro and in vivo.

Description

Docket No. 6045.0586WO | S23-094 STATISTICAL CHARGE-ALTERING RELEASABLE TRANSPORTERS FOR NUCLEIC ACID DELIVERY STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with Government support under contract 2002933 awarded by the National Science Foundation and under contract CA245533 awarded by the National Institutes of Health. The Government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority under 35 U.S.C. § 119 to U.S. Provisional Application No.63 / 531,362 filed August 8, 2023, the contents of which are hereby incorporated by reference in their entirety. BACKGROUND

[0003] Nucleic acids are polyanionic, polar, and relatively large molecules on the order of about 13 kilodaltons, compared for example to small molecule therapeutics which are generally about 1 kilodalton or less. These properties of nucleic acids prevent or significantly hinder their unassisted passage through the nonpolar lipid membranes of cells and tissues. In addition, nucleic acids, especially RNAs, are susceptible to enzymatic degradation. Two main strategies have developed to address these difficulties with delivery of nucleic acid based therapeutic agents. One approach is the development of noncharged and nonbiodegradable nucleic acid surrogates. Another approach is the development of delivery vehicles that facilitate delivery of the nucleic acids across lipid membranes. Delivery vehicles have included lipids, peptides, and aptamers as well as cationic polymers.

[0004] Efficient delivery vehicles for transporting nucleic acids into target cells and tissues, and where the nucleic acid encodes a polypeptide or protein, expression of the encoded polypeptide or protein, are needed to realize the full potential of nucleic acid-based technologies, including RNA-based vaccines and immunotherapies. BRIEF SUMMARY

[0005] Provided herein are polymer-based nucleic acid delivery vehicles derived from lipid carbonate monomers and cationic aminoester monomers, and related compositions and methodsDocket No. 6045.0586WO | S23-094 for transporting nucleic acids into cells. In aspects, the polymers are in the form of statistical copolymers.

[0006] In one aspect, provided is a compound of Formula I, R1-X-Y-[(L1)x1-stat-(L2)x2-stat-(A2)y2-stat-(A1)y1]-R2 (Formula I) wherein R1 is substituted or unsubstituted C1-C24 alkyl, C1-C24 heteroalkyl, cycloalkyl, optionally 3-6 membered cycloalkyl, heterocycloalkyl, optionally 3-6 membered heterocycloalkyl, aryl, heteroaryl, or alkylaryl, optionally benzyl or methylbenzyl; X is a bond or linking group; Y is a bond, O, S, or N; L1and L2are each a lipid carbonate monomer unit selected from a lipid-functionalized methyl-trimethylene carbonate (MTC) unit, or a lipid-functionalized ethyl-trimethylene carbonate (ETC) unit; A1and A2are each independently an alpha aminoester (AAE) monomer unit having a structure ofwherein RA is H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2,-(CH2)4-NH2,-CH2-C6H4- OH, -(CH2)2-S-CH3, -CH2-C6H5 or -CH(CH3)2; x1 and y1 are each independently greater than zero and less than 1,000;Docket No. 6045.0586WO | S23-094 x2 and y2 are each independently zero or less than 1,000; and R2is H, C2-C24alkylcarbonyl, alkenylcarbonyl, substituted or unsubstituted alkyl or alkenyl, including pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and octadecenyl, optionally where the alkyl or alkenyl is substituted with one or more of acyl, C2-C6alkyl, and carboxylato.

[0007] The compound may also include where x2 is zero and the compound is represented by Formula II: R1-X-Y-[(L1)x1-stat-(A2)y2-stat-(A1)y1]-R2(Formula II), where R1, X, Y, L1, A1, A2, R2, x1, y1, and y2 are as defined above.

[0008] In some aspects, the compound is represented by Formula II-1: 1are as a or C1- C30 alkyl, which may be fully saturated, mono- or polyunsaturated, and RA1 and RA2 are each independently H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl; optionally RA1and RA2are each independently H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2,- (CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S-CH3, -CH2-C6H5 or -CH(CH3)2.

[0009] The compound may also include where y2 is zero and the compound is represented by Formula III: R1-X-Y-[(L1)x1-stat-(L2)x2-stat-(A1)y1]-R2 (Formula III), where R1, R2, X, Y, L1, L2, A1, R2, x1, x2, and y1 are as defined above.

[0010] In some aspects, the compound is represented by Formula III-1:Docket No. 6045.0586WO | S23-094 1a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated, and RA1 is H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl; optionally RA1 is H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2,-(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2- S-CH3,-CH2-C6H5or -CH(CH3)2.

[0011] In some aspects, the compound is represented by Formula III-2: 2are as are octyl, dodecyl, octadecyl; methyl, ethyl, propyl, or butyl substituted C2-C12 alkyl, haloalkyl, or alkylcarbonyl; substituted or unsubstituted C2-C12 alkoxycarbonyl; linolenyl, linoleoyl, or oleoyl; C12-C20alkenylcarbonyl; aryl or heteroaryl; substituted 5 membered cycloheteroalkenyl; or substituted or unsubstituted dilinolenyl, dilinoleoyl, or dioleoyl; and RAis H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl; optionally RA is H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S- CH3,-CH2-C6H5or -CH(CH3)2.

[0012] The compound may also include where x2 and y2 are both zero, and the compound is represented by Formula IV: R1-X-Y-[(L1)x1-stat-(A1)y1]-R2(Formula IV), where R1, X, Y, L1, A1, R2, x1 and y1 are as defined above.

[0013] In some aspects, the compound is represented by Formula IV-1:Docket No. 6045.0586WO | S23-094 1C1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated, and RA1is H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl; optionally RA1is H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S-CH3, -CH2-C6H5 or - CH(CH3)2.

[0014] In some aspects, the compound is represented by Formula IV-2: 2are as dodecyl, octadecyl; methyl, ethyl, propyl, or butyl substituted C2-C12 alkyl, haloalkyl, or alkylcarbonyl; substituted or unsubstituted C2-C12 alkoxycarbonyl; linolenyl, linoleoyl, or oleoyl; C12-C20 alkenylcarbonyl; aryl or heteroaryl; substituted 5 membered cycloheteroalkenyl; or substituted or unsubstituted dilinolenyl, dilinoleoyl, or dioleoyl; and RAis H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl; optionally RAis H, methyl, ethyl, -CH2-NH2, -(CH2)2- NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S-CH3, -CH2-C6H5 or -CH(CH3)2.

[0015] In accordance with aspects of any of the foregoing, the sum of x1, x2, y1, and y2 may be from 5-1,000, 5-800, 5-600, 5-300, 5-200, or 5-100. In some aspects, the sum of x1, x2, y1, and y2 is from 50-1,000, 50-800, 50-600, 50-300, 50-200, or 50-100.

[0016] In accordance with aspects of any of the foregoing, the compound may include where R1is substituted C1-C24alkyl, C1-C24heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl. In some aspects, the compound may also include R1 is substituted or unsubstitutedDocket No. 6045.0586WO | S23-094 heteroaryl. In some aspects, the compound may also include where R1 is substituted or unsubstituted benzyl or phenyl. In some aspects, the compound may also include where the substituent is hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C5-C20 aryloxy, acyl, acyloxy, C2-C24 alkoxycarbonyl, amino, alkylamido, C2-C24 alkylcarbonato, C6-C20 arylcarbonato, carboxy, carboxylato, carbamoyl, alkylcarbamoyl, thiocarbamoyl, carbamido, formyl, thioformyl, mono- and di-(C1-C24alkyl)-substituted amino, mono- and di-(C5-C20aryl)- substituted amino, C2-C24 alkylamido, C5-C20 arylamido, sulfo, sulfonato, C1-C24 alkylsulfanyl, arylsulfanyl, C1-C24alkylsulfinyl, C5-C20arylsulfinyl, C1-C24alkylsulfonyl, C5-C20arylsulfonyl, C1-C24alkyl, C2-C24alkenyl, C5-C30aryl, or C6-C30aralkyl.

[0017] In accordance with aspects of any of the foregoing, the compound may also include where R1 is a molecule useful for in vivo imaging or a ligand moiety that binds to a cell-surface receptor, where R1is joined to the copolymer via a linking group. In some aspects, the compound may include where R1 is a fluorophore, optionally difluoroboron-β-diketonate (BDK), a “Black Hole Quencher” dye, or a naphthalene derivative, including dansyl derivatives such as 5-(dimethylamino)-N-(2-hydroxyethyl)naphthalene-1-sulfonamide. In some aspects, the compound may also include where R1is a ligand moiety, optionally a saccharide, a disaccharide, an oligosaccharide, a liposaccharide, a lipid, a peptide, an antibody, biotin, a biotin derivative, or fingolimod.

[0018] In accordance with aspects of any of the foregoing, the compound may also include where X is substituted or unsubstituted alkylene, heteroalkylene, alkenylene, arylene, heteroarylene, aralkylene, or alkarylene. In some aspects, the compound may also include where X is alkylene, heteroalkylene, alkenylene, arylene, heteroarylene, aralkylene, or alkarylene substituted with one or more functional groups. In some aspects, the one or more functional groups is oxo, amine, carbonyl, carbonate, or sulfonamide. In some aspects, the compound may also include where X is substituted or unsubstituted heteroalkylene comprising one or more heteroatoms selected from O, S, or N.

[0019] In accordance with aspects of any of the foregoing, the compound may also include where RAis H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2,-(CH2)4-NH2,-CH2-C6H4- OH, -(CH2)2-S-CH3,-CH2-C6H5or -CH(CH3)2.

[0020] In one aspect, a method of transfecting a nucleic acid into a cell in vitro or ex vivo, the method includes contacting the cell in vitro or ex vivo with the composition or pharmaceutical composition of any one of claims 21-26.Docket No. 6045.0586WO | S23-094

[0021] In one aspect, a method of delivering a nucleic acid to a cell in a subject, the method includes administering to the subject the composition or pharmaceutical composition of any one of claims 21-26.

[0022] Also provided are compositions and pharmaceutical compositions including nanoparticles of the compound non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In some aspects, the nucleic acid is RNA or DNA. In some aspects, the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing. In some aspects, the nucleic acid is a therapeutic agent or the nucleic acid encodes a therapeutic agent.

[0023] The composition or pharmaceutical composition may also include where the composition is a vaccine.

[0024] Also provided are methods of transfecting a nucleic acid into a cell in vitro or ex vivo, the methods including contacting the cell in vitro or ex vivo with a composition or pharmaceutical composition including nanoparticles of the compound non-covalently complexed with a nucleic acid or a plurality of different nucleic acids.

[0025] Also provided are methods of delivering a nucleic acid into a cell in a subject, the methods including administering to the subject a composition or pharmaceutical composition as described herein.

[0026] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims. BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG.1 is a bar graph showing luminescence of HeLa cells transfected in vitro with a series of exemplary compounds of Formula IV-1 (SC-1, SC-2, SC-3, SC-4, SC-7) complexed with firefly luciferase (fLuc) mRNA and a block copolymer (BC-1) complexed with fLuc mRNA as control.

[0028] FIG.2 is a bar graph showing luminescence of HEK293T cells transfected in vitro with two exemplary compounds of Formula IV-1 (SC-1, SC-6) complexed with Fluc mRNA, or reference block copolymers (BC-ONA, BC-2, BC-3) complexed with Fluc mRNA.Docket No. 6045.0586WO | S23-094

[0029] FIG.3A is a bar graph showing protein expression (fluorescence) in A549 cells transfected with representative compounds of the invention (SC-6, SC-10, SC-11) or reference block copolymers (BC-2, BC-3, BC-4, BC-ONA) complexed with mRNA encoding enhanced green fluorescent protein (eGFP) where protein expression is measured as eGFP fluorescence intensity (left axis, bar graphs) and the percentage of cells expressing eGFP is indicated by black dots (%, right axis).

[0030] FIG.3B is a bar graph showing mRNA uptake (fluorescence) in A549 cells transfected with the same compounds and reference block copolymers as in FIG.3A where uptake is measured by Cy5 fluorescence intensity (left axis, bar graphs) and the percentage of Cy5 positive cells (%, black dots, right axis).

[0031] FIG.4 is a bar graph showing bioluminescence (luminescence intensity) in mice following intravenous injection of representative compounds and reference block copolymers.

[0032] FIG.5 is a bar graph showing bioluminescence (luminescence intensity) in mice following intravenous injection of representative compounds and a reference block copolymers illustrating organ specific expression in spleen (5 bracketed bars, left) and lung (2 bracketed bars, right).

[0033] FIG.6 is a bar graph showing bioluminescence (luminescence intensity) in mice following intravenous injection of mRNA complexed with representative compounds and a reference block copolymers illustrating organ specific expression in spleen and liver.

[0034] FIG.7A is a line graph of size (Z avg diameter, nm) versus time for two compounds of the invention and a reference block copolymer.

[0035] FIG.7B is a line graph of charge (Zeta potential, mV) versus time for two compounds of the invention and a reference block copolymer.

[0036] FIG.8A shows bioluminescence in mice following intravenous injection of mRNA complexed with two representative compounds and a reference block copolymer.

[0037] FIG.8B is a bar graph showing in vivo protein expression of the same two compounds as in FIG.8A compared to protein expression elicited by a block copolymer previously shown to exhibit high expression. DETAILED DESCRIPTION

[0038] The disclosure provides compounds in the form of statistical copolymers derived from lipid carbonate monomers and cationic aminoester monomers which have unique andDocket No. 6045.0586WO | S23-094 unexpected properties compared to similar copolymers having monomers arranged in a block distribution. The compounds disclosed herein are effective to transport nucleic acids into cells in sufficient amounts to support high levels of encoded protein expression, including organ specific protein expression, where similar block copolymers are ineffective. In addition, when formulated with mRNA, the high molecular weight statistical copolymers form unexpectedly stable nanoparticles compared to similar block copolymers. The statistical copolymers described here have the further advantage of being easier to prepare than similar block copolymers.

[0039] Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and / or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

[0040] The term “alkyl,” by itself or as part of another substituent, refers to an acyclic branched or unbranched hydrocarbon group containing from about 1 to 24 carbon atoms (C1‑C24) or from 1 to 18 carbon atoms (C1‑C18), which may be fully saturated, mono‑ or polyunsaturated and can include mono‑, di‑ and multivalent radicals. Examples of saturated alkyl groups include methyl, ethyl, n‑propyl, isopropyl, n‑butyl, t‑butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2‑propenyl, crotyl, 2‑isopentenyl, 2‑(butadienyl), 2,4‑pentadienyl, 3‑(1,4‑pentadienyl), ethynyl, 1‑ and 3‑propynyl, 3‑butynyl, and the higher homologs and isomers. In some aspects, an unsaturated alkyl group may be a linolenyl or oleyl. “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, including where two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group. For example, a substituted alkyl group may include a carbonyl (- C(=O)-) moiety. The term “heteroalkyl” refers to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in more detail below. If not otherwiseDocket No. 6045.0586WO | S23-094 indicated, the term “alkyl” includes unsubstituted, substituted, and / or heteroatom-containing alkyl substituents.

[0041] The term “alkoxy” refers to an alkyl group bound through a single, terminal ether linkage which may be represented as -O-alkyl, where alkyl is as defined above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. The term “alkylthio” refers to a group -S-alkyl.

[0042] The term “heteroalkyl”, which is shorthand for “heteroatom-containing alkyl”, refers to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom. Similarly, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, and the term “heteroaryl” refers to “aryl” substituents that are heteroatom-containing. In this context, “heteroatom-containing” refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, such as nitrogen (N), oxygen (O), sulfur (S), phosphorus (P) or silicon (Si), and more typically in the context of the present disclosure with N, O, or S. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl groups are provided below.

[0043] In some aspects, the heteroatom is O, N, or S. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2- S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, -CH=CH- N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3,R-S-S-R’, and RO-S(O)x-OR’. In some aspects, a heteroalkyl moiety may include one, two, three, four, or five heteroatom (e.g., O, N, S). In some aspects, a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S).

[0044] The term “alkenyl” refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, octadecenyl, eicosenyl, and the like. In some aspects, alkenyl groups may contain 2 to about 18 carbon atoms (C2‑C18) or 2 to 12 carbon atoms (C2‑C12). The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the term “heteroalkenyl” refers to alkenyl in which at least one carbon atom is replaced with a heteroatom. A heteroalkenylDocket No. 6045.0586WO | S23-094 may optionally include more than one double bond and / or one or more triple bonds in additional to the one or more double bonds.

[0045] The term “alkylene” refers to a di-radical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. In aspects, an alkylene substituent group may be methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)6-), octylene (-(CH2)8-) and the like. Similarly, the terms “alkenylene”, “alkynylene”, “arylene”, “aralkylene”, and “alkarylene” refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively. In some aspects, the aralkylene group is substituted on the alkylene moiety or the arylene moiety (e.g. at carbons 2, 3, 4, or 6) with a functional group. In some aspects, the aralkylene group has the formula:In the context of the present disclosure, alkylene groups are utilized as linking groups. Accordingly, these and other di-radical groups may be referred to herein as “linkers”, “linker groups”, “linker substituents”, “linking groups” or “linking substituents”. The alkylene, alkenylene, alkynylene, arylene, aralkylene, and alkarylene groups may also contain one or more functional groups. The term “functional group” in this context refers to di-radical moieties that contain one or more functional groups such as an oxo (-O-, such as in an ether linkage), amine (-NR-), carbonyl (-C(=O)-), carbonate, and the like. In some aspects, the functional group of a functional linker may be selected from oxo, ‑N3, ‑CN, ‑CHO, ‑OH, ‑NH2, ‑COOH, ‑CONH2, ‑NO2, ‑SH, ‑SO2CH3 ‑SO3H, , ‑OSO3H, ‑SO2NH2, ‑NHNH2, ‑ONH2, ‑NHC(O)NHNH2, substituted or unsubstituted C1‑C5 alkyl or substituted or unsubstituted heteroalkyl. In embodiments, the alkylarylene is unsubstituted.

[0046] The term "amino" is used herein to refer to the group -NZ1Z2 wherein Z1 and Z2 are hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.Docket No. 6045.0586WO | S23-094

[0047] The terms “cycloalkyl” and “heterocycloalkyl” refer to cyclic versions of the “alkyl” and “heteroalkyl” groups defined above. Cycloalkyl and heterocycloalkyl are not aromatic. In aspects, a cycloalkyl group includes monocyclic hydrocarbon ring systems containing from 3 to 9 carbon atoms (C3-C9), one or more of which may be replaced with a heteroatom, and where such groups can be saturated or unsaturated, but not aromatic. The term “3 to 6 membered” in reference to a cycloalkyl or heterocycloalkyl refers to the number of atoms, i.e. carbon atoms or carbon and heteroatoms, in the monocyclic ring. For example, the term “3 to 6 membered cycloalkyl or heterocycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, heterocyclopropyl, heterocyclobutyl, heterocyclopentyl, and heterocyclohexyl. In aspects, a cycloalkyl group includes a bicyclic or multicyclic cycloalkyl ring system where multiple rings are fused together, where at least one of the fused rings is a cycloalkyl ring and the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkyl ring of the multiple rings. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3- piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. In embodiments, the cycloalkyl is fully saturated. In aspects, the cycloalkyl is monounsaturated. In embodiments, the cycloalkyl is polyunsaturated. In aspects, the heterocycloalkyl is fully saturated. In embodiments, the heterocycloalkyl is monounsaturated. In aspects, the heterocycloalkyl is polyunsaturated.

[0048] The term “acyl” means, unless otherwise stated, ‑C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0049] The term “aryl” refers to cyclic groups that contain at least one aromatic ring, for example a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl). In some aspects of the present disclosure, the aryl group contains 6, 9 or 10 atoms such as phenyl, biphenyl,Docket No. 6045.0586WO | S23-094 naphthyl, diphenylether, diphenylamine, benzophenone, indanyl, anthracenyl, 1,2- dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within an aryl ring of the multiple rings. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the term “heteroaryl” refers to an aryl substituent in which at least one carbon atom is replaced with a heteroatom, as described in more detail below. If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0050] The term “aralkyl” refers to an alkyl group with an aryl substituent, and the term “alkaryl” refers to an aryl group with an alkyl substituent, where “alkyl” and “aryl” are as defined above. In general, aralkyl and alkaryl groups may contain from 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms or from 6 to 12 carbon atoms.

[0051] The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings). A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4- oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the aboveDocket No. 6045.0586WO | S23-094 noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents as described herein.

[0052] An “arylene” and a “heteroarylene,” alone or as part of another substituent, means a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0053] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0054] The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.

[0055] The term “substituted” as in “substituted alkyl”, “substituted aryl”, and the like, refers to at least one hydrogen atom bound to a carbon or other atom that is replaced with one or more non-hydrogen substituents in the alkyl, aryl, or other moiety. The term “substituted or unsubstituted” preceding a list, as in “substituted or unsubstituted C1-C24 alkyl, C1-C24 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl” is intended to modify each member of the list, as in "“substituted or unsubstituted C1-C24alkyl, substituted or unsubstituted C1-C24 heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted alkylaryl”. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24alkoxycarbonyl (-(CO)-O-alkyl), C6-C20aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), mono-substituted C1-C24alkylcarbamoyl (- (CO)-NH(C1-C24alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24alkyl)2), mono- substituted arylcarbamoyl (--(CO)--NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH- (CO)-NH2), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24alkyl)-substituted amino, mono- and di-(C5-C20aryl)-substituted amino, C2-C24alkylamido (-Docket No. 6045.0586WO | S23-094 NH-(CO)-alkyl), C5-C20 arylamido (-NH-(CO)-aryl), imino (-CR=NH where R is hydrogen, C1- C24alkyl, C5-C20aryl, C6-C20alkaryl, C6-C20aralkyl, etc.), alkylimino (-CR=N(alkyl), where R is hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R is hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), sulfonamide (-SO2NH), C1-C24alkylsulfanyl (-S-alkyl or “alkylthio”), arylsulfanyl (-S-aryl or “arylthiol”), C1-C24alkylsulfinyl (-(SO)-alkyl), C5-C20arylsulfinyl (-(SO)-aryl), C1-C24alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl); and the hydrocarbyl moieties C1- C24alkyl (including C1-C18alkyl, C1-C12alkyl, C1-C8alkyl and C1-C6alkyl), C2-C24alkenyl (including C2-C18alkenyl, C1-C12alkenyl, C1-C8alkenyl, and C1-C6alkenyl), C5-C30aryl (including C5-C20 aryl, C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and C6-C12 aralkyl). In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above- mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.

[0056] The suffix “ene” added on to any of the above groups means that the group is divalent, i.e. inserted between two other groups.

[0057] The symbol “ ” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0058] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. Compounds

[0059] Provided are compounds in the form of statistical copolymers derived from two different types of monomers, lipid carbonate monomers and cationic aminoester monomers. The corresponding units of the copolymers derived from these monomers are referred to asDocket No. 6045.0586WO | S23-094 “constitutional units” or “monomer units” or simply “units”. In some aspects, two different types of lipid carbonate monomer unit and / or cationic aminoester monomer unit may be included in the copolymer such that the copolymers described here may comprise, e.g., from 2- 4 different types of constitutional units derived from 2-4 different types of monomer.

[0060] Below, the statistical copolymers provided herein are compared against other polymeric compounds, including block copolymers of similar structure, meaning that the same or similar constitutional units are shared but the units are distributed as blocks of identical monomer units, rather than statistically. The nomenclature poly(A-stat-B) or polyA-block- polyB is adopted herein to differentiate these two types of copolymers, in accordance with Hodge et al., Pure Appl. Chem.202092(5):797-813.

[0061] The polymer formulas described herein are presented as graphical representations of an “ideal” form of the polymer. For example, while the graphical representations may present the repeating monomer units as connected to each other in the same orientation, e.g., “head-to- tail”, it is understood that in the actual polymer there may also be some “head-to-head” and / or “tail-to-tail” dyads.

[0062] Provided are compounds of Formula I or Formula Ia: R1-X-Y-[(L1)x1-stat-(L2)x2-stat-(A2)y2-stat-(A1)y1]-R2 (Formula I). R1-X-Y-[(A1)y1-stat-(A2)y2-stat-(L2)x2-stat-(L1)x1]-R2(Formula Ia). where R1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; X is a bond or a linking group as defined herein; Y is a bond, O, S, or N; L1 and L2 are each independently a lipid carbonate monomer unit; A1and A2are each independently an aminoester monomer unit; x1 and y1 are each independently greater than zero and less than 1,000;Docket No. 6045.0586WO | S23-094 x2 and y2 are each independently zero or less than 1,000; and R2is H, C2-C24alkylcarbonyl, alkenylcarbonyl, substituted or unsubstituted alkyl or alkenyl, including pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and octadecenyl, optionally where the alkyl or alkenyl is substituted with one or more of acyl, C2-6alkyl, and carboxylato.

[0063] In an aspect, x2 is zero, and the compounds are represented by Formula II or Formula IIa: R1-X-Y-[(L1)x1-stat-(A2)y2-stat-(A1)y1]-R2(Formula II) R1-X-Y-[(A1)y1-stat-(A2)y2-stat-(L1)x1]-R2 (Formula IIa), where R1, X, Y, L1, A1, A2, R2, x1, y1, and y2 are as defined above and as further defined infra.

[0064] In an aspect, y2 is zero, and the compounds are represented by Formula III or Formula IIIa: R1-X-Y-[(L1)x1-stat-(L2)x2-stat-(A1)y1]-R2(Formula III) R1-X-Y-[(A1)y1-stat-(L2)x2-stat-(L1)x1]-R2(Formula IIIa), where R1, X, Y, L1, L2, A1, R2, x1, x2, and y1 are as defined above and as further defined infra.

[0065] In an aspect, x2 and y2 are both zero, and the compounds are represented by Formula IV or Formula IVa: R1-X-Y-[(L1)x1-stat-(A1)y1]-R2(Formula IV) R1-X-Y-[(A1)y1-stat-(L1)x1]-R2(Formula IVa), where R1, X, Y, L1, A1, R2, x1 and y1 are as defined above and as further defined infra.

[0066] In aspects of Formulas I-IV or Ia-IVa, R1 is substituted or unsubstituted C1-C24 alkyl, C1-C24heteroalkyl, C3-C12cycloalkyl, C3-C12heterocycloalkyl, aryl, or heteroaryl. In some aspects, R1 is substituted or unsubstituted benzyl, methyl benzyl, or phenyl. In aspects, theDocket No. 6045.0586WO | S23-094 substituent is hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C5-C20 aryloxy, acyl, acyloxy, C2-C24alkoxycarbonyl, amino, alkylamido, C2-C24alkylcarbonato, C6-C20arylcarbonato, carboxy, carboxylato, carbamoyl, alkylcarbamoyl, thiocarbamoyl, carbamido, formyl, thioformyl, mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)- substituted amino, C2-C24alkylamido, C5-C20arylamido, sulfo, sulfonato, C1-C24alkylsulfanyl, arylsulfanyl, C1-C24alkylsulfinyl, C5-C20arylsulfinyl, C1-C24alkylsulfonyl, C5-C20arylsulfonyl; and the hydrocarbyl moieties C1-C24 alkyl (including C1-C18 alkyl, C1-C12 alkyl, C1-C8alkyl and C1-C6alkyl), C2-C24alkenyl (including C2-C18alkenyl, C1-C12alkenyl, C1-C8alkenyl, and C1-C6alkenyl), C5-C30aryl (including C5-C20aryl, C5-C12aryl), and C6-C30aralkyl (including C6-C20 aralkyl, and C6-C12 aralkyl).

[0067] In aspects of Formulas I-IV or Ia-IVa, R1 comprises one or more nucleophilic groups which may include alcohol, diol, amine, diamine, polyamine, aryl, azide, carboxylato (e.g., - COO-), ether, hydroxyl, phenyl, polyol, sulfhydryl, disulfhydryl, polysulfhydryl, and combinations thereof.

[0068] In aspects of Formulas I-IV or Ia-IVa, R1is a molecule useful for in vivo imaging including a fluorophore such as difluoroboron-β-diketonate (BDK), a “Black Hole Quencher” dye including BHQ-1, BHQ-2, BHQ-3, and derivatives thereof, including 2-[N-(2- hydroxyethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2- nitrophenyl)diazenyl]phenyl]diazenyl]anilino]ethanol, or a naphthalene derivative, including dansyl derivatives such as 5-(dimethylamino)-N-(2-hydroxyethyl)naphthalene-1-sulfonamide. In some aspects of Formulas I-IV or Ia-IVa, R1is a ligand moiety that binds to a cell-surface receptor such as a saccharide, a disaccharide, an oligosaccharide, a liposaccharide, a lipid, a peptide, an antibody, or a small organic molecule such as biotin and biotin derivatives including biotin pentanediol, any of which may be joined to the copolymer via a linking group as defined herein. In some aspects, R1is the small molecule fingolimod (FTY720) joined to the copolymer via a linking group.

[0069] In aspects of Formulas I-IV or Ia-IVa, X is a linking group which may be a substituted or unsubstituted alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, alkenylene, arylene, heteroarylene, aralkylene, or alkarylene. In some aspects, X is alkylene, heteroalkylene, alkenylene, arylene, heteroarylene, aralkylene, or alkarylene substituted with one or more functional groups such as oxo, amine, carbonyl, carbonate, or sulfonamide.Docket No. 6045.0586WO | S23-094

[0070] In aspects of Formulas I-IV or Ia-IVa, X is substituted or unsubstituted alkylene or heteroalkylene comprising one or more heteroatoms selected from O, S, or N. In some aspects, X is substituted or unsubstituted methylene or ethylene or heteromethylene or heteroethylene substituted with O, S, or N, and combinations thereof. In some aspects, X is methoxy (-CH2O-), ethoxy (-CH2CH2O-), -OCH2CH2O-, -NHCH2CH2O-, -SO2NHCH2CH2O-.

[0071] In aspects of Formulas I-IV or Ia-IVa, L1and L2are each independently a lipid- functionalized methyl-trimethylene carbonate (MTC) unit having a structure ofwhich may be fully saturated, mono- or polyunsaturated. In some aspects, RL is independently stearyl, oleyl, linoleyl, dodecyl, nonenyl, isoprenoid, 2-hexyloctanyl, 2-ethylhexanyl, 2-octyldodecyl or cholesterol.

[0072] In aspects of Formulas I-IV or Ia-IVa, L1 and L2 are each independently a lipid- functionalized ethyl-trimethylene carbonate (ETC) unit derived from an allyl carbonate (AC) as discussed in more detail infra, where the ETC unit has a structure ofDocket No. 6045.0586WO | S23-094 and where RK is octyl, dodecyl, octadecyl; methyl, ethyl, propyl, or butyl substituted C2- C12alkyl or haloalkyl, such as (a) or (b);or unsubstituted C2-C12alkoxycarbonyl, such as (c), ;methyl, ethyl, propyl, or butyl substituted C2-C12 alkylcarbonyl, such as ;ethoxy linolenate, such asDocket No. 6045.0586WO | S23-094 (j);(i); ;or C12-C20alkenylcarbonyl, such as (h'), or .

[0073] such as n are independently 0-26.including hydroxyalkyl, aminoalkyl, and halo substituted alkyl, such as , where m and n are independently 0-26.some or substituted 5 membered cycloheteroalkenyl, includingDocket No. 6045.0586WO | S23-094 tolyl, pyridinyl, or imidazolyl.

[0076] is substitutedalkoxycarbonyl, such as, , ,alkoxy linolenate, orDocket No. 6045.0586WO | S23-094 In some aspects, RK is alkyl substituted or unsubstituted dilinolenyl, dilinoleyl, or dioleyl, for example

[0078] In aspects of Formulas I-IV or Ia-IVa, A1and A2are each independently an alpha aminoester (AAE) monomer unit having a structure ofwherein RA is H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, including thio substituted alkyl, hydroxy substituted aryl, and cresyl.Docket No. 6045.0586WO | S23-094

[0079] In some aspects, RA is H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, - (CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S-CH3,-CH2-C6H5or -CH(CH3)2.

[0080] In some aspects, the AAE unit has a cyclic structure such as .

[0081] I-IV or Ia-IVa, x1, x2, y1, and y2 are each independently 5- 1,000, 5-800, 5-600, 5-300, 5-200, 5-100, 5-50, or 5-25. In some aspects, x1, x2, y1, and y2 are each independently 50-1,000, 50-900, 50-800, or 50-600. In some aspects, x1, x2, y1, and y2 are each independently 50-500, 50-400, 50-300, 50-200, or 50-100. In some aspects, x1, x2, y1, and y2 are each independently 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. In some aspects, x1, x2, y1, and y2 are each independently 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100. In some aspects, X2 or Y2, or both, is zero.

[0082] In aspects of Formulas I-IV or Ia-IVa, the sum of x1, x2, y1, and y2 is from 5-1,000, 5-800, 5-600, 5-300, 5-200, or 5-100. In some aspects, the sum of x1, x2, y1, and y2 is from 50-1,000, 50-800, 50-600, 50-300, 50-200, or 50-100.

[0083] In aspects, the compound is represented by (Formula II-1) in which L1 is an MTC monomer unit and A1 and A2 are each an AAE monomer unit: 1.where R1, R2, x1, y1 and y2 are as defined above or as defined in Table 1; RL1 is as defined above for RL or as defined in Table 1; and RA1 and RA2 are each independently as defined above for RA, or as defined in Table 1.

[0084] Table 1: Representative statistical copolymers of Formula II-1. # Compound R1RL1x1 RA2y2 RA1y1 R2unitsDocket No. 6045.0586WO | S23-094 SC-13 benzyl oleyl 31 H 10 (CH2)3NH2 3 COCH3 44 SC-14 benzyl oleyl 31 H 23 (CH2)3NH26 COCH360 SC-15 benzyl oleyl 42 H 24 (CH2)4NH2 4 COCH3 70

[0085] In some aspects, the compound is represented by Formula III-1 in which L1 and L2 are each an MTC unit and A1is an AAE unit: 1.RL2 are each independently as defined above for RL, or as defined in Table 2; and RA1is as defined above for RAor as defined in Table 2.

[0086] Table 2: Representative statistical copolymers of Formula III-1. Compound R1RL1x1 RL2x2 RA1y1 R2# units SC-10 benzyl oleyl 6 nonenyl 7 H 10 H 23 SC-11 benzyl oleyl 21 nonenyl 22 H 33 H 76 SC-21 benzyl oleyl 5 nonenyl 6 H 8 H 19

[0087] In some aspects, the compound is represented by Formula IV-1 in which L1is an MTC unit and A1 is an AAE unit:Docket No. 6045.0586WO | S23-094 1.above for RL or as defined in Tables 3a and 3b; and RA1 is as defined above for RA or as defined in Tables 3a and 3b.

[0088] Table 3: Representative statistical copolymers of Formula IV-1. # Compound R1RL1x1 RA1y1 R2units SC-1 benzyl dodecyl 13 H 12 H 25 SC-2 benzyl dodecyl 14 H 13 H 27 SC-3 benzyl dodecyl 16 H 14 H 30 SC-4 benzyl dodecyl 30 H 28 H 58 SC-5 benzyl dodecyl 39 H 37 H 78 SC-6 benzyl dodecyl 50 H 49 H 99 SC-7 benzyl dodecyl 132 H 127 H 259 SC-8 benzyl dodecyl 14 (CH2)3NH2 4.5 COCH3 18.5 SC-9 benzyl dodecyl 58 (CH2)3NH2 14 COCH3 72 SC-12 benzyl dodecyl 13 H 12 H 25Docket No. 6045.0586WO | S23-094 SC-22 benzyl dodecyl 13 (CH2)3NH2 4 COCH3 17 SC-23 benzyl hexyloctanyl 17 H 13 H 30 SC-24 benzyl hexyloctanyl 16 CH2C6H4OH 3 H 19 SC-25 benzyl hexyloctanyl 16 CH2C6H5 3 H 19SC-26 benzyl oleyl 18 CH2C6H55 H 23 SC-27 benzyl oleyl 16 CH2C6H4OH 4 H 20 SC-28 benzyl oleyl 15 H 13 H 28 SC-29 benzyl oleyl 19 CH310 H 29 SC-30 benzyl dodecyl 13 CH2C6H57 H 20 SC-31 benzyl nonenyl 13 CH2C6H5 11 H 24 SC-32 benzyl dodecyl 13 CH2C6H4OH 6 H 19 SC-33 benzyl nonenyl 15 CH2C6H4OH 9 H 24 SC-34 benzyl dodecyl 13 (CH2)3NH2 3 H 16 SC-35 benzyl nonenyl 15 (CH2)3NH2 4 H 19 SC-36 benzyl hexyloctanyl 12 H 14 H 26 SC-37 benzyl octyldodecyl 10 H 12 H 22 SC-38 benzyl nonenyl 13 H 12 H 25Docket No. 6045.0586WO | S23-094 SC-39 benzyl hexyloctanyl 17 (CH2)3NH2 5 H 22 SC-40 benzyl octyldodecyl 16 (CH2)3NH25 H 21 SC-41 benzyl oleyl 16 (CH2)3NH2 5 H 21 SC-42 benzyl octyldodecyl 13 CH2C6H4OH 11 H 24 SC-43 benzyl ethylhexanyl 16 (CH2)3NH24 H 20 SC-44 benzyl ethylhexanyl 13 CH2C6H4OH 9 H 22 SC-45 benzyl ethylhexanyl 14 CH2C6H5 9 H 23 SC-46 benzyl ethylhexanyl 12 H 15 H 27 SC-47 benzyl oleyl 12 (CH2)3NH23 COCH315

[0089] In some aspects, the compound is represented by Formula III-2 in which L1 and L2 are each an ETC unit and A1is an AAE unit: 2.where R1, R2, x1, x2 and y1 are as defined above; RK1 and RK2 are each independently as defined above for RK; and RAis as defined above.

[0090] In some aspects, the compound is represented by Formula IV-2 in which L1and L2are each an ETC unit and A1 is an AAE unit:Docket No. 6045.0586WO | S23-094 2.defined in Table 4, where “4MeBzyl” refers to 4-methylbenzyl.

[0091] Table 4: Representative statistical copolymers of Formula IV-2. Compound R1RKx1 RAy1 R2# units SC-17 4MeBzyl dodecyl 15 H 15 H 30 SC-18 4MeBzyl h 10 H 10 h' 20 SC-19 4MeBzyl i 11 H 11 i' 22 SC-20 4MeBzyl j 11 H 11 j' 22

[0092] The following block copolymers were made as reference materials and utilized for comparative studies with the statistical copolymers described herein.

[0093] Table 5: Representative block copolymers of Formula IV-1. Compound R1 RL1 x1 RA1 y1 R2 # units BC-1 benzyl dodecyl 14 H 11 H 25 BC-2 benzyl dodecyl 13 H 12 H 25 BC-3 benzyl dodecyl 45 H 65 H 110Docket No. 6045.0586WO | S23-094 BC-4 benzyl dodecyl 37 H 34 H 71 BC-6 benzyl dodecyl 15 (CH2)3NH25 COCH320 BC-7 benzyl dodecyl 12.5 CH2C6H4OH 10 COCH3 22.5 BC-29 benzyl dodecyl 15 (CH2)2SCH3 5 H 20

[0094] Table 6: Representative block copolymers of Formula IV-2. Compound R1 RK x1 RA y1 R2 # units BC-17 4MeBzyl octyl 11 H 12 H 23 BC-18 4MeBzyl dodecyl 11 H 12 H 23 BC-19 4MeBzyl octadecyl 12 H 12 H 24 BC-20 4MeBzyl a 11 H 12 H 23 BC-21 4MeBzyl b 11 H 12 H 23 BC-22 4MeBzyl d 11 H 12 H 23 BC-23 4MeBzyl e 11 H 12 H 23 BC-24 4MeBzyl f 13 H 12 f' 25 BC-25 4MeBzyl g 12 H 11 g' 23 BC-26 4MeBzyl h 11 H 11 h' 22Docket No. 6045.0586WO | S23-094 BC-27 4MeBzyl i 12 H 12 i' 24 BC-28 4MeBzyl j 12 H 12 j' 24

[0095] Table 7: Representative block copolymers of Formula III-1. Compound R1RL1x1 RL2x2 RA1y1 R2# units BC-ONA benzyl oleyl 5 nonenyl 5 H 8 H 18

[0096] Table 8: Structures of representative compounds Name StructureDocket No. 6045.0586WO | S23-094Synthetic Methods

[0097] Examples of synthesis of the statistical copolymers described herein, as well as certain precursors and monomers is described in the following paragraphs. Synthesis of Tyrosine monomer

[0098] O-1.0), NaHCO3(1.581g, , 18.07 mmol, 2.6 equiv) and 20.6 mL MeCN were added to a 100 mL round bottomDocket No. 6045.0586WO | S23-094 flask with a stirbar and heated to reflux. After stirring for 20 mins, 2-Bromoethanol (1.042g, 8.34 mmol, 1.2 equiv) was added in one portion. The reaction was refluxed for 20.5 hours. After cooling to room temperature, the mixture was diluted in acetone and filtered through celite. The reaction was concentrated to a yellow oil and then resuspended in 7.7 mL anhydrous MeOH. Under nitrogen atmosphere, Boc2O (1.517g, 6.95 mmol, 1.0 equiv) was added and left to stir at room temperature for 23 hour. The reaction was concentrated and purified by column chromatography (3:1 DCM:EtOAc) to yield 1.1161 g colorless / pale yellow oil (40.6% yield).

[0099] While2.75 mmol, 1 equiv) was added with TsOH (0.068 g, 0.36 mmol, 0.13 equiv) and 72.0 mL PhMe to a 250 mL round bottom flask with a stirbar. Once the oil bath was at temperature, the reaction mixture was placed into the bath and a reflux condenser was attached. The reaction was left to reflux for 40 minutes. Then the supernatant was separated and concentrated to yield a colorless solid crude product. The crude product was purified by column chromatography (5% EtOAc / DCM). Yield: 0.6806 g white solid (68.2% yield; 27% total yield over 3 steps). Synthesis of methionine monomer. was charged with a stir bar, 1.50 g of L-methionine methyl ester hydrochloride (S1a, 7.51 mmol, 1.00 equiv), 1.40 g of NaHCO3(16.7 mmol, 2.20 equiv.), and 23 mL of acetonitrile ([S1a] = 0.33 M). The reaction flask was equipped with a refluxing condenser cooled with a water jacket. The mixture was refluxed while stirring for fifteen minutes at 86°C before 0.59 mL of bromoethanol (8.26 mmol, 1.10 equiv.) was added in one portion. The reaction wasDocket No. 6045.0586WO | S23-094 allowed to stir while refluxing for two days. During the course of this time, reaction progression was tracked using proton NMR. Once ~90% of the bromoethanol was consumed, the flask was removed from heat, allowed to cool to room temperature, and the crude material was filtered through a pad of celite. The filtrate was collected and concentrated to dryness under reduced pressure. The crude material (S1b) was carried forward without purification to synthesize S2.

[0101] Synthesis of methyl N-(tert-butoxycarbonyl)-N-(2-hydroxyethyl)-L-methioninate (S2). All of the crude reaction mixture from the synthesis of S1b (1.56 g) was suspended in 7.50 mL of MeOH ([S1b] = 1.0 M) and stirred for 5 minutes before 1.98 mL of di-tert-butyl dicarbonate was added in one portion (9.00 mmol, 1.15 equiv.). The reaction stirred overnight before it was concentrated to dryness under reduced pressure to give a yellow oil as the crude product. The crude material was purified using column chromatography. The crude material was dissolved in minimal DCM, loaded onto a silica gel column, and eluted using a 4:1 ratio of DCM:EtOAc (v / v). In this solvent mixture, a TLC of the crude material was found to exhibit six spots with Rf= 0, 0.17, 0.28, 0.48, 0.83, and 0.90 (Rf= 0.48 is the desired product). Fractions containing the desired product were combined and concentrated to dryness under reduced pressure to yield 0.820 g of the desired product S2 as a slightly yellow oil (36% yield over 2 steps). The structure of S2 was confirmed by proton and carbon NMR.

[0102] Synthesis of tert-butyl (S)-3-(2-methylthio)ethyl)-2-oxomorpholine-4-carboxylate (M). A 250 mL round bottom flask was charged with a stir bar and 83 mL of toluene ([S2] = 0.025 M) to which 0.650 g of S2 (2.11 mmol, 1.00 equiv.) was added. The flask was equipped with a reflux condenser cooled with a water jacket and an oil bath was heated to 100ºC. Then, 72.4 mg of tosyl acid monohydrate (0.381 mmol, 0.180 equiv.) was added to the reaction flask, and the flask was put into the 100ºC oil bath. The reaction was allowed to proceed for 30 minutes at which point it was removed from heat and concentrated under reduced pressure to dryness. The crude material was loaded onto a silica gel column and the desired product was eluted using 12:1 DCM: EtOAc (Rf = 0.74 for the desired product). Collection of the relevant fractions yielded 0.356 g of M (61% yield) as a slightly yellow oil. The structure of the desired product was confirmed by proton and carbon NMR. The monomer, M, was stored in a -20ºC freezer until used for polymerizations.

[0103] Synthesis of phenylalanine monomerDocket No. 6045.0586WO | S23-094

[0104] phenylalaninate

[0105] L-phenylalanine methyl ester hydrochloride (15.00g, 69.5 mmol, 1.0 equiv), NaHCO3 (15.19g, , 180.8 mmol, 2.6 equiv) and 300 mL MeCN were added to a 1 L round bottom flask with a stirbar and heated to reflux. After stirring for 20 mins, 2-Bromoethanol (10.43g, 83.46 mmol, 1.2 equiv) was added in one portion. The reaction was refluxed for 20.5 hours. After cooling to room temperature, the mixture was diluted in acetone and filtered through celite. The reaction was concentrated to a golden oil and then resuspended in 70 mL anhydrous MeOH. Under nitrogen atmosphere, Boc2O (15.18g, 69.6 mmol, 1.0 equiv) was added and left to stir at room temperature for 24 hour. The reaction was concentrated and purified by column chromatography (9:1 DCM:EtOAc) to yield 9.517 g colorless / pale yellow oil (34.6% yield).

[0106]

[0107] While preheating oil bath to 110 °C, the intermedia (9.517 g, 29.43 mmol, 1 equiv) was added with TsOH (0.728 g, 3.83 mmol, 0.13 equiv) and 294.3 mL PhMe to a round bottom flask with a stirbar. Once the oil bath was at temperature, the reaction mixture was placed into the bath and a reflux condenser was attached. The reaction was left to reflux for 40 minutes. The supernatant was separated and concentrated to yield colorless solid crude product. The crude product was purified by column chromatography (100% DCM). Yield: 5.28 g white solid (61% yield; 21% total yield over 3 steps).

[0108] Synthesis of alanine, valine, DAPA, DABA monomer

[0109] The syntheses of the alanine, valine, DAPA, and DABA monomers follows the exact procedure as the phenylalanine monomer synthesis except for the differences in respective starting materials, i.e., 1-alanine methyl ester hydrochloride for the alanine monomer, valine methyl ester hydrochloride for the valine monomer, H-Dab(Boc) methyl ester hydrochloride for the DABA monomer, and H-Dap(Boc) methyl ester hydrochloride for the DAPA monomer.Docket No. 6045.0586WO | S23-094

[0110] The methyl ester hydrochloride (1.0 equiv), NaHCO3 (2.6 equiv) and 300 mL MeCN were added to a 1 L round bottom flask with a stirbar and heated to reflux. After stirring for 20 mins, 2-Bromoethanol (1.2 equiv) was added in one portion. The reaction was refluxed for 20.5 hours. After cooling to room temperature, the mixture was diluted in acetone and filtered through celite. The reaction was concentrated to a golden oil and then resuspended in 70 mL anhydrous MeOH. Under nitrogen atmosphere, Boc2O (1.0 equiv) was added and left to stir at room temperature for 24 hour. The reaction was concentrated and purified by column chromatography. The intermedia was added with TsOH (0.13 equiv) and 250 mL PhMe to a round bottom flask with a stirbar. Once the oil bath was at temperature, the reaction mixture was placed into the bath and a reflux condenser was attached. The reaction was left to reflux for 40 minutes. The supernatant was separated and concentrated to yield colorless solid crude product. The crude product was purified by column chromatography.

[0111] As discussed above, one advantageous aspect of the statistical CART polymers described herein is that both the lipid carbonate monomers and the aminoester monomercan be polymerized simultaneously at 25 °C in a single step. Note that more than two different types of monomer can be used. In the scheme below only two are monomers are represented for clarity, a lipid carbonate monomer and an alpha aminoester monomer. However, for example, two different lipid carbonate monomers and / or two different aminoester monomers may be used, as discussed and exemplified herein.illustrate the process. A 2-mL reaction vial equipped with a screw cap, a PTFE / silicon septum, and a small-sized magnetic stir bar was evacuated and backfilled with nitrogen, to which benzyl alcohol (R1-OH) stock solution was prepared by injecting a 3x amount of 1,5,7- triazabicyclo[4.4.0]dec-5-ene (TBD) and benzyl alcohol diluted with anhydrous DCM. The mixture was vortexed for 30 s to achieve homogeneity. A stock solution of all types of monomers in DCM was prepared under nitrogen atmosphere. Added one third of the initiator stock solution to the monomer stock solution and stirred for 25 minutes. The reaction was quenched by acetic acid / acetic anhydride. The mixture was further stirred for 15 minDocket No. 6045.0586WO | S23-094 before analysis. The resulting crude material was dissolved in DCM and dialyzed against MeOH overnight (3.5K cellulose). Concentrated the material remaining after dialysis to a colorless residue.1H NMR samples were prepared by directly diluting a small quantity with CDCl3. GPC samples were prepared by diluting the remaining with anhydrous THF to ~5 mg / mL and filtered through a syringe filters (Nylon, 0.2 um). Comparative Example: Synthesis of Block Ornithine CARTs at low temperature

[0113] In contrast to the one-step polymerization at room temperature that is possible for the statistical copolymers, the lipid carbonate monomers and morpholinone monomers of block- CARTs must be polymerized sequentially in two steps. For substituted morpholinone monomers where R3≠ H (e.g., R3= any hydrocarbyl or substituted hydrocarbyl), the substituted morpholinones can only be polymerized at lower temperatures, such as -78°C, and a third end-capping step with acetic anhydride is necessary to prevent the polymer from depolymerizing.

[0114] The synthesis of a block-CART in which R2 is ornithine is provided as a comparative example. Under a nitrogen atmosphere, benzyl alcohol (R1-OH) stock solution was prepared by injecting a 7x amount of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and benzyl alcohol diluted with anhydrous DCM in a 2-mL reaction vial equipped with a screw cap, a PTFE / silicon septum, and a small-sized magnetic stir bar. The mixture was stirred for 5 min to achieve homogeneity. MTC dodecyl monomer was added to a separate 2-mL reaction vial and dissolved in anhydrous DCM. Ornithine-morpholinone was weighed out in a separate 2-mL reaction vial. Acetic anhydride was weighed out in another separate reaction vial and diluted with anhydrous DCM. Added one seventh of the initiator stock solution to the reaction vial containing MTC dodecyl monomer; stirred for 7 min. Added the solid Ornithine-morpholinone to the reaction vial and stirred until dissolved. The reaction vial was then cooled to –78°C and stirred for 15 min. The polymerization was quenched with the pre-weighed acetic anhydride solution. The resulting crude material was dissolved in DCM and dialyzed against MeOH overnight (3.5K cellulose). Concentrated the material remaining after dialysis to a colorless residue.1H NMR samples were prepared by directly diluting a small quantity with CDCl3. GPC samples were prepared by diluting the remaining with anhydrous THF to ~5 mg / mL and filtered through a syringe filters (Nylon, 0.2 um). Synthesis of compounds of Formulas III-2 and IV-2Docket No. 6045.0586WO | S23-094

[0115] Synthesis of allyl carbonate, which is used as the initiator for synthesis of compounds of Formulas III-2 and IV-2, may be performed as follows.

[0116] diol (121g, 0.694mol) was dissolved in 700mL dichloromethane in a 2L round bottom flask. While stirring, the suspension of CDI (146.3g, 0.902 mol) in 300mL dichloromethane was added slowly by pipette in 1 hour. After addition, the reaction volume was reduced to 250mL by rotary evaporator. The resulting orange-brown mixture was washed with deionized water (1L) followed by 1M HCl (3 x 200mL) and deionized water (3 x 200mL). The organic layer was dried with MgSO4 (20g) and filtered through a filter funnel. All volatiles were then removed in vacuo to result in desired product as a slightly yellow liquid. (111g, 80% yield)1H NMR (500 MHz, cdcl3) δ 5.85 (ddt, J = 17.3, 10.3, 5.6 Hz, 1H), 5.25 (dq, J = 17.2, 1.6 Hz, 1H), 5.19 (dq, J = 10.4, 1.4 Hz, 1H), 4.32 (d, J = 11.0 Hz, 2H), 4.13 (d, J = 11.0 Hz, 2H), 3.97 (dt, J = 5.6, 1.4 Hz, 2H), 3.39 (s, 2H), 1.52 (q, J = 7.6 Hz, 2H), 0.91 (t, J = 7.6 Hz, 3H).13C NMR (126 MHz, cdcl3) δ 148.60, 134.07, 117.55, 72.86, 72.51, 68.35, 35.56, 23.42, 7.43.

[0117]

[0118] PTFE / silicon septum, and a small-sized magnetic stir bar was evacuated and backfilled with nitrogen, to which initiator stock solution was prepared by injecting a 3x amount of TBD and benzyl alcohol diluted with anhydrous DCM. The mixture was vortexed for 30 s to achieve homogeneity. A stock solution of all types of monomers (# of type can be greater than 2) in DCM was prepared under nitrogen atmosphere. Added one third of the initiator stock solution to the monomer stock solution and stirred for 25 minutes. The reaction was quenched by acetic acid.Docket No. 6045.0586WO | S23-094 The mixture was further stirred for 15 min before analysis. Dissolved the crude material in DCM and dialyzed against MeOH overnight (3.5K cellulose). Concentrated the material remaining after dialysis to result in desired polymer A as a colorless residue. 1H NMR samples were prepared by directly diluting a small quantity with CDCl3. GPC samples were prepared by diluting the remaining with anhydrous THF to ~5 mg / mL and filtered through a syringe filters (Nylon, 0.2 um). All microsyringes were washed by DCM for three times before use.

[0119] Thiol-to mol of total alkene groups) and AIBN (5 mol% relative to alkene) in dry THF and heated at 55oC for 4 hours. The reaction mixture was dialyzed against MeOH overnight (3.5K cellulose). Concentrated the material remaining after dialysis to result in desired polymer B as a colorless residue. 1H NMR samples were prepared by directly diluting a small quantity with CDCl3. GPC samples were prepared by diluting the remaining with anhydrous THF to ~5 mg / mL and filtered through a syringe filters (Nylon, 0.2 um). All microsyringes were washed by DCM for three times before use.total mol of -OH groups), DMAP (0.5 eq relative to total mol of -OH groups) and R3COOH (1.2 eq relative to total mol of -OH groups) in DCM in a 20mL scintillation vial and stirred for 3 hours at room temperature. The reaction mixture was dialyzed against MeOH overnight (3.5K cellulose). Concentrated the material remaining after dialysis to result in desired acid- functionalized product as a colorless residue. 1H NMR samples were prepared by directly diluting a small quantity with CDCl3. GPC samples were prepared by diluting the remaining with anhydrous THF to ~5 mg / mL and filtered through a syringe filters (Nylon, 0.2 um). All microsyringes were washed by DCM for three times before use.Docket No. 6045.0586WO | S23-094

[0121] Synthesis for compounds of Formulas IV-3

[0122] , thionylchloride (2 eq) in DCM(30 mL) was added slowly in nitrogen atmosphere under an ice bath. The reaction mixture was stirred in an ice bath for 1 hour and then for an additional hour at room temperature. The solution was slowly added to a diethanolamine (5 eq) DCM (50 mL) solution in an ice bath. The reaction was brought to ambient temperature and stirred overnight (16 h). The reaction was washed three times with 1M HCl, Na2HCO3solution and brine. The organic phase was separated, dried over Na2SO4, filtered, and concentrated in vacuo. The intermedia was further purified with chromatography.a was KBr (1 eq), NaHCO3 (1.5 eq), TEMPO (0.1 eq), and 100 mL DCM. Intermedia (1 eq) was then added using a 50 mL DCM to transfer. The resultant orange slurry was stirred vigorously at room temperature and bleach was added in a dropwise fashion. The reaction was monitored by thin layered chromatography (TLC). When the reaction was complete, about100 mL water was added, the orange organic layer was removed, and the aqueous phase was extracted with DCM (2 x 100 mL). The combined organic layers were washed with 150 mL each of water, saturated sodium bicarbonate, and brine. The organic layer was then dried over MgSO4, filtered, and concentrated to yield the crude product, which was purified further by chromatography. Compositions

[0124] Also provided are compositions, including pharmaceutical compositions, comprising a compound as described herein and a carrier or excipient, including a pharmaceutically acceptable carrier or excipient.Docket No. 6045.0586WO | S23-094

[0125] In aspects, the composition or pharmaceutical composition comprises a compound of Formula I-IV, Formula Ia-IVa, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In aspects, the complexes condense to form nanoparticles ranging in size from 50-500 nanometers (nm) in diameter or from 100-300 nm. In this context, size refers to the mean particle size (Z). Thus, the disclosure also provides compositions or pharmaceutical compositions comprising nanoparticles of a compound of Formula I-IV, Formula Ia-IVa, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids.

[0126] In aspects, provided is a composition or pharmaceutical composition comprising a compound of Formula I-IV, Formula Ia-IVa, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids, wherein the compound is SC-1, SC- 2, SC-3, SC-4, SC-6, SC-7, SC-8, SC-9, SC-10, SC-11, SC-12, SC-13, SC-14, SC-15, SC-17, SC-18, SC-21, SC-22, SC-23, SC-24, SC-25, SC-26, SC-27, SC-28, SC-29, SC-30, SC-31, SC- 33, SC-35, SC-36, SC-37, SC-38, SC-39, SC-40, SC-40, SC-41, SC-42, SC-43, SC-44, SC-45, SC-46, or SC-47.

[0127] In aspects, the nucleic acid is an RNA or DNA. In aspects, the nucleic acid is messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing. In aspects, the nucleic acid is mRNA or saRNA.

[0128] In aspects, the nucleic acid is a therapeutic agent. In aspects, the nucleic acid encodes a therapeutic agent.

[0129] In aspects, the nucleic acid includes one or more vectors, which may be eukaryotic expression vectors, bacterial plasmid vectors or viral vectors. In some aspects where the vector is a eukaryotic expression vector, the vector may include (a) a first polynucleotide encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence in the genome of eukaryotic cell, and (b) a second polynucleotide encoding a Cas9 protein, optionally wherein the Cas9 protein is codon optimized for expression in the cell. In aspects, the first (a) and second (b) polynucleotides are located in the same or different vectors. In aspects, the nucleic acid comprises a CRISPR RNA (crRNA), optionally wherein the crRNA is in the same plasmid vector as the first nucleotide sequence. In aspects, the nucleic acid comprises a transactivatingDocket No. 6045.0586WO | S23-094 RNA (tracrRNA). In aspects, the tracrRNA is optionally in the same vector as the second nucleotide sequence.

[0130] In aspects, the nucleic acid includes (a) a first polynucleotide encoding a transposase; and (b) a second polynucleotide comprising a nucleic acid sequence of a gene of interest flanked by a transposase recognition site. In aspects, the first (a) and second (b) polynucleotides are located in the same or different vectors. In accordance with this aspect, the transposase recognizes and excises a genomic sequence of interest.

[0131] In general, a compound of Formula I-IV, Formula Ia-IVa, or subformula thereof, is complexed with nucleic acid in an amount effective to produce a theoretical charge ratio of cationic compound to anionic nucleic acid of from about 2:1 to about 30:1 (cation:anion). In aspects, the theoretical charge ratio is 2:1, 3:1, 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1. Theoretical (+ / -) charge ratios are calculated as moles of ammonium cations to moles of phosphate anions, assuming full amine protonation and phosphate deprotonation. In some aspects, the charge ratio (+ / -) is 3:1, or 5:1. Complexation of nucleic acid with a compound described herein may take place in the presence of a coordinating metal such as Zn+2, Mg+2, Ca+2; a dynamic non- covalent cross linker such as a carbohydrate; a counterion such as Cl-, AcO-, succinate, or citrate; or a solubility modulator such as a lipid or a polyethyleneglycol (PEG), or any combination thereof.

[0132] In other aspects, the composition or pharmaceutical composition comprises a compound of Formula I-IV, Formula Ia-IVa, or a subformula thereof, without a nucleic acid cargo, for example where the nucleic acid is to be complexed at a later time such as before administration of the pharmaceutical composition to a subject.

[0133] In some aspects, the composition is a vaccine. In aspects where the composition is a vaccine, the composition may further include an immunological adjuvant. The immunological adjuvant can include, but is not limited to, agonists of Toll-like Receptors (TLRs), agonists of the STING pathway, agonistic antibodies against CD40, OX40, CTLA4, PD1, or PD1-L, Freund’s adjuvant, bryostatins, PKC modulators, and ligands for CD40, OX40, CD137, PD1, CTLA4 and any combinations thereof.

[0134] Pharmaceutically acceptable carriers or excipients include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum,Docket No. 6045.0586WO | S23-094 gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Additional acceptable carriers, excipients, or stabilizers may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0135] In some aspects, the composition or pharmaceutical composition may include a cryoprotectant agent. Non-limiting examples of cryoprotectant agents include a glycol (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combinations thereof.

[0136] In some aspects, the composition can be lyophilized with a cryoprotectant agent, such as described above. Such lyophilized compositions are stable at room temperature for at least 20 days and at -20 °C or 4 °C, the composition is stable for at least 40 days.

[0137] Accordingly, in an aspect provided is a stable lyophilized composition or pharmaceutical composition comprising a compound of Formula I-IV, Formula Ia-IVa, or subformula thereof, and a cryoprotective agent, optionally selected from a glycol, dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, or any combinations thereof, wherein the composition is stable at room temperature for at least 20 days, or at -20 °C or 4 °C for at least 40 days, and wherein the compound of Formula I-IV, Formula Ia-IVa, or subformula thereof retains its activity as a nucleic acid delivery vehicle. In some aspects, the stable lyophilized composition is formulated with 5% sucrose. Methods for Nucleic Acid DeliveryDocket No. 6045.0586WO | S23-094

[0138] The disclosure also provides methods for delivery of nucleic acids to cells and / or tissues in vitro, ex vivo, or in vivo. In aspects, the methods comprising contacting a target cell or tissue with a composition comprising nanoparticulate particles of a nucleic acid complexed with a compound of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof. In aspects, the methods comprise administering to a subject a composition comprising nanoparticulate particles of a nucleic acid complexed with a compound of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof, including a pharmaceutical composition or vaccine. Administration may be according to any suitable route, for example, parenteral, including e.g., intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intracranial, etc., or transmucosal, including e.g., buccal, nasal, sublingual, transdermal, etc. In aspects, the subject is a mammal, for example a human, a non-human primate, a murine (i.e., mouse and rat), a canine, a feline, or an equine. In embodiments, the subject is a human.

[0139] In an aspect, provided is a method of transfecting a nucleic acid into a cell in vitro or ex vivo, the method comprising contacting the cell in vitro or ex vivo with a compound of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In aspects, the complexes are in the form of nanoparticles. In aspects, the compound is SC-1, SC-2, SC-3, SC- 4, SC-6, SC-7, SC-8, SC-9, SC-10, SC-11, SC-12, SC-13, SC-14, SC-15, SC-17, SC-18, SC-21, SC-22, SC-23, SC-24, SC-25, SC-26, SC-27, SC-28, SC-29, SC-30, SC-31, SC-33, SC-35, SC- 36, SC-37, SC-38, SC-39, SC-40, SC-40, SC-41, SC-42, SC-43, SC-44, SC-45, SC-46, or SC- 47.

[0140] In an aspect, provided is a method of delivering a nucleic acid to a cell in a subject, the method comprising administering to the subject the composition or pharmaceutical composition comprising non-covalent complexes of a compound of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof, with the nucleic acid. In aspects, the complexes are in the form of nanoparticles. In aspects, the compound is SC-1, SC-2, SC-3, SC-4, SC-6, SC-7, SC-8, SC- 9, SC-10, SC-11, SC-12, SC-13, SC-14, SC-15, SC-17, SC-18, SC-21, SC-22, SC-23, SC-24, SC-25, SC-26, SC-27, SC-28, SC-29, SC-30, SC-31, SC-33, SC-35, SC-36, SC-37, SC-38, SC- 39, SC-40, SC-40, SC-41, SC-42, SC-43, SC-44, SC-45, SC-46, or SC-47.

[0141] In an aspect, provided is a method of delivering a nucleic acid to the spleen of a subject, where the method includes administering to the subject a composition or pharmaceutical composition including complexes of a compound of Formula I-IV or aDocket No. 6045.0586WO | S23-094 compound of Formula Ia-IVa, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In aspects, the compound is SC-2, SC-7, or SC- 21.

[0142] In an aspect, provided is a method of delivering a nucleic acid to the lung of a subject, where the method includes administering to the subject a composition or pharmaceutical composition including complexes of a compound of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In aspects, the compound is SC-22.

[0143] In accordance with any of the foregoing methods, the nucleic acid may be RNA or DNA. In some aspects, the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing.

[0144] In aspects, the nucleic acid may be a therapeutic nucleic acid or the nucleic acid may encode one or therapeutic agents, for example a cytokine, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

[0145] In some aspects, a method of transfecting a nucleic acid into a cell as described herein may be part of a method for gene editing or genetic engineering. For example, one or more nucleic acids may be transfected using the methods described herein in a CRISPR-based system or a transposon-based system for gene editing or genetic engineering. Accordingly, one or more nucleic acids may be transfected according to the methods described here, which nucleic acids may be located on one or more vectors. For example, the one or more nucleic acids may comprise a vector having a first nucleotide sequence encoding a CRISPR-Cas system guide RNA (gRNA) that hybridizes with a target sequence in the genome of a target cell and a second nucleotide sequence encoding a Cas9 protein. Alternatively, the gRNA and Cas9 protein can be located on different vectors, or either the gRNA or Cas9 protein may be produced in the target cell. The one or more nucleic acids may also comprise a CRISPR RNA (crRNA) and / or transactivating RNA (tracrRNA), each of which may be located on the same or a different vector as the gRNA and / or Cas9 encoding sequence.Docket No. 6045.0586WO | S23-094

[0146] In another aspect, the one or more nucleic acids may comprise a sequence encoding a transposase and a nucleic acid sequence of a gene of interest flanked by a transposase recognition site, which may be located on the same or different vectors.

[0147] In some aspects, a compound of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof, may be complexed with one or more nucleic acids encoding one or more antigenic or immunogenic epitopes or peptides which may form a vaccine composition. In some aspects, a mixture of two or more different compounds of Formula I-IV or a compound of Formula Ia-IVa, or subformula thereof, may be complexed with one or more nucleic acids encoding one or more antigenic or immunogenic epitopes or peptides.

[0148] The present inventors unexpectedly discovered that statistical co-polymers of high molecular weight were effective to transport nucleic acids into cells both in vitro and in vivo, where for mRNA the encoded protein was also expressed with high efficiency in vitro and in a tissue specific manner in vivo, as discussed in the following paragraphs. This was in contrast to expectations based on similar polymers of high molecular weight having a block distribution of monomers, which are ineffective both in transport and protein expression in vitro and in vivo.

[0149] HeLa cells were transfected with a series of compounds of Formula IV-1 complexed with firefly luciferase (fLuc) mRNA in a 10:1 charge ratio. Protein expression measured as luminescence is shown in FIG.1 for representative compounds. These data show that both high molecular weight (SC-4 and SC-7) and low molecular weight (SC-1, SC-2, SC-3) compounds exhibit comparable or better levels of protein expression than a representative block copolymers (BC-1) of similar structure. Also tested in Hela cells were compounds SC-21, SC- 22, BC-1, and BC-6. Data is shown in Table 10 below.

[0150] Table 10: in vitro protein expression measured as average (n=6) luminescence (arbitrary units) including standard deviations (std) for representative compounds. Compound avg std cell line SC-1 2141 321 HeLa kyoto SC-2 1907 331 HeLa kyoto SC-3 2983 426 HeLa kyotoDocket No. 6045.0586WO | S23-094 SC-4 687 101 HeLa kyoto SC-6 320 75 HEK 293T SC-7 1434 253 HeLa kyoto SC-8 438 60 HEK 293T SC-9 473 69 HEK 293T SC-10 442 103 HEK 293T SC-11 620 117 HEK 293T SC-12 724 208 HEK 293T SC-13 1575 535 MDA-MB-231 SC-14 414 117 MDA-MB-231 SC-21 1257 223 HeLa kyoto SC-22 551 171 HeLa kyoto BC-1 930 138 HeLa kyoto BC-2 458 223 HEK 293T BC-3 20 10 HEK 293T BC-6 426 90 HeLa kyoto BC-ONA 527 384 HEK 293TDocket No. 6045.0586WO | S23-094

[0151] Representative compounds of low and high molecular weight or low and high molecular weight block copolymers of similar structure were transfected with fLuc mRNA into HEK293T cells. Protein expression was measured as luminescence. FIG.2 shows that, unexpectedly, both high (SC-6, DP 99) and low (SC-12, DP 25) molecular weight compounds of Formula Formula IV-1 resulted in effective protein expression. In contrast, only the low molecular weight (BC-2, DP 25 and BC-ONA, DP 18) block copolymers elicited protein expression. The high molecular weight block copolymer (BC-3, DP 110) was ineffective. Also tested in HEK293T cells were compounds SC-11, SC-9, and BC-ONA. Data is shown above in Table 10.

[0152] Both high and low molecular weight statistical copolymers elicited high levels of protein expression from mRNA delivered into cells in vitro while similar high molecular weight block copolymers were ineffective. FIG.3A shows protein expression in A549 cells transfected with representative compounds including high molecular weight compounds (SC-6, DP 99, SC-11, DP 76) and a low molecular weight compound (SC-10, DP 23), or high (BC-3, DP 110, BC-4, DP 71) and low (BC-2, DP 25, BC-ONA, DP 18) molecular weight block copolymers, each complexed with mRNA encoding enhanced green fluorescent protein (eGFP). Protein expression is measured as eGFP fluorescence.

[0153] Statistical copolymers of both high and low molecular weight were able to deliver high amounts of mRNA into cells in vitro. In contrast, only the low molecular weight block copolymers were effective to transport nucleic acid into cells. FIG.3B shows mRNA uptake in the same A549 cells transfected with the same compounds as FIG.3A. In FIG.3B mRNA uptake is measured as Cy5 fluorescence.

[0154] Statistical copolymers were also effective for protein expression in vivo, showing comparable or higher levels of protein expression compared to reference block copolymers that elicit high in vivo protein expression. FIG.4 shows protein expression in mice following retro- orbital injection of fLuc mRNA complexed with representative statistical copolymers (SC-2, SC-7, SC-21, SC-1, SC-4) or reference block copolymers (BC-ONA, BC-1), measured as luminescence in photons / second (p / s). All formulations had a 10:1 (+ / -) charge ratio. Mice were imaged after 6 hours.

[0155] Additional in vivo expression data is provided in Table 11 below. In these experiments, complexes of fLuc mRNA with compound were administered by intramuscular injection. EachDocket No. 6045.0586WO | S23-094 formulation was divided into two doses which were each injected into a separate flank of the same mouse. Anesthetized mice were monitored for 4 hours after injection. Prior to imaging, mice were treated with substrate d-luciferin via intraperitoneal injection. Mice were imaged after 6 minutes on a Spectral instruments Imaging Kino imager with a 30 second exposure time. Results are reported as average (n=2 mice) with standard deviation (std).

[0156] Table 11: in vivo protein expression measured as average luminescence (p / s) including standard deviations (std) for representative compounds complexed with fLuc mRNA. Charge ratios are indicated. Compound avg std charge ratio SC-13 2.55E+07 1.06E+07 5:1SC-14 2.69E+07 1.31E+07 1:1 SC-15 8.29E+06 6.00E+04 5:1SC-10 6.02E+07 1.37E+07 5:1SC-11 1.07E+08 8.90E+06 2:1SC-47 3.23E+06 9.60E+05 3 BC-2 4.64E+07 3.85E+07 10:1BC-3 6.11E+06 3.02E+06 10:1BC-6 2.50E+07 1.06E+07 1:1 BC-7 1.30E+07 4.71E+06 25:1BC-ONA 9.41E+07 2.70E+07 2:1Docket No. 6045.0586WO | S23-094

[0157] Additional experiments were performed with a different RNA cargo, a self-amplifying fLuc RNA (fLuc saRNA). In these experiments, complexes of fLuc saRNA with compound were administered by intramuscular injection. Each formulation was divided into two doses which were each injected into a separate flank of the same mouse. Mice were imaged on day 10 after injection (day 0). Prior to imaging, mice were treated with substrate d-luciferin via intraperitoneal injection. Mice were imaged after 6 minutes on a Spectral instruments Imaging Kino imager with a 30 second exposure time. Results are reported as average (n=2 mice) with standard deviation (std).

[0158] Compared to mRNA, self-amplifying RNA has a delayed peak expression as well as a prolonged expression time, which accounts for the difference in imaging times after administration of the mRNA complexes versus the saRNA complexes.

[0159] Table 12: in vivo protein expression measured as average luminescence (p / s) including standard deviations (std) for representative compounds complexed with fLuc saRNA. Charge ratios are indicated. Compound avg std charge ratio BC-ONA 1.28E+07 3.56E+06 10:1SC-23 3.03E+07 5.60E+06 7:1SC-24 1.14E+06 5.71E+05 7:1SC-25 3.56E+06 3.48E+05 7:1SC-26 2.76E+07 3.66E+06 7:1SC-27 1.07E+07 4.43E+06 7:1SC-28 8.75E+06 7.17E+06 7:1SC-29 1.37E+06 7.63E+05 7:1Docket No. 6045.0586WO | S23-094 SC-30 1.58E+07 3.92E+06 7:1 SC-31 2.21E+07 7.67E+06 7:1 SC-33 9.31E+06 4.88E+06 7:1 SC-35 1.22E+07 6.39E+06 7:1 SC-36 4.08E+07 2.35E+07 7:1 SC-37 4.23E+06 8.07E+04 7:1 SC-38 1.84E+07 1.29E+06 7:1 SC-39 5.26E+06 3.17E+05 7:1 SC-40 5.91E+05 4.63E+05 7:1 SC-41 1.56E+05 2.64E+04 7:1 SC-42 2.10E+05 1.29E+05 7:1 SC-43 6.01E+06 2.32E+06 7:1 SC-44 1.51E+05 4.40E+04 7:1 SC-45 3.41E+05 2.32E+05 7:1 SC-46 1.09E+05 2.08E+04 7:1 SC-47 2.38E+08 1.25E+07 7:1

[0160] Statistical copolymers also showed organ specific protein expression in vivo. As shown in FIG.5, compounds containing N-hydroxyethyl glycine monomer units exhibited high proteinDocket No. 6045.0586WO | S23-094 expression in the spleen (SC-2, SC-7, SC-21) and a compound containing N-hydroxyethyl- ornithine monomer units (SC-22) exhibited high protein expression in the lungs. The sequence of monomer units did not significantly alter the organ tropism.

[0161] Similar tropism for the spleen and lung was observed for a block copolymers of similar structure, BC-1, containing N-hydroxyethyl glycine monomer units, and BC-6, containing N- hydroxyethyl-ornithine monomer units.

[0162] FIG.6 illustrates the high protein expression in spleen versus liver elicited by compounds including N-hydroxyethyl glycine monomer units (SC-2, SC-7). Similar expression was seen with a representative block copolymer (BC-ONA). Protein expression in liver was lower than that in spleen by more than two orders of magnitude. Results are shown for independent, duplicate injections of SC-2, SC-7.

[0163] Another surprising property of the statistical copolymers was the stability of the nanoparticles formed when the compounds were complexed with nucleic acid. FIG.7A shows the change in diameter (measured as hydrodynamic radii) of the particles over time. Duplicate runs are illustrated for compounds SC-1, SC-7, and a reference block copolymer, BC-1. As illustrated in the figure, the nanoparticles formed from complexes of SC-1 or SC-7 retained their size over time while those formed from the representative block copolymer did not.

[0164] FIG.7B shows the change in zeta potential of the nanoparticles over time, illustrating a similar stability in surface charge of the particles formed from complexes of SC-1 or SC-7 compared to the reference block copolymer, BC-1. The initial size and charges are given in the table below.

[0165] Table 13. Initial average diameter and charge of nanoparticles Compound Z-avg (nm) zeta potential (mV0 BC-1 164 + / - 14 32 + / - 7 SC-1 214 + / - 3 39 + / - 2 SC-7 185 + / - 7 68 + / - 5

[0166] Compounds of Formula IV-2 elicited unexpectedly high in vivo protein expression. As shown in FIG.8A, compounds SC-17 and SC-18 complexed with fLuc mRNA elicited robustDocket No. 6045.0586WO | S23-094 protein expression in mice following intravenous injection. In contrast, a representative block copolymer did not elicit any protein expression.

[0167] FIG.8B shows in vivo protein expression for compounds SC-17 and SC-18 complexed with fLuc mRNA compared to a reference block copolymer, BC-ONA, previously shown to elicit high protein expression.

[0168] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0169] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical sciences.

[0170] The terms “oligomer” and “polymer” are used interchangeably herein to refer to a compound that has a plurality of repeating subunits, which may be referred to as blocks or monomer units, or simply as monomers. The terms “co-oligomer” or “copolymer” are used interchangeably herein to refer to an oligomer or polymer that includes two or more different types of monomers.

[0171] The term “initiator” refers to a compound that initiates a polymerization reaction. Thus, the initiator is typically incorporated at the end of the synthesized polymer. For example, a plurality of monomer molecules can be reacted with an initiator to provide a polymer or copolymer. The initiator can be present on at least one end of the resulting polymer or copolymer and not constitute a repeating monomer unit of the polymer.

[0172] The term “small organic molecule” refers to a small organic compound, including heteroorganic and organometallic compounds, having a molecular weight less than about 1,000 grams per mole, or less than about 500 grams per mole and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0173] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.Docket No. 6045.0586WO | S23-094

[0174] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details that are not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0175] In the claims, the term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. The transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the recited embodiment. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

Claims

Docket No. 6045.0586WO | S23-094 CLAIMS What is claimed is:

1. A compound of Formula I, R1-X-Y-[(L1)x1-stat-(L2)x2-stat-(A2)y2-stat-(A1)y1]-R2 (Formula I) wherein R1 is substituted or unsubstituted C1-C24 alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl; X is a bond or linking group; Y is a bond, O, S, or N; L1 and L2 are each a lipid carbonate monomer unit selected from either a lipid- functionalized methyl-trimethylene carbonate (MTC) unit, or a lipid-functionalized ethyl- trimethylene carbonate (ETC) unit; A1 and A2 are each independently an alpha aminoester monomer unit having a structure ofor substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4- OH, -(CH2)2-S-CH3,-CH2-C6H5or -CH(CH3)2; x1 and y1 are each independently greater than zero and less than 1,000; x2 and y2 are each independently zero or less than 1,000; and R2is H, C2-C24alkylcarbonyl, alkenylcarbonyl, substituted or unsubstituted alkyl or alkenyl, including pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and octadecenyl, optionally where the alkyl or alkenyl is substituted with one or more of acyl, C2-C6alkyl, and carboxylato.Docket No. 6045.0586WO | S23-094 2. The compound of claim 1, wherein x2 is zero and the compound is represented by Formula II: R1-X-Y-[(L1)x1-stat-(A2)y2-stat-(A1)y1]-R2 (Formula II), where R1, X, Y, L1, A1, A2, R2, x1, y1, and y2 are as defined in claim 1.

3. The compound of claim 2, wherein the compound is represented by Formula II-1: or unbranchedC1-C30 alkyl, which may be fully saturated, mono- or polyunsaturated, and RA1 and RA2 are each independently H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4- OH, -(CH2)2-S-CH3, -CH2-C6H5 or -CH(CH3)2.

4. The compound of claim 1, wherein y2 is zero and the compound is represented by Formula III: R1-X-Y-[(L1)x1-stat-(L2)x2-stat-(A1)y1]-R2 (Formula III), where R1, R2, X, Y, L1, L2, A1, x1, x2, and y1 are as defined in claim 1.

5. The compound of claim 4, wherein the compound is represented by Formula III-1:independently a branched or unbranched C1-C30alkyl, which may be fully saturated, mono- or polyunsaturated, and RA1 is H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4- OH, -(CH2)2-S-CH3,-CH2-C6H5or -CH(CH3)2.Docket No. 6045.0586WO | S23-094 6. The compound of claim 4, wherein the compound is represented by Formula III-2:independently octyl, dodecyl, octadecyl; methyl, ethyl, propyl, or butyl substituted C2-C12 alkyl, haloalkyl, or alkylcarbonyl; substituted or unsubstituted C2-C12alkoxycarbonyl; linolenyl, linoleoyl, or oleoyl; C12-C20alkenylcarbonyl; aryl or heteroaryl; substituted 5 membered cycloheteroalkenyl; or substituted or unsubstituted dilinolenyl, dilinoleoyl, or dioleoyl; and RAis H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S- CH3, -CH2-C6H5 or -CH(CH3)2.

7. The compound of claim 1, wherein x2 and y2 are both zero, and the compound is represented by Formula IV: R1-X-Y-[(L1)x1-stat-(A1)y1]-R2 (Formula IV), where R1, X, Y, L1, A1, R2, x1 and y1 are as defined in claim 1.

8. The compound of claim 7, wherein the compound is represented by Formula IV-1:C1- C30 alkyl, which may be fully saturated, mono- or polyunsaturated, and RA1 is H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -Docket No. 6045.0586WO | S23-094 (CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S-CH3, -CH2-C6H5 or - CH(CH3)2.

9. The compound of claim 7, wherein the compound is represented by Formula IV-2: whereoctadecyl; methyl, ethyl, propyl, or butyl substituted C2-C12 alkyl, haloalkyl, or alkylcarbonyl; substituted or unsubstituted C2-C12alkoxycarbonyl; linolenyl, linoleoyl, or oleoyl; C12-C20alkenylcarbonyl; aryl or heteroaryl; substituted 5 membered cycloheteroalkenyl; or substituted or unsubstituted dilinolenyl, dilinoleoyl, or dioleoyl; and RAis H, methyl, ethyl, aminoalkyl, substituted alkyl, or substituted aryl, optionally H, methyl, ethyl, -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S- CH3, -CH2-C6H5 or -CH(CH3)2.

10. The compound of any one of claims 1 to 9, the sum of x1, x2, y1, and y2 is from 5-1,000, 5-800, 5-600, 5-300, 5-200, or 5-100.

11. The compound of claim 10, wherein the sum of x1, x2, y1, and y2 is from 50-1,000, 50- 800, 50-600, 50-300, 50-200, or 50-100.

12. The compound of any one of claims 1 to 11, wherein R1is substituted C1-C24alkyl, C1-C24heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl.

13. The compound of claim 12, R1 is substituted or unsubstituted heteroaryl.

14. The compound of claim 12, wherein R1is substituted or unsubstituted benzyl or phenyl.Docket No. 6045.0586WO | S23-094 15. The compound of any one of claims 12 to 14, wherein the substituent is hydroxyl, sulfhydryl, C1-C24alkoxy, C2-C24alkenyloxy, C5-C20aryloxy, acyl, acyloxy, C2-C24alkoxycarbonyl, amino, alkylamido, C2-C24 alkylcarbonato, C6-C20 arylcarbonato, carboxy, carboxylato, carbamoyl, alkylcarbamoyl, thiocarbamoyl, carbamido, formyl, thioformyl, mono- and di-(C1-C24alkyl)-substituted amino, mono- and di-(C5-C20aryl)-substituted amino, C2-C24alkylamido, C5-C20arylamido, sulfo, sulfonato, C1-C24alkylsulfanyl, arylsulfanyl, C1-C24alkylsulfinyl, C5-C20 arylsulfinyl, C1-C24 alkylsulfonyl, C5-C20 arylsulfonyl, C1-C24 alkyl, C2- C24alkenyl, C5-C30aryl, or C6-C30aralkyl.

16. The compound of any one of claims 1 to 15, wherein R1is a molecule useful for in vivo imaging or a ligand moiety that binds to a cell-surface receptor and R1 is joined to the copolymer via a linking group.

17. The compound of claim 16, wherein R1is a fluorophore, optionally difluoroboron-β- diketonate (BDK), a “Black Hole Quencher” dye, or a naphthalene derivative, including dansyl derivatives such as 5-(dimethylamino)-N-(2-hydroxyethyl)naphthalene-1-sulfonamide.

18. The compound of claim 16, wherein R1is a ligand moiety, optionally a saccharide, a disaccharide, an oligosaccharide, a liposaccharide, a lipid, a peptide, an antibody, biotin, a biotin derivative, or fingolimod.

19. The compound of any one of claims 1 to 18, wherein X is substituted or unsubstituted alkylene, heteroalkylene, alkenylene, arylene, heteroarylene, aralkylene, or alkarylene.

20. The compound of claim 19, wherein X is alkylene, heteroalkylene, alkenylene, arylene, heteroarylene, aralkylene, or alkarylene substituted with one or more functional groups.

21. The compound of claim 20, wherein the one or more functional groups is oxo, amine, carbonyl, carbonate, or sulfonamide.

22. The compound of any one of claims 19 to 21, wherein X is substituted or unsubstituted heteroalkylene comprising one or more heteroatoms selected from O, S, or N.

23. The compound of any one of claims 1 to 22, wherein RA is H, methyl, ethyl, -CH2-NH2, - (CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2,-CH2-C6H4-OH, -(CH2)2-S-CH3, -CH2-C6H5 or - CH(CH3)2Docket No. 6045.0586WO | S23-094 24. A composition or pharmaceutical composition comprising nanoparticles of the compound of any one of claims 1 to 23 non-covalently complexed with a nucleic acid or a plurality of different nucleic acids.

25. The composition or pharmaceutical composition of claim 24, wherein the nucleic acid is RNA or DNA.

26. The composition or pharmaceutical composition of claim 25, wherein the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing.

27. The composition or pharmaceutical composition of any one of claims 24 to 26, wherein the nucleic acid is a therapeutic agent or the nucleic acid encodes a therapeutic agent.

28. The composition or pharmaceutical composition of any one of claims 24 to 27, wherein the composition is a vaccine.

29. A method of transfecting a nucleic acid into a cell in vitro or ex vivo, the method comprising contacting the cell in vitro or ex vivo with the composition or pharmaceutical composition of any one of claims 24 to 28.

30. A method of delivering a nucleic acid to a cell in a subject, the method comprising administering to the subject the composition or pharmaceutical composition of any one of claims 24 to 28.

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