Cationic polymers and polymer conjugates for delivery of biological agents

Abstract

Provided herein are cationic polymers containing titratable end groups, which polymers are able to bind negatively -charged biological agents (e.g., macromolecules, such as RNA) by electrostatic interactions. The titratable end groups act as proton sponges at mildly acidic pH, allowing endosomal escape of polymers containing the titratable end groups and bound biological agents. Also provided are conjugates between said cationic polymers and polymers containing nitrophenylboronic acid (NPBA) and polyethylene glycol (PEG). Also provided are nanoparticles containing said cationic polymers and said conjugates of cationic polymer and NPBA-PEG. The NPBA-PEG polymers optionally comprise one or more targeting agents, such as antibodies, which are capable of directing the nanoparticles to a cellular target such as a T-cell. Nanoparticles can also comprise, in addition to a biological agent and / or a targeting agent, a therapeutic agent, such as a large-molecule therapeutic.

Description

CATIONIC POLYMERS AND POLYMER CONJUGATESFOR DELIVERY OF BIOLOGICAL AGENTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority U.S. Provisional Appl. No. 63 / 588,162, filed Oct. 5, 2023, which is incorporated by reference in its entirety.FIELD

[0002] The present disclosure is in the field of polymers, and nanoparticles comprising said polymers, for use in delivery of biological agents; for example, therapeutic molecules, particularly negatively-charged molecules such as nucleic acids.BACKGROUND

[0003] Therapeutic uses of RNA include the use of large RNAs, such as messenger RNA (mRNA), for adoptive immunotherapy and smaller RNAs, such as miRNAs, for treatment of tumors.

[0004] Adoptive immunotherapy is a process in which a customized T-cell population, directed to a target molecule (such as an antigen), is generated by expressing, in a T-cell, a nucleic acid encoding a binding site for the target molecule; e.g., a chimeric antigen receptor (CAR). Certain methods for adoptive immunotherapy require isolation and enrichment of T-cells from a subject; transfection of the isolated T-cells with a nucleic acid (e.g., a mRNA) encoding the binding site for the target molecule; and infusion of the transfected T-cells back into the subject; all of which steps are complicated and involve losses of material.

[0005] An alternative strategy is to introduce a nucleic acid encoding a CAR into a subject, such that the nucleic acid is targeted to a T-cell. In these methods, the introduced mRNA enters the target cell via receptor-mediated endocytosis and is deposited into an endosome of the target T-cell, from which it must escape to gain access to the cytoplasm of the T-cell, where it can be translated into the CAR. These methods suffer from a number of problems. First, the integrity of the mRNA must be maintained after its delivery into the subject. Second, the mRNA must be effectively and specifically targeted to the desired type of T-cell. Third, once inside the T-cell, theintroduced mRNA must be able to escape the endosome and gain access to the cytoplasm of the T-cell.

[0006] Other therapeutic uses of RNA include treatment of tumors by RNA interference (RNAi), using small RNAs that regulate gene expression, such as siRNA or miRNA. For example, siRNA has attractive features for use as a therapeutic, including: (i) the ability to target essentially any gene (thus, all targets are in principle druggable), (ii) potent, single-digit, picomolar ICso’s (concentration required for 50% inhibition) for mRNA inhibition in well-designed siRNAs, (iii) chemical modifications and sequence designs that can minimize off-target effects and immune stimulation without compromising potency and target specificity, and (iv) a catalytic RNAi mechanism of action, resulting in extended siRNA inhibition of mRNA target expression. In these cases, once again, the therapeutic RNA must remain stable from degradation, be directed effectively to the target cell, and gain access to the correct cellular compartment.

[0007] The therapeutic utility of RNA molecules (both large RNAs such as mRNAs and smaller RNAs such as siRNAs and miRNAs) has been limited due to the lack of effective delivery vehicles (which are able to direct the RNA to its intended target cell and prevent degradation of the RNA en route') and by the sequestration of introduced RNAs in a cellular compartment in which they are not biologically active (i.e., the endosome).

[0008] Attempts to address these problems have included the use of cationic polymers as RNA delivery vehicles, and co-administration of endosomolytic agents (such as chloroquine) to facilitate release of RNA-polymer complexes from the endosome. See, for example, Parayath et al. (2020) Nature Communications 11: 6080-6096 (https: / / doi.org / 10.1038 / s41467-020-19486-2). However, even these methods have provided only moderate efficiency in delivering biologically active RNA to cells in vivo.SUMMARY

[0009] Accordingly, new polymers, new targeting agents, and new methods for endosomal release are all required to facilitate the use of RNA molecules as therapeutics. The present disclosure provides cationic polymers, suitable for delivery of negatively -charged biological agents, such as nucleic acids; and nanoparticles containing said cationic polymers. The polymers also contain chemical groups that act as proton sponges, so that nanoparticles containing saidpolymers, when internalized by a cell into an endosome, are able to exit from the endosome and enter the cytoplasm of a cell.

[0010] Also provided are conjugates of a first cationic polymer with one or more second polymer(s) containing nitrophenylboronic acid (NPBA) and polyethylene glycol (PEG), with the second polymer being bound to the cationic polymer through a reversible (pH-sensitive) covalent complexation between NPBA group(s) of the second polymer and vicinal diols present in the cationic polymer. The second polymer can optionally contain a targeting agent that is conjugated to the PEG-containing end of the NPBA-PEG polymer. The second polymer can also optionally contain a second biological agent and / or a therapeutic agent (e.g., an antibody) that is conjugated to the PEG-containing end of the second polymer.

[0011] Accordingly, provided herein are cationic polymers comprising titratable end groups. Also provided are cationic polymers comprising titratable end groups additionally comprising biological agents, such as, for example, polynucleotides such as mRNA and small RNA molecules such as, for example, siRNA. Also provided are conjugates of (a) a cationic polymer comprising titratable end groups and a biological agent with (b) one or more NPBA / PEG- containing polymers, optionally comprising a second biological agent and / or a targeting molecule and / or a therapeutic molecule.

[0012] Also provided are method for making the polymers and polymer conjugates described herein. Also provided are methods for treatment of diseases and disorders, for example, malignant disorders such as cancers, using the polymers and polymer conjugates described herein.

[0013] In certain aspects, the invention provides a cationic polymer described by the structure of Formula (II):wherein:End Group A and End Group B independently comprise an amine-containing group with a pKa between 4 and 7 inclusive; and cMAP iswherein: m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is independently at each occurrence 0, 1, 2, 3, 4, or 5; and p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 50,000 Da.

[0014] In certain embodiments, in the cationic polymer of Formula (II), m is 4, 5, or 6. In certain embodiments, in the cationic polymer of Formula (II), the amine-containing group is an imidazole group. In certain embodiments, in the cationic polymer of Formula (II), End Group A is:End Group B is:wherein:Li and L2 are linking groups; and k is independently at each occurrence 0, 1, 2, 3, 4, or 5.

[0015] In certain embodiments, Li and L2 are independently at each occurrence:-(Co-6alkylene)-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-,-(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-,-(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or-(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.

[0016] In certain embodiments, Li is -C-C(=O)- and L2 is -C(=O)-C-.

[0017] In certain embodiments, in the cationic polymer of Formula (II), m is 5; n is 1; p is 4-40; k is 1;Li is -C-C(=O)- ; andL2 is -C(=O)-C-

[0018] In certain embodiments, in the cationic polymer of Formula (II), further comprises a biological agent. In certain embodiments, the biological agent is a nucleic acid. In certain embodiments, the nucleic acid is a RNA molecule. In certain embodiments, the RNA molecule is a messenger RNA (mRNA) molecule or a siRNA molecule. In certain embodiments, the mRNA molecule encodes a chimeric antigen receptor (CAR). In certain embodiments, the chimeric antigen receptor is targeted to CD19 or B-cell maturation antigen (BCMA). In certain embodiments, the nucleic acid is bound to the polymer by electrostatic interactions between the backbone of the polynucleotide and amidine groups of the cMAP. In certain embodiments, the biological agent is covalently bound to the cationic polymer. In certain embodiments, the biological agent is bound through a ether, ester, amide or carbamate linkage to the cMAP.

[0019] In certain aspects, the invention provides a polymer conjugate comprising:(a) the cationic polymer comprising a biological agent; and(b) one or more boronic acid-containing polymers comprising a structure ofFormula (III):wherein: the cationic polymer and the boronic acid-containing polymer are reversibly linked to one another by one or more borate condensation linkages between the one or more boronic acidmoiety or moi eties of Formula (III) and one or more pairs of vicinal diols of the cMAP portion of the cationic polymer of Formula (II); the spacer is selected from any linear or branched organic group;L3 is a linking group, or linking groups, between the one or more boronic acid moi eties and the spacer;L4is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages; q is independently at each occurrence 0, 1, 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the foregoing.

[0020] In certain embodiments, in the polymer conjugate of Formula (III), q is 1. In certain embodiments, in the polymer conjugate of Formula (III), r is 2. In certain embodiments, in the polymer conjugate of Formula (III), s is 50-300. In certain embodiments, in the compounds of Formula (III), t is 1. In certain embodiments, in the polymer conjugate of Formula (III), L3 and L4are independently at each occurrence:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-, -(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.

[0021] In certain embodiments, in the polymer conjugate of Formula (III), the spacer is an organic group bearing at least one terminal amine and at least one terminal carboxylic acid.

[0022] In certain embodiments, in the polymer conjugate of Formula (III), wherein the spacer has the formula -NH-CH-Ri-C(=O)-, wherein Ri is an amino acid functional group.In certain embodiments, in the polymer conjugate of Formula (III), wherein(a) Ri is the functional group of lysine;(b) L3is -C(=O)-NH-; and(c) L4is -C(=O)-NH-.

[0023] In certain embodiments, in the polymer conjugate of Formula (III), wherein the spacer is a lysyl group; q is 1; r is 2; s is 50-300; t is 1; for the first instance of r, L3is -C(=O)-NH-; for the second instance of r, L3is -C(=O)-NH-;L4is -C(=O)-NH-; andXi is -CH3or -C-C(=O)-OH.In certain embodiments, the conjugate comprises a plurality of molecules of Formula III, wherein:(a) in at least one of the molecules of Formula III, Xi is -CH3; and(b) in at least one of the molecules of Formula III, Xi comprises a targeting agent.

[0024] In certain embodiments, in the polymer conjugate of Formula (III), wherein the conjugate further comprises one or more targeting molecules. In certain embodiments, the targeting molecule is selected from the group consisting of a protein, a peptide, an aptamer, and a ligand for a cellular receptor. In certain embodiments, the protein is an antibody or an antibody fragment. In certain embodiments, the antibody or antibody fragment is directed to CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR). In certain embodiments, the cellular receptor is CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR). In certain embodiments, the ligand for a cellular receptor is transferrin. In certain embodiments, the targeting molecule is a covalent conjugate of Xi, suchthat Xi has the structure -C-C(=O)-NH-targeting molecule. In certain embodiments, the polymer conjugate further comprises a biological agent. In certain embodiments, the biological agent is an agent described above.

[0025] In certain embodiments, the invention provides a nanoparticle comprising a polymer conjugate provided herein.

[0026] In certain embodiments, the invention provides a pharmaceutical composition comprising the nanoparticles and a pharmaceutically acceptable carrier or excipient.

[0027] In certain embodiments, the invention provides a method for the treatment of a disease or disorder in a subject, the method comprising administering the pharmaceutical composition comprising the polymer conjugates provided herein to the subject. In certain embodiments, the disease or disorder is a malignancy. In certain embodiments, the malignancy is selected from the group consisting of a hematologic malignancy and a solid tumor. In certain embodiments, the hematologic malignancy is a B-cell malignancy. In certain embodiments, the B- cell malignancy is a leukemia or a lymphoma. In certain embodiments, the solid tumor is a breast tumor, a lung tumor, an ovarian tumor, a gastric tumor, a colorectal tumor, a pancreatic tumor, a primary brain tumor or a brain metastasis.DETAILED DESCRIPTION

[0028] The present disclosure may be understood more readily by reference to the following description taken in connection with the accompanying Examples, all of which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of any claimed invention. Similarly, unless specifically otherwise stated, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosure herein is not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement. Throughout this text, it is recognized that the descriptions refer to compositions and methods of making and using said compositions. That is, where the disclosure describes or claims a feature or embodiment associated with a composition or a method of making or using a composition, it is appreciated that such a description or claim is intended to extend these features or embodiment toembodiments in each of these contexts (i.e., compositions, methods of making, and methods of using).

[0029] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a material” is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.

[0030] When a value is expressed as an approximation by use of the descriptor “about,” it will be understood that the particular value forms another embodiment. In general, use of the term “about” indicates approximations that can vary depending on the desired properties sought to be obtained by the disclosed subject matter and is to be interpreted in the specific context in which it is used, based on its function. The person skilled in the art will be able to interpret this as a matter of routine. In some cases, the number of significant figures used for a particular value may be one non-limiting method of determining the extent of the word “about.” In other cases, the gradations used in a series of values may be used to determine the intended range available to the term “about” for each value. Where present, all ranges are inclusive and combinable. That is, references to values stated in ranges include every value within that range.

[0031] It is to be appreciated that certain features of the disclosure which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or specifically excluded, each individual embodiment is deemed to be combinable with any other embodiment(s) and such a combination is considered to be another embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself, combinable with others.

[0032] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.” Likewise, a termsuch as Cl -3 alkyl also includes, as separate embodiments, Cl alkyl, C2 alkyl, C3 alkyl, Cl -2 alkyl, and C2-3 alkyl.

[0033] Reference to alcohols, aldehydes, amines, carboxylic acids, ketones, or other similarly reactive functional groups also includes their protected analogs. For example, reference to hydroxy or alcohol also includes those substituents wherein the hydroxy is protected by an acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), P-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methoxymethyl (MOM), monomethoxytrityl (MMT), p-methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl (e.g., trimethyl silyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso- propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS)), or ethoxyethyl (EE) group. Reference to amines also includes those substituents wherein the amine is protected by a tert-butyloxy carbonyl (Boc), carbobenzyloxy (Cbz), p-methoxybenzyl carbonyl (Moz or MeOZ), 9- fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), carbamate, p- methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-m ethoxyphenyl (PMP), tosyl (Ts), or sulfonamide (Nosyl & Nps) group. Reference to substituent containing a carbonyl group also includes those substituents wherein the carbonyl is protected by an acetal or ketal, acylal, or dithiane group. Reference to substituent containing a carboxylic acid or carboxylate group also includes those substituents wherein the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert-butyl ester, an ester of 2, 6-di substituted phenol (e.g. 2,6- dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), a silyl ester, an orthoester, or an oxazoline. Protecting groups for imidazole groups (e.g., histidine) include trityl (Trt); monomethoxytrityl (Mmt); 4-methyltrityl (Mtt); benzyloxymethyl (Bom); and 2,4-dinitrophenyl (Dnp).Abbreviations

[0034] The following abbreviations and acronyms may be used in this disclosure:Ab: antibodyACN: acetonitrileARS: alizarin red SAsp: aspartate, aspartic acidBBB: blood-brain barrierBoc: tert-butyloxycarbonylBoc-Lys(Boc)-OSu: bis-Boc-L-lysine-N-hydroxysuccinimide esterBom: benzyloxymethylBTB: blood-tumor barrierCAB: cabazitaxelCAR: chimeric antigen receptorCbz: carboxybenzylCPME: cyclopentyl methyl etherCPT: camptothecinC V : column volume cMAP: cationic mucic acid polymer, cationic mucic acid-containing polymer cP: cationic polymerDa: daltonDCM: dichloromethane, methylene chlorideDF: diafiltrationDIC: N,NEdiisopropylcarbodiimideDIPEA: N,NEdiisopropylethyamine di SPA-PEG: di(succinimidyl propionate)-PEGDLS: dynamic light scatteringDMA: dimethylacetamideDMAP: 4-dimethylaminopyridineDMF: N,N-dimethyl-formamideDMSO: dimethyl sulfoxideDnp: 2,4-dinitrophenylEDC: 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodiimide hydrochlorideEEDQ: N-ethoxy carbonyl -2-ethoxy-l,2-dihydroquinolineEGFR: epidermal growth factor receptorESI: electrospray ionizationEtOAc: ethyl acetateFPLC: fast protein liquid chromatography g: gramGABA: gamma-amino butyric acidGly: glycine, glycylGPC: gel permeation chromatography h: hourHBSS: Hank’s balanced salt solutionHC1: hydrochloric acidHEPES: N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acidHex: hexanoic acid, w-caproic acidHIC: hydrophobic interaction chromatographyHOPO: hydroxypyridine-N-oxideHPLC: high-performance liquid chromatographyHPLC-UV: HPLC with ultraviolet absorbance detectionIm: imidazole, imidazoliumIm-cMAP-Im: cationic mucic acid flanked by imidazole moietiesIm.Na: 4-imidazoleacetic acid, sodium salt hydrateIPA: isopropyl alcohol kDa: kilodaltonL: literLAR: linker-to-antibody ratioMAM: polymerizable mucic acid monomerMAP: mucic acid polymerMeOH: methanolMES: 2-(N-morpholino)ethanesulfonic acid, 2-ethanesulfonic acid mg: milligram mL: milliliter mmol: millimoleMmt: monomethoxytrityl mol: mole mPEG: methoxy polyethylene glycol mRNA: messenger ribonucleic acid MTBE: methyl tert-butyl etherMtt: 4-methyltritylMWCO: molecular weight cut-offN-Boc diamine: N-Boc-ethylenediamineNHS: N-hydroxy succinimideNMM: N-methylmorpholineNP: nanoparticleNPBA: 3-carboxy-5-nitrophenylboronic acid, nitrophenylboronic acidPAA: poly(amino acid)PAMAM: poly(amidoamine)PBAE: poly(P-amino ester)PBS: phosphate-buffered salinePd / C: palladium on carbon catalystPEG: polyethylene glycolPEGm: methoxy polyethylene glycolPent: pentanoic acid; n-valeric acidPEG: polyethylene oxidePES: poly ethersulfonePEI: polyethyleneiminePFP: pentaflurorphenyl(PFPO)2O: bis(pentafluorophenyl) carbonatePrOAc: isopropyl acetatePVDF: polyvinylidene fluoride, poly vinylidene difluoridePyAOP: (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphateRNA: ribonucleic acid siRNA: short interfering ribonucleic acid, silencing ribonucleic acidSN38 : 7-ethyl- 10-hydroxy-camptothecinSpP: strands per particleTBDPS: tert-butyldiphenylsilane; tert-butyldiphenylsilylTBDP S Cl : tert-butly 1 (chi oro)dipheny 1 si 1 aneTEA: triethylamineTf: holo-transferrinTfR: transferrin receptorTFA: trifluoroacetate, trifluoroacetic acidTHF: tetrahydrofuranTIPS: triisopropyl silaneTras: trastuzumab (Herceptin)Trt: tritylUF / DF: ultrafiltration & diafiltration pg: microgram pL: microliter pmol: micromoleUV: ultravioletVai: valine, valylReaction components

[0035] The procedures described herein include standard solvents and catalysts as are known in the art. Although particular compounds (e.g., acids, bases, coupling agents) are recited in the disclosure, it is clear to one of skill in the art that different reagents can be used.

[0036] To that end, exemplary solvents include but are not limited to chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-di chloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-m ethyltetrahydrofuran), alcohols (e.g., methanol (MeOH), ethanol (EtOH), isopropanol (IP A), tert-butanol), alkanes (e.g., pentane, hexanes, heptanes), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethyl sulfoxide, dimethyl formamide, acetone, N- methyl-2-pyrrolidone, ), and polar protic solvents (e.g., water, ethanol, acetic acid, propionic acid).

[0037] Exemplary acids include but are not limited to mineral acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).

[0038] Exemplary bases include but are not limited to amino bases (e.g. 1,4- diazabicyclo[2.2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert-butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammonium hydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butyl magnesium chloride), pyridines (e.g., 4-dimethylaminopyridine, 2,6- lutidine, pyridine), carbonates e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and hydrides (e.g., sodium hydride, calcium hydride, potassium hydride).

[0039] Exemplary coupling agents include but are not limited to carbodiimide reagents (e.g., N,Ntdiisopropylcarbodiimide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,NEdi cyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1- hydroxy-7-azabenzotriazole, 6-chl oro-1 -hydroxy benzotri azole, N-hydroxy succinimide, 1- hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2- (hydroxyimino)acetate), anhydride-based or -forming reagents (e.g., ditertbutyl carbonate, acetic anhydride, 2-ethoxy-l -ethoxy carbonyl- 1,2-dihydroquinoline, ethyl chloroformate), acylazoles (e.g., carbonyl diimidazole), acid halide generating reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriphenylphosphonium dihydrogen trifluoride), phosphonium salt coupling reagents (e.g., benzotriazol-l-yloxytris(diemthylamino)phosphonium hexafluorophosphate), tetramethyl aminium reagents (e.g., 2-(2-oxo-l(2H)-pyridyl-l,l,3,3- tetramethyluronium tetrafluoroborate), aminium reagents (e.g., 2-chloro-l,3- dimethylimidazolidinium hexafluorophosphate), oxyma salts (e.g., l-((l-cyano-2-ethoxy-2- oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), antimonate uranium salts (e. , benzotriazol- l-yloxuy-N,N-dimethyl-methaniminium hexachloroantimonate), organophosphorus reagents (e.g, diethylcyanophosphonate, 1 -oxo- chlorophospholane, 2-propanephosphonic acid anhydride), triazine based reagents (e.g., 2-chloro- 4,6-dimethoxy-l,3,5-triazine), organosulfur reagents (e.g, pentafluorophenyl-4- nitrobenzenesulfonate), pyridinium reagents (e.g., 2-chloro-l -methylpyridinium iodide), polymer bound reagents (e.g., polymer supported N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline).Cationic polymers with titratable end groups

[0040] Provided herein are cationic polymers comprising titratable end groups. At appropriate pH values, these end groups act as proton sponges, facilitating endosomal release of, e.g., polymers and nanoparticles containing these titratable end groups.

[0041] Accordingly, provided herein are cationic polymers having a structure described byFormula I:wherein:End Groups A and B independently comprise an organic moiety with a pKa between 4 and 7 inclusive;End Group A and End Group B can be the same or different; cP is a cationic polymer comprising a plurality of monomers; and the size and repeat number of the monomer is sufficient to provide a number average molecular weight for the polymer in a range from about 500 Da to about 50,000 Da. In certain embodiments, cP is a cationic mucic acid-containing polymer (cMAP). Other cationic polymers include polyethyleneimines (PEIs), poly(P-amino esters) (PBAEs), poly(amidoamines) (PAMAMs), cationic polyacrylates, cationic poly(amino acids) (PAAs) and cationic glycopolymers (e.g, chitosan). In certain embodiments, cP is a homopolymer. In other embodiments, cP is a heteropolymer in which at least one of the monomer subunits is cationic. In certain embodiments, the organic moiety with a pKa between 4 and 7 inclusive is an amine- containing group.

[0042] In certain embodiments, the cationic polymer (cP) is a polyethyleneimine (PEI) group. Polyethyleneimine or polyaziridine is a hydrophilic, cationic polymer with repeating units composed of an amine group and a two-carbon aliphatic CH2-CH2 spacer. Polyethyleneimines can be linear or branched. Linear polyethyleneimines (z.e., [-NH-CH2-CH2]n) can contain both primary (RI-NH2) and secondary (R1-NH-R2) amines; branched PEIs contain primary (RI-NH2), secondary (R1-NH-R2) and tertiary (R1-N(R2)-R3) amine groups. Branched PEI can be synthesized by cationic ring-opening polymerization of aziridine. An exemplary structure of a branched PEI is shown below:Cationic mucic acid polymers (cMAPs)

[0043] In certain embodiments, the cationic polymer (cP) portion of Formula l is a cationic mucic acid polymer (cMAP). In additional embodiments, the cationic mucic acid polymer has the structure:wherein: m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is independently at each occurrence 0, 1, 2, 3, 4, or 5; and p is the number of repeat units and is sufficient to provide a number average molecular weight for the cP portion of the structure of Formula I in a range from about 500 Da to about 50,000 Da. In additional embodiments, m is any integral value between 1 and 50 inclusive. In additional embodiments, n is, independently at each occurrence, any integral value between 1 and 20 inclusive. In additional embodiments, p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 5,000,000 Da. In additional embodiments, in is any integral value between 1 and 50 inclusive and n is, independently at each occurrence, any integral value between 1 and 20 inclusive and p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 5,000,000 Da.

[0044] Cationic mucic acid polymers, and methods for their synthesis, have been described in International Patent Application Publication No. WO 2017 / 003668; US Patent Application Publication No. US 2021 / 0261739; and in U.S. Patents 10,287,401; 10,717,825 and 11,041,050;the disclosures of which are incorporated herein by reference in their entireties, for the purposes of describing the synthesis, structure and properties of cationic mucic acid polymers.Titratable End Groups

[0045] In certain embodiments, the titratable end group is an amine-containing end group.In additional embodiments, the amine-containing end group is an imidazole group:At pH values between 4 and 7, the imidazole group becomes protonated:Thus, within an endosome, the imidazole group acts as a proton sponge, preventing acidification of the endosome and leading to rupture of the endosome and escape of the imidazole-containing polymer into the cytoplasm of the cell.

[0046] Accordingly, in certain embodiments of the cationic polymers disclosed herein, End Group A and / or End Group B is an imidazole-containing group. Exemplary imidazole- containing end groups include:andwherein:Li and L2 are linking groups; and k is independently at each occurrence 0, 1, 2, 3, 4, or 5. In additional embodiments, k can, independently at each occurrence, have any integral value between 1 and 20 inclusive.

[0047] In certain embodiments, amine-containing cationic polymers have a structure described by Formula II:wherein:End Group A is:wherein: k is independently, at each occurrence, 0, 1, 2, 3, 4, or 5; and Li is a linking group;End Group B is:wherein L2 is a linking group; and cMAP iswherein: m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is independently at each occurrence 0, 1, 2, 3, 4, or 5; and p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 50,000 Da. In certain embodiments, p is between 4 and 40. Such polymers, containing a cationic mucic acid polymer flanked by imidazole groups, are denoted Im-cMAP-Im polymers for the purposes of this disclosure.

[0048] In additional embodiments, m is any integral value between 1 and 50 inclusive. In additional embodiments, n is, independently at each occurrence, any integral value between 1 and 20 inclusive. In additional embodiments, p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 5,000,000 Da. In additional embodiments, r is any integral value between 1 and 20 inclusive. In additional embodiments, m is any integral value between 1 and 50 inclusive and n is, independently at eachoccurrence, any integral value between 1 and 20 inclusive and p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 5,000,000 Da and r is any integral value between 1 and 20 inclusive.

[0049] In additional embodiments, End Group A and End Group B can comprise protected imidazole groups. In these embodiments, End group A is:and End Group B is:wherein PGi and PG2 are protecting groups. Protecting groups for the secondary amine of the imidazole group are known in the art. Exemplary, but non-limiting protecting groups, for either PGi, PG2 or both include trityl (Trt), monomethoxytrityl (Mmt), 4-methyltrityl (Mtt), 2,4- dinitrophenyl (Dnp) and benzyloxymethyl (Bom).

[0050] In certain embodiments, the imidazole-containing end group is a histidinyl group. In these embodiments, End Group A is:and End Group B is:

[0051] In additional embodiments, the imidazole-containing end group is a protected histidinyl group. In these embodiments, End Group A is:and End Group B is:wherein PGi and PG2 are protecting groups as described above for protecting the secondary amine of imidazole end groups.

[0052] In additional embodiments, End Group A and / or End Group B comprise other amine-containing groups, titratable in the pH range between about 4 and about 7 such as PEI and / or chitosan. In further embodiments, End Group A can be any of imidazole, protected imidazole, PEI, histidine, protected histidine, or chitosan and End Group B can be any of imidazole, protected imidazole, PEI, histidine, protected histidine, or chitosan.Linking groups between cMAP and amine-containing end groups

[0053] The linking groups (Li and L2) between the titratable (e.g., amine-containing) end groups and the remainder of the cationic polymer (e.g., cMAP) can be any organic group suitable for the purpose. For example, amides, esters, carbamates, thioesters, disulfides, anhydrides and / or ethers can be used as linking groups. Accordingly, in certain embodiments, Li and L2 can independently be any of-(Co-6alkylene)-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-, -(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.Nitrophenylboronic acid (NPBA)-containing polyethylene glycol (PEG) polymers

[0054] In certain embodiments, cationic polymers with titratable end groups, as described above, are conjugated to polyethylene glycol (PEG) polymers containing one or more nitrophenylboronic acid (NPBA) moieties. For example, a NPBA-PEG polymer can have a structure as shown in Formula III, in which the NPBA portion of the polymer is to the left of the spacer in the formula, and the PEG portion of the polymer is to the right of the spacer:wherein:L3 is a linking group, or linking groups, between the one or more boronic acid moieties and the spacer;L-i is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages;Spacer is a moiety containing at least one terminal amine and / or at least one terminal carboxylic acid; q is independently at each occurrence 0, 1, 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is a terminal functional group.

[0055] In certain embodiments, Xi is either of:(a) -C1-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the groups of (a); orXi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such thatXi has the structure -C-C(=O)-NH-targeting molecule. In additional embodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

[0056] In certain embodiments, the spacer is an amino acid. In a preferred embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, L4 is -C(=O)-NH-, Xi is -CH3, and the spacer is a lysyl group. In an additional embodiment, q is 1, r is 2, s is 50-350, t is 1, L3 is -C(=O)-NH-, L4 is -C(=O)-NH-, Xi is -C-C(=O)-OH, and the spacer is a lysyl group.

[0057] A NPBA-PEG polymer can be synthesized, for example, by combining an amine- functionalized PEG (e.g., PEG2k, PEG3.5k, PEG5k, PEGlOk) with a carboxy NPBA to form a NPBA-PEG conjugate joined by an amide linkage.Linking groups in NPBA-PEG Polymers

[0058] The s and L4 linkers shown in Formula III can be any organic moiety suitable for the purpose. For example, L3 and L4 can be independently, at each occurrence, one or more of the following groups:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-,-(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-, -(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-ealkyl ene)-NH-C (=0)-0-(Co-6alkyl ene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.Additional linking groups include, for example, amides, esters, carbamates, thioesters, disulfides, anhydrides and / or ethers.Spacers in NPBA-PEG Polymers

[0059] The spacer moiety shown in Formula III can be any organic moiety suitable for the purpose. In certain embodiments, the spacer is an organic group bearing at least one terminal amine and at least one terminal carboxylic acid. In additional embodiments, an amino acid, or portion thereof, is used as a spacer molecule in the NPBA-PEG polymers disclosed herein. The twenty essential amino acids have the general structure:in which Ri is a functional group that is specific to each particular amino acid. Functional groups for the twenty essential amino acids are given in Table 1. Chemical structures for a number of non-essential amino acids are given in Table 2.Table 1: Essential amino acid functional groupsLegend: The left column provides the name of the amino acid and its three-letter and one-letter abbreviations. The right column shows the chemical structure of the functional group (Ri) for each ammo acid, except for proline.* For proline, the structure of the entire amino acid is shown, since Ri cannot be represented independently.Table 2: Non-essential amino acids and their chemical structures

[0060] In certain embodiments, lysine is used as a spacer for attaching two NPBA moi eties to a PEG-containing polymer, as shown in Formula IV below:wherein: s is 20-1200; andXi is(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the groups of (a); orXi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-targeting molecule. In additional embodiments, a therapeutic molecule (as described elsewhere herein) is a covalentconjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)- NH-therapeutic molecule.

[0061] Additional moieties that can be used as spacers include y-shaped polyethylene glycol groups, branched polyethylene glycol groups, and branched polyethyleneimine groups.Terminal functional groups in NPBA-PEG Polymers

[0062] The terminal functional group of a NPBA-PEG polymer (denoted Xi in Formulas III and IV) can be any moiety suitable for the purpose. In certain embodiments, Xi comprises a reactive group, such as an amine, an alcohol or a carboxylic acid. In additional embodiments, Xi is -CH3. Additional terminal groups include, for example, -C(i-6) alkyl; optionally substituted with -OH, -COOH, -C(=O)-O-(alkyl), -C(=O)-O-(aryl), -NH2, -NH(alkyl), -N(alkyl)2, -NH(aryl) or - N(aryl2).

[0063] In additional embodiments, Xi comprises a targeting agent or targeting molecule, as described elsewhere in this disclosure A targeting molecule is generally attached to a NPBA- PEG polymer, in which Xi contains a reactive group, by conversion of the reactive group (e.g, - COOH) to an activated derivative (e.g., pentafluorophenyl (PFP) ester), and reaction of the activated derivative with the targeting molecule. The exemplary PFP ester moiety is known to react with amino groups in proteins, particularly antibodies. Pham, G.H et al. (2018) ChemBioChem 19:790-804.

[0064] In further embodiments, Xi comprises a therapeutic agent, as described elsewhere in this disclosure. A therapeutic agent is generally attached to a NPBA-PEG polymer, in which Xi contains a reactive group, by conversion of the reactive group (e.g., -COOH) to an activated derivative (e.g., pentafluorophenyl (PFP) ester), and reaction of the activated derivative with the targeting agent. The exemplary PFP ester moiety is known to react with amino groups in proteins, particularly antibodies. Pham, G.H e / a / . (2018) ChemBioChem 19:790-804.

[0065] A NPBA-PEG polymer comprising two NPBA moieties, a lysine spacer and a - COOH reactive group ((NPBA)2-Lys-PEG-COOH) is activated, for reaction with a targeting agent (such as, for example, an antibody) or with a therapeutic agent (such as, for example, an antibody), by forming a PFP ester derivative of the NPBA-PEG polymer. In these embodiments, bis(pentafluorophenyl) carbonate is combined with a (NPBA)2-Lys-PEG-COOH polymer to forma NPBA-PEG-pentafluorophenyl ester (e.g., (NPBA)2-Lys-PEG-COO-PFP), wherein s is defined as above in Formula IV.1. DCMThe NPBA-PEG-pentafluorophenyl ester is then reacted with the targeting agent or with the therapeutic agent to form a NPBA-PEG polymer with a covalently bound-targeting agent or a NPBA-PEG polymer with a covalently bound therapeutic agent.

[0066] In certain embodiments, s is between 2 and 1,200 or any integral values therebetween; e.g., between 100 and 300, between 20 and 300, between 120 and 180, and / or between 140 and 160.

[0067] In certain embodiments, the value of s is such that the PEG (i.e. -CH2-CH2-O-) portion of the compound has a number average molecular weight of between about 2 kDa and about 15 kDa; e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.Polymer conjugates

[0068] In certain embodiments, the present disclosure provides polymer conjugates comprising a first polymer which is a cationic polymer (such as, for example, cMAP or Im-cMAP- Im) and one or more second polymers which are NPBA-PEG polymers. Exemplary polymer conjugates have a structure as shown in Formula V below:wherein:Li and L2 are independently, at each occurrence:-(Co-6alkylene)-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-,-(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-;L3 and L4 are independently, at each occurrence:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-,-(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-;Spacer is a moiety containing at least one terminal amine and / or at least one terminal carboxylic acid, an amino acid, a y-shaped polyethylene glycol group, a branched polyethylene glycol group, and a branched polyethyleneimine group; m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is independently at each occurrence 0, 1, 2, 3, 4, or 5;p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 50,000 Da; k is independently at each occurrence 0, 1, 2, 3, 4 or 5; q is independently at each occurrence 0, 1, 2, 3 or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is any of:(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), - C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the groups of (a); orXi comprises a targeting molecule or a therapeutic molecule. In certain embodiments, a targeting molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-targeting molecule. In additional embodiments, a therapeutic molecule (as described elsewhere herein) is a covalent conjugate of one of the functional groups of (a) above, such that Xi has the structure -C-C(=O)-NH-therapeutic molecule.

[0069] In certain embodiments, polymer conjugates comprise, in addition to a cationic polymer and a biological agent, one or more NPBA-PEG polymers, optionally comprising a targeting agent and / or a therapeutic agent. In these embodiments, a NPBA-PEG polymer can comprise a targeting agent as part of Xi. Also in these embodiments, a NPBA-PEG polymer can comprise a therapeutic agent as part of Xi. Also in these embodiments, a NPBA-PEG polymer can comprise a methyl group (-CH3) as Xi. In these embodiments, nanoparticles can comprise any combination of NPBA-PEG polymers having a targeting agent as part of Xi, a therapeutic agent as part of Xi, or with Xi being -CH3. For example, a nanoparticle can comprise a cationic polymer with a bound biological agent (e.g., a RNA; e.g., a mRNA or a siRNA), one or more NPBA-PEG polymers comprising a targeting agent as part of Xi and one or more NPBA-PEG polymers in which Xi is -CH3.

[0070] In certain embodiments, a polymer conjugate as disclosed herein comprises a cationic polymer (e.g., a cMAP or an Im-cMAP-Im) and a plurality of NPBA-PEG polymers, in some of which Xi comprises a targeting agent and in some of which Xi is -CH3.

[0071] In certain embodiments, a polymer conjugate as disclosed herein comprises a cationic polymer (e.g., a cMAP or an Im-cMAP-Im) and a plurality of NPBA-PEG polymers, in some of which Xi comprises a therapeutic agent and in some of which Xi is -CH3.

[0072] In additional embodiments, a polymer conjugate as disclosed herein comprises a cationic polymer (e.g., a cMAP or an Im-cMAP-Im) and a plurality of NPBA-PEG polymers, in some of which Xi comprises a targeting agent, in some of which Xi comprises a therapeutic agent, and in some of which Xi is -CH3.Biological agents

[0073] In certain embodiments, nanoparticles as disclosed elsewhere herein comprise a cationic polymer and a biological agent. In certain embodiments, the cationic polymer is Im- cMAP-Im. Biological agents include amino acids, peptides, polypeptides, nucleotides, and nucleic acids, such as oligonucleotides and polynucleotides. Exemplary nucleic acids include DNAs such as genomic DNA, cDNA, plasmids and antisense oligonucleotides. Additional exemplary nucleic acids include RNAs, such as, for example, mRNA, siRNA, circular RNA, shRNA, mi RNA, antisense RNA, guide RNA, and tracr RNA. Any of the biological agents mentioned above can be linear, circular or branched (e.g., cyclic peptides, or circular nucleic acids, such as circular RNA).

[0074] In additional embodiments, the biological agent can be negatively charged, such as a nucleic acid. In further embodiments, the nucleic acid can be DNA, RNA or a peptide nucleic acid (PNA). Biological agents that are RNA can be low molecular weight RNAs such as antisense RNA, miRNA and siRNA, which can be introduced into a target cell to regulate (e.g., repress) expression of an endogenous target gene; or high molecular weight RNAs such as mRNAs, which can be used to encode an exogenous protein in a target cell. In addition, a biological agent can be any of a guide RNA and / or a tracr RNA, and / or a RNA encoding the Cas9 nuclease, for use in gene editing using the CRISPR-Cas9 system. Biological agents can also be DNA molecules encoding any of the RNAs described above.

[0075] Biological agents that are nucleic acids, such as RNA, will bind electrostatically to the cationic portion (e.g., the cMAP portion) of the polymer or polymer conjugate, upon contact of the RNA with the polymer (or polymer conjugate) in aqueous solution. See Example 9. Nucleic acids bind to the cationic polymer (e.g., cMAP) through electrostatic interactions between thenegatively-charged backbone of the nucleic acid and positively-charged groups in the cationic polymer (e.g., the amidine groups in the cMAP).Targeting Molecules

[0076] In certain embodiments, cMAPs as described herein contain, instead of or in addition to a biological agent (such as a nucleic acid; e.g., a mRNA or a siRNA), a targeting molecule or targeting agent. Targeting molecules are known in the art and include, for example, small molecules (e.g, vitamins, e.g., folate), saccharides (e.g., mannose, allose, altrose, glucose, gulose, idose, galactose, talose, di saccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g, antibodies, proteins that bind cell-surface receptors such as transferrin), nucleic acids (e.g., aptamers), peptoids and peptide nucleic acids (PNAs).

[0077] For example, cMAPs (and nanoparticles made from cMAPs) can comprise the targeting molecule transferrin, which targets the polymer or polymer conjugate (or a nanoparticle formed from the polymer or the polymer conjugate) to endothelial cells of the blood-brain barrier or the blood-tumor barrier. In these embodiments, the targeting molecule is attached to a NPBA- PEG polymer by way of reaction with a PFP-activated ester, as described elsewhere herein. Inasmuch as primary amine groups in proteins react with the PFP-activated ester, attachment of a polypeptide (such as, for example, holo-transferrin) to the NPBA-PEG-pentafluorophenyl ester is achieved by combining the ester with holo-transferrin. Progress of the reaction is monitored by high-performance liquid chromatography (HPLC), and the monoPEGylated transferrin conjugate is purified by hydrophobic interaction chromatography (HIC), based on the differential binding of monoPEGylated transferrin compared to species with higher degrees of PEGylation (e.g., diPEGylated and triPEGylated species). This method prevents loss of iron from transferrin during purification, as occurred using previous methods, thereby preserving the holo-transferrin structure.

[0078] In additional embodiments, a targeting molecule is a protein, a peptide, an aptamer, or a ligand for a cellular receptor. In certain embodiments, the cellular receptor is CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR).

[0079] A targeting molecule that is a protein can be, for example, an antibody or fragment thereof. In certain embodiments, a targeting molecule is an antibody directed to CD3, an antibody directed to CD4, an antibody directed to CD8, an antibody directed to the epidermal growth factor receptor (EGFR) or an antibody directed to the TfR.

[0080] In further embodiments, for targeting to a tumor cell, one or more small molecule targeting molecules can be used. These include, but are not limited to, carbohydrates (such as, for example, fructose, glucose, mannose, levan; or a derivative thereof), vitamins (such as, for example, riboflavin, biotin, thiamine, vitamin B12, and folic acid), and amino acids (such as, for example, glycine, alanine, arginine lysine and tryptophan). Additional small molecule targeting agents include adenosine, anisamide, anacardic acid, phenylboronic acid and methotrexate. See, for example, Kaur et al. (2023) J. Controlled Release 355:417-433.

[0081] In certain embodiments, a targeting molecule is covalently coupled to a NPBA- PEG polymer, as described elsewhere herein. For example, a targeting molecule can be part of the Xi moiety of the NPBA-PEG polymer, e.g., as a covalent conjugate of one of the functional groups that are specified for Xi elsewhere herein.Targeting Molecules for use in mRNA-based Therapies

[0082] To target a nanoparticle to a T-cell, the targeting molecule can be an antibody directed against a T-cell surface molecule. For example, a targeting molecule can be an antibody directed against a cluster of differentiation (CD) molecule present on the surface of the desired T- cell. In certain embodiments, nanoparticles targeted to the CD8 antigen (e.g., by using an anti- CD8 antibody as the targeting molecule) target cytotoxic (CD8+) T-cells. Other T-cell surface molecules include CD3 and CD4. Accordingly, nanoparticles targeted to the CD3 and / or CD4 antigens (e.g., by using anti-CD3 and / or anti-CD4 antibodies as the targeting molecule or molecules) also target T-cells.

[0083] Nanoparticles can also be targeted to cells of the immune system other than T-cells. For example, CD19 and CD20 are B cell surface markers. Accordingly, to target a nanoparticle to a B cell, a molecule (such as ,e.g., an antibody) that binds to CD 19 or CD20 can be used as a targeting molecule. To target a nanoparticle to a natural killer (NK) cell, a molecule (such as ,e.g. , an antibody) that binds to CD33 or CD56 can be used as a targeting molecule. To target a nanoparticle to a macrophage, a molecule (such as ,e.g., an antibody) that binds to CD74 can be used as a targeting molecule. To target a nanoparticle to a dendritic cell, a molecule (such as ,e.g., an antibody) that binds to CD11 can be used as a targeting molecule. Surface markers for cells of the immune system are known in the art; therefore, additional targeting molecules will be apparent to the skilled artisan.

[0084] In additional embodiments, nanoparticles can be targeted to red blood cells (erythrocytes) and their precursors (e.g., reticulocytes, erythroblasts, normoblasts) for treatment of, e.g., sickle cell anemia, thalassemia, hemophilia, cystic fibrosis and other erythrocyte-related disorders as are known in the art. Erythrocyte surface molecules to which targeting molecules can be directed include blood group antigens (A, B and O), Rh factor (D antigen), anion transport protein (band 3), Glycophorin A (CD235a), acetylcholinesterase, AMP deaminase, BGP1, CD36, CD47, transferrin receptor (CD71) and others known to those of skill in the art.Biological Agents for use in mRNA-based Therapies

[0085] For use in adoptive immunotherapies, a nanoparticle can comprise a mRNA encoding a chimeric antigen receptor (CAR). In certain embodiments, a mRNA biological agent encodes a CAR directed to the CD19 antigen, a surface marker for B-cells. CD19-targeted CARs can be used for treatment of, among other malignancies, B-cell acute lymphoblastic leukemia, B- cell non-Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma and multiple myeloma. In additional embodiments, a biological agent is a DNA molecule encoding a CAR; e.g., a DNA molecule encoding a CAR directed to CD 19.

[0086] In other embodiments, a mRNA biological agent encodes a CAR directed to the B- cell maturation antigen (BCMA), which is also a B-cell surface marker. CARs directed against BCMA can be used for the treatment of, among other maladies, multiple myeloma. In additional embodiments, a biological agent is a DNA molecule encoding a CAR directed to BCMA.

[0087] Nanoparticles targeted to T cells can contain, as a biological agent a mRNA encoding a molecule that recognizes (e.g., binds to, such as, for example, an antibody or a CAR) one or more of CD19, BCMA, TRAIL, CEACAM5, EPCAM, EGFR, R0R1, MET, CD133, CD70, SIRPa, FGFR1, CD44, CD133, TNFRSF17, IL3RA, SDC1, EGFRvIII, MUC1, FAP, CD44, MS4A1, CD22, PDCD1, CA9, CD174, TNFRSF8, CD33, CD38, EPHA2, CD274, FOLR1, SLAMF7, CD5, NCAM1, ErbB2, KDR, L1CAM, GD2, ULBP1, ULBP2, IL1RAP, GPC3, IL13RA2, MSLN, FOLH1, CD23, CD276, CSPG4, TEM1, GPNMB and PSCA. Additional targets are known to those of skill in the art.

[0088] Nanoparticles targeted to B cells can contain, as a biological agent, for example, a mRNA encoding an antibody directed to an antigen of choice; a mRNA encoding an immunomodulatory antibody, or a mRNA encoding interleukin-2 (IL2).

[0089] Nanoparticles targeted to NK cells can contain, as a biological agent, for example, a chimeric antigen receptor (CAR) such as, for example, a CAR directed to CD 19 or a CAR directed to BCMA or any of the CARs mentioned elsewhere in this disclosure.

[0090] Nanoparticles targeted to macrophages can contain, as a biological agent, a mRNA encoding IRF5, a mRNA encoding IKKp, or a mRNA encoding interleukin-12 (IL12), for example.

[0091] Nanoparticles targeted to dendritic cells can contain, as a biological agent, for example, a mRNA encoding interleukin-2 (IL2) or a mRNA encoding interleukin- 12 (IL 12), for example.

[0092] Nanoparticles targeted to erythrocytes and their precursors can contain, as a biological agent, for example, a mRNA encoding Factor VIII (e.g., for treatment of hemophilia), a mRNA encoding a-globin (e.g., for treatment of a thalassemia), a mRNA encoding P-globin (e.g., for treatment of P-thalassemia and / or sickle-cell disease), a mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (e.g., for treatment of cystic fibrosis) or any other mRNA encoding a protein useful in treating an erythrocyte -related disorder, as are known to those of skill in the art.Targeting Molecules for use in siRNA-based Therapies

[0093] RNA interference methods can be used to treat malignant cells (e.g., solid tumor cells and malignant myeloid cells) by delivering one or more siRNA or miRNA molecules to the malignant cell to repress the expression of certain genes; e.g., oncogenes.

[0094] Tumor types that are treated by delivery of siRNA to a malignant cell include solid tumors expressing EGFR (many tumor types), TIR / CD71 (many tumor types), HER2 (e.g., breast tumors, gastric tumors), HER3 (e.g., breast tumors, lung tumors), TROP2 (e.g., breast tumors, lung tumors), PSMA (e.g., prostate tumors), LIV-1 (e.g., breast tumors), FRa / FOLRl (e.g., ovarian tumors, lung tumors, breast tumors), FGFR2 (e.g., gastric tumors), DLL3 (e.g., lung tumors), c- MET (e.g., lung tumors), MUC1 (e.g., colorectal tumors, pancreatic tumors), CEACAM5 (e.g., colorectal tumors, lung tumors), B7-H3 (e.g., lung tumors, prostate tumors), B7-H4 (e.g., breast tumors, lung tumors, ovarian tumors), Nectin-4 (e.g., urothelial tumors, breast tumors), Claudin 18.2 (e.g., gastric tumors, pancreatic tumors), Claudin 6 (e.g., ovarian tumors), ROR1 (e.g., breast tumors, lung tumors), R0R2 (e.g., ovarian tumors, lung tumors, melanoma), EpCAM (many tumortypes), NaPi2b (e.g., ovarian tumors, lung tumors), CDH6 (e.g., renal cell carcinoma, ovarian tumors), integrin beta-6 e.g., colorectal tumors, lung tumors, gastric tumors, cervical tumors) , tissue factor (e.g., cervical tumors), CD228 (e.g., melanomas), PDGF (many tumor types), PTK7 (many tumor types) and mesothelin / MSLN (many tumor types).

[0095] Hematological malignancies that are treated by delivery of siRNA include, for example, those in which the malignant cells express CD74 (e.g., B cells, T cells and macrophages), CD30 (e.g., B cells), CD33 (e.g., T cells, NK cells), CD22 (e.g., B cells), CD25 (e.g., T cells), CD70 (e.g., B cells), CD19 (e.g., B cells) and CD20 (e.g., B cells).

[0096] Nanoparticles containing siRNA can be targeted to the appropriate tumor by including, as a targeting agent, a molecule (e.g., an antibody) that binds to one or more of the markers mentioned in the two preceding paragraphs. In some cases, the targeting agent also serves as a therapeutic agent. For example, antibodies to the following tumor markers can also act as therapeutic molecules: EGFR, HER2, HER3, FGFR2, Claudin 18.2, Claudin 6, EpCAM, PDGF, PTK7, mesothelin / MSLN, CD74, CD30, CD33, CD22, CD25, CD70, CD19 and CD20.Biological Agents for siRNA-based Therapies

[0097] Exemplary, biological agents for use in RNA interference methods are siRNAs targeted to certain cancer-related genes (e.g., oncogenes) such as, for example, c-myc, c-myb, mdm2, PKA-I, Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases, telomerase, PDGF / sis, erb-B, fos, jun, mos, src, the Bcr / Abl fusion gene, EGFR, BRAF, RRM2, HER2, VEGF, FGFR2, K-RAS, Claudin 18.2, EpCAM, STAT3, mesothelin / MSLN and BCRP.

[0098] In additional embodiments siRNA directed to EGFR sequences (siEGFR) is used as a biological agent in a nanoparticle used for treatment of EGFR-expressing tumors. In another embodiment, siBRAF and / or siRRM2 is used as a biological agent(s) in a nanoparticle used for treatment of CD71 (TfR)-expressing tumors. In another embodiment, siHER2 is used as a biological agent in a nanoparticle used for treatment of HER2-expressing tumors. In another embodiment, siVEGF is used as a biological agent in a nanoparticle used for treatment of FRa / FOLRl -expressing tumors, CEACAM5-expressing tumors, ROR1 -expressing tumors ROR2-expressing tumors and NaPi2b- expressing tumors. In another embodiment, siFGFR2 is used as a biological agent in a nanoparticle used for treatment of FGFR2-expressing tumors. Inanother embodiment, siK-Ras is used as a biological agent in a nanoparticle used for treatment of MUC 1 -expressing tumors and Claudin 18.2-expressing tumors. In another embodiment, siClaudin 18.2 is used as a biological agent in a nanoparticle used for treatment of Claudin 18.2-expressing tumors. In another embodiment, siEpCAM is used as a biological agent in a nanoparticle used for treatment of EpCAM-expressing tumors. In another embodiment, siSTAT3 is used as a biological agent in a nanoparticle used for treatment of CD228-expressing tumors. In another embodiment, siMSLN is used as a biological agent in a nanoparticle used for treatment of mesothelin / MSLN- expressing tumors.

[0099] In further embodiments for treatment of hematological malignancies (e.g., blood cell cancers) siBCRP is used as a biological agent in a nanoparticle used for treatment of cancers in which the malignant cells express CD19 and / or CD20.Antisense-based therapies

[0100] In additional embodiments, antisense RNA directed to an oncogene (various oncogenes are mentioned elsewhere in this disclosure) can be delivered to a malignant cell using the nanoparticles disclosed herein. The sequence of the antisense RNA can be determined based on the sequence of the target oncogene; as oncogene sequences are known in the art. The targeting molecule used in such nanoparticles can be determined based surface markers expressed by the malignant cell, since surface markers that are characteristic of particular malignant cells are known in the art.Therapeutic Molecules

[0101] In certain embodiments, polymer conjugates, as described herein (e.g., conjugates of NPBA-PEG and Im-cMAP-Im), comprise a therapeutic molecule (e.g., a large molecule therapeutic). For example, a therapeutic molecule can be part of the Xi moiety of the NPBA-PEG polymer, e.g., as a covalent conjugate of one of the functional groups that are specified for Xi elsewhere herein. Large molecule therapeutics are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and bioactive proteins) and nucleic acids.

[0102] Exemplary therapeutic polypeptides include, but are not limited to, antibodies such as trastuzumab (Herceptin, an anti-Her2 antibody), anti-Her3, anti-Trop2, anti-PSMA, anti-LIV- 1, anti-FOLRl, anti-DLL3, anti-PDGF, anti-FRalpha, anti-PTK7, anti-mesothelin, anti-c-MET,anti-MUCl, anti-CD70, anti-CD74, anti-CD30, anti-CD33, anti-FLT3, anti-CD22, anti-CD20, and anti-CD19, anti-CD4, anti-CD8, and antibodies directed againstB7-H3, B7-H4, Nectin-4, Claudin 18.2, R0R1, R0R2, BCMA, EpCAM, Claudin 6, CDH6, CEACAM5, and Integrin beta-6.

[0103] Certain polypeptide therapeutics, such as antibodies directed to tumor markers like those exemplified in the previous paragraph, are also used at targeting molecules.

[0104] For example, NPBA-PEG / Im-cMAP-Im polymer conjugates (and nanoparticles made from these conjugates) can comprise the anti-Her2 antibody trastuzumab (also known as Herceptin). In these embodiments, the antibody is attached to a NPBA-PEG polymer by way of reaction with a PFP-activated ester, as described elsewhere in this disclosure.

[0105] Inasmuch as the primary amine groups in proteins react with PFP- activated esters, attachment of trastuzumab to the NPBA-PEG-pentafluorophenyl ester is achieved by reacting the NPBA-PEG-pentafluorophenyl ester with trastuzumab. Progress of the reaction is monitored by HPLC and the monoPEGylated trastuzumab conjugate is purified by HIC, based on the differential binding of monoPEGylated trastuzumab compared to species containing higher degrees of PEGylation e.g., diPEGylated and triPEGylated species).Formation of nanoparticles

[0106] To obtain nanoparticles containing a cationic polymer and bound nucleic acid, the nucleic acid (at a concentration of 0.1-10 mg / mL) is mixed with the cationic polymer (or a polymer conjugate comprising a cationic polymer and a NPBA-PEG polymer, with the NPBA-PEG polymer optionally comprising a targeting agent and / or a therapeutic agent) in an aqueous solution (e.g., phosphate buffer) at a charge ratio (+ / -) of between 0.5: 1 and 5: 1. In certain embodiments, the charge ratio is between 1 : 1 and 3: 1. The solution is then filtered and can be frozen until further use.

[0107] The size of the nanoparticles (i.e., cross-sectional diameter) ranges from about 10 to about 300nm in diameter. Exemplary nanoparticle diameters are 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 220 nm, 240 nm, 250 nm, 260 nm, 275 nm, 290 nm, 330 nm, or any value within the range of 20-300 nm. Specific embodiments can also describe these nanoparticles as having diameters in a range of from about 20 nm to about 40 nm, from about 40 nm to about 80 nm, from about 80 nm to about 120 nm, from about 120 nm to about 180 nm, fromabout 180 nm to about 240 nm, from about 240 nm to about 300 nm, or a combination of two or more of these ranges. In certain embodiments, the nanoparticles are spherical and have a diameter of 30-40 nm as determined by DLS and Cryo-transmission electron microscopy.Treatment methods

[0108] Also provided are methods for treating malignancies with the nanoparticles disclosed herein. For example, nanoparticles that contain mRNA encoding a CAR directed to CD 19, and further comprising a targeting agent directed to CD8, can be used for the treatment of hematological malignancies such as, for example, B-cell acute lymphoblastic leukemia, B-cell non-Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma and multiple myeloma. This type of nanoparticle generates a CD8+cytotoxic T-cell that is targeted to the CD 19 antigen found on the surface of certain malignant white blood cells (e.g., B-cells).

[0109] In other embodiments, nanoparticles that contain a mRNA encoding a CAR directed to the B-cell maturation antigen (BCMA) can be used for the treatment of, among other maladies, multiple myeloma.

[0110] Other targets for adoptive immunotherapy (i.e., CAR T-cell therapy) are described elsewhere in this disclosure.Formulations, kits, and routes of administration

[0111] Therapeutic compositions comprising nanoparticles as disclosed herein are also provided. Such compositions typically comprise the nanoparticles and a pharmaceutically acceptable carrier or excipient. Supplementary active compounds can also be incorporated into nanoparticle compositions.

[0112] The therapeutic compositions disclosed herein are useful for, inter alia, treating cancer, cancer metastases and other disorders. Accordingly, a “therapeutically effective amount” of a composition comprising nanoparticles is any amount that reduces symptoms or, e.g., stimulates tumor regression. For example, dosage amounts of nanoparticles can vary from about 0.1-20 mg / kg body weight, or from about 0.5 to 2.0 mg / kg body weight or from about 1-5 mg / kg body weight or from about 1 mg / kg body weight to about 10 mg / kg body weight or more (or any integral or decimal value therebetween); with a frequency of administration of, e.g., hourly, twice per day, once per day, twice per week, once per week, twice per month, once per month, dependingupon, e.g., body weight, route of administration, severity of disease, etc. Thus, a therapeutically effective amount can comprise a plurality of administrations of the same amount, or different amounts, of nanoparticles. In certain embodiments, a single administration of nanoparticles is a therapeutically effective amount.

[0113] In certain embodiments, nanoparticles are administered at a dosage of 3-100 mg nanoparticle (or any integral or decimal value therebetween) per square meter of the surface area of the body of the subject, as is typical for dosages of nucleic acid-containing therapeutics. For example, dosages can be any of 5, 10, 20, 30, 40 50, 60, 70, 80, 90, 100 mg / m2; or from 10-90, 20-80, 30-70 or 40-60 mg / m2.

[0114] Various pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds ), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0115] The nanoparticles described herein can be suspended in a physiologically compatible carrier for administration. As used herein, the term “physiologically compatible carrier” refers to a carrier that is compatible with the nanoparticles and with any other ingredients of the formulation, and is not deleterious to the recipient thereof. Those of skill in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include water e.g., pH 4 water), phosphate-buffered saline, Hank’s balanced salt solution+ / -glucose (HBSS), and multiple electrolyte solutions such as, e.g., Plasma-LyteTM A (Baxter).

[0116] The volume of a nanoparticle suspension administered to a subject will vary depending on the site of administration, treatment goal and number of nanoparticles in solution. Typically the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, a “therapeutically effective amount” or “effective amount” refers to the number of administered nanoparticles which are required to effect treatment of the particular disorder; i.e., to produce a reduction in the amount and / or severity of the symptoms associated with that disorder. For example, in the case of a cancer metastasis to the brain, administration of a therapeuticallyeffective amount of nanoparticles results in reduction and / or reversal of the symptoms of metastasis; e.g, regression of the metastatic tumor. Therapeutically effective amounts vary with the type and extent of brain damage, and can also vary depending on the overall condition of the subject.

[0117] The disclosed therapeutic compositions can also include pharmaceutically acceptable materials, compositions or vehicle, such as a liquid or solid fdler, diluent, excipient, solvent or encapsulating material, i.e., carriers. These carriers can, for example, stabilize the nanoparticles and / or facilitate the retention of the nanoparticles in the body. Each carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically- acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol and polyethylene glycol; polyols, such as glycerin, sorbitol and mannitol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer® solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0118] Nanoparticles can be administered to a subject by any suitable route, including, but not limited to, inhalation, topically, nasally, orally, parenterally (e.g., intravenously, intraperitoneally, intravesically or intrathecally) or rectally in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard practice. Administration of the compounds described herein can be carried out using any method known in the art. For example, administration may be transdermal, parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intraci sternal, intraperitoneal, intracerebroventricular, intrathecal, intranasal, aerosol,by suppositories, or by oral administration. A pharmaceutical composition of the nanoparticles described herein can be for administration by injection, or for oral, pulmonary, nasal, transdermal, or ocular administration.

[0119] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intra-arterial, intra-ocular, intra-cranial, sub- meningeal, or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible coating designed for slow-release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. The dosage of the compositions of the disclosure will vary according to the extent and severity of the need for treatment, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

[0120] In additional embodiments, the compositions described herein are delivered intracranially at or near a site of brain injury or metastasis. Such localized delivery allows for the delivery of the composition non-systemically, thereby reducing the body burden of the composition as compared to systemic delivery. Local delivery can be achieved, for example, by intra-cranial injection, or through the use of various medically implanted devices including, but not limited to, stents and catheters, or can be achieved by inhalation, phlebotomy, or surgery. Methods for coating, implanting, embedding, and otherwise attaching desired agents to medical devices such as stents and catheters are established in the art and contemplated herein.

[0121] Another aspect of the present disclosure relates to kits for carrying out the administration of nanoparticles, optionally in combination with another therapeutic agent, to a subject. In one embodiment, a kit comprises a composition of nanoparticles formulated in a pharmaceutical carrier, suitable for administration, e.g., by injection.Synthetic methods

[0122] Also provided are methods for the synthesis of cationic mucic acid -containing polymers (cMAPs) as shown in the Examples provided elsewhere in this disclosure.EXAMPLESExample 1: Synthesis of N-Boc-Protected Mucic Acid Ethylenediamine

[0123] A reactor was charged with mucic acid (18.0 g, 86 mmol). Methanol (MeOH, 400 mL) and concentrated sulfuric acid (H2SO4, 1.4 mL) were added. The mixture was heated to reflux (85 °C) overnight (~18 hours) under constant stirring. After ~18 hours, triethylamine (TEA, 32 mL) was added and the mixture was allowed to stir at reflux (85 °C) for 60 minutes. To the reaction mixture was added N-Boc-ethylenediamine (N-Boc diamine, 30.0 g, 187 mmol) dissolved in 50 mL of MeOH. The reaction was stirred at reflux (85 °C) overnight (~18 hours). The reaction mixture was allowed to cool to room temperature, fdtered, and washed with MeOH. The collected solids were then taken into 450 mL of MeOH and refluxed under constant stirring for 1 hour. The reaction mixture was allowed to cool to room temperature, filtered under vacuum, and washed with MeOH. Collected solids were dried overnight under vacuum to afford N-Boc-protected mucic acid ethylenediamine (26.1 g, 62 % yield). 'HNMR (600 MHz, DMSO-r / 6): 7.71 (t, 2H), 6.81 (t, 2H), 5.13 (d, 2H), 4.35 (q, 2H), 4.09 (d, 2H), 3.77 (q, 2H), 3.12 (m, 4H), 2.98 (m, 4H), 1.36 (s, 18).mucic acid mucic acid dimethyl esterN-Boc-protected mucic acid ethylenediamineExample 2: Synthesis of Mucic Acid Ethylenediamine

[0124] A reactor was charged with N-Boc-protected mucic acid ethylenediamine (25.9 g, 52 mmol). 3 M aqueous hydrochloric acid (HC1, 466 mL) in MeOH was added slowly under constant stirring. Following slow addition of HC1, the reaction flask was moved to an orbital shaker and shaken overnight at room temperature. The reaction slurry was filtered, and the solids were washed with MeOH three times (150 mL). Collected solids were dried overnight for severalhours under vacuum at 35 °C to afford mucic acid ethylenediamine (18.0 g, 94 % yield). 'H NMR (600 MHz, DMSO-t / 6): 7.97-7.84 (m, 8H), 5.30 (d, 2H), 4.58 (d, 2H), 4.16 (d, 2H), 3.82(m, 2H), 3.39-3.32 (m, 4H), 2.85 (m, 4H).N-Boc-protectect mucic acid ethylenediamine mucic acid ethylenediamineExample 3: Synthesis of cationic mucic acid polymer (cMAP)

[0125] Mucic acid ethylenediamine (202 mg, 0.55 mmol) was added to a glass vial with a stir bar. A 0.5 M sodium carbonate solution in nanopure water (Na2COs, 2 mL) was added to the vial, and the solution was stirred for 5 min. Dimethyl suberimidate (DMS) (150 mg, 0.55 mmol) was then added to the mixture, and the reaction was stirred for 23 h at 25 °C. I N HCI (4 mL) was added dropwise acidify the reaction mixture. The resulting solution was dialyzed, using a prewashed 1 kDa MWCO Spectra Por membrane, against 1 L of nanopure water (pH 3), for three changes. Product was isolated and lyophilized to dryness to afford cMAP (110 mg, 35 % yield) as a white solid, which was stored under argon at -20 °C. ’H NMR (500 MHz, DMSO-t / 6): 9.61-8.78, 7.91, 5.41, 4.55, 4.17, 3.83, 3.57, 3.28, 2.81-2.26, 1.61, 1.28.1 05 M Na2CO3mucic acid ethylenediamine DMS

[0126] After polymerization, cMAP polymers can have a number of terminal structures, as shown below. The polymer can contain amine groups at both ends (top); an amine group atone end and a methoxy group at the other end (middle) or methoxy groups at both ends (bottom).A small amount of terminal carboxylic acid is also generated from hydrolysis of one end ofDMS.

[0127] For cMAP synthesized by this method, p is about 14, giving the polymer an average molecular weight of about 7,500 Da, determined as the arithmetic mean of the number average molecular weight and the weight average molecular weight. These values will change if different reaction conditions (e.g., a different ratio of monomers) or different purification methods (e.g., a membrane with a different molecular weight cut-off) are used.Example 4: Synthesis of an imidazole-containing cMAP (Im-cMAP-Im)

[0128] Because a significant amount of polymer chains in the cMAP mixture contain amine groups at both ends (see above), it is possible to attach suitably derivatized (e.g., carboxyl-terminated) end groups to the cMAP polymer. For example, titratable end groups containing imidazole units can be covalently bound to a cMAP polymer using the following procedure.

[0129] Imidazole is conjugated to the cMAP polymer described in Example 3 by amidation of the primary amines at the end of the polymer with 4-imidazoleacetic acid. In a typical procedure, cMAP (200 mg, 45 pmol) is dissolved in 900 uL of 25 mM 2-ethanesulfonic acid (MES, pH 6.5) buffer to which is added 4-imidazoleacetic acid, sodium salt hydrate (Im.Na, 66 mg, 0.45 mmol). This solution is used to dissolve l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) hydrochloride (171 mg., 0.89 mmol). N-hydroxysuccinimide (NHS) (5.2 mg, 45 pmol), dissolved in 300 pL of 25 mMMES, pH 6.5 buffer, is added immediately to the cMAP solution. The resulting solution is stirred for 24 hours at room temperature and dialyzed against water using a pre-washed 1 kDa molecular weight cut-off(MWCO) Spectra Por membrane. The solution is lyophilized to dryness to afford Im-cMAP-Im as a solid.MES, pH 6 5

[0130] As stated in Example 3, p is about 14 for Im-cMAP-Im synthesized by this method.Example 5: Synthesis of (NPBA)2-Lys-PEGm

[0131] A reactor was charged with amine PEGm (NEE-PEGsooom, 300 mg, 0.06 mmol) and bis-Boc-L-lysine-N-hydroxysuccinimide ester (Boc-Lys(Boc)-OSu, 75 mg, 0.17 mmol). The reactor was purged with argon, and methylene chloride (DCM) was added to achieve an amine PEGm concentration of 0.2 g / mL. N,N-diisopropylethylamine (DIPEA, 35 pL, 0.18 mmol) was added, and the reaction was stirred at 19-25°C for 16-22 hours. The reaction mixture was dried under vacuum. The solids were dissolved in dimethyl sulfoxide (DMSO) to obtain a homogenous solution with a concentration of about 0.05 g amine PEGm / mL. The product was purified by dialysis using a pre-washed 2 kDa MWCO Spectra Por membrane. Dialysis was performed three times against DMSO and three times against Milli-Q water (each exchange is into about 125x of the product solution volume). The product solution was frozen and lyophilized for at least 72 hours to yield purified Boc-Lys(Boc)-PEGm (Boc-Lys(Boc)-PEGsooom, 291 mg, 91 % yield) as a solid. The solid was stored at < -20°C in glass vials under argon.

[0132] A reactor was charged with Boc-Lys(Boc)-PEGm (201 mg, 0.45 mmol), purged with argon, and vented with a needle. Triisopropyl silane (TIPS), DCM, and trifluoroacetic acid (TFA) were added at a volume ratio of 5:72:23, with TFA added dropwise, to achieve a Boc- Lys(Boc)-PEGm concentration of 0.08 g Boc-Lys(Boc)-PEGm / mL of liquid volume (TIPS :DCM: TFA). The reaction was stirred under vent at 19-25 °C for 16-22 hours. DCM and TFA were removed under vacuum. The solids were dissolved in a minimum amount of DCM and added dropwise to methyl tert-butyl ether (MTBE, about 60x the DCM volume), and the suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and supernatant liquid was decanted. These steps (dissolving in DCM, adding MTBE, stirring, centrifuging and decanting) were repeated, and the isolated solid was dried under vacuum to remove excess solvent. Optionally, the solid was dissolved in a minimum amount of Milli-Q water and lyophilized for at least 72 hours to yield purified Lys-PEGm (Lys-PEGsooom, 124 mg, 64 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.

[0133] A reactor was charged with Lys-PEGm (100 mg, 0.020 mmol), and was purged with argon. DCM was added to achieve a Lys-PEGm concentration of about 0.1 g Lys-PEGm / mL DCM. DIPEA (10 pL, 0.059 mmol) was added, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 10 minutes). A separate reactor was charged with (7-azabenzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP, 29 mg, 0.056 mmol) and 3-carboxy-5-nitrophenylboronic acid (NPBA, 59 mg, 0.28 mmol). The reactor was purged with argon, and DCM was added to achieve a NPBA concentration of 0.08 g NPBA / mL. To this suspension, N,N-dimethylformamide (DMF) was added dropwise, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 0.18 volume of DCM volume and 15 minutes). DIPEA (97 pL, 0.56 mmol) was added drop wise resulting in color change of the solution to yellow. The Lys-PEGm was transferred dropwise to the NPBA solution, and the reaction mixture was stirred at 19-25 °C for 16-22 hours, then dried under vacuum. 0.5 N hydrochloric acid (HC1) was added to achieve a NPBA concentration of about 0.03 g NPBA / mL, and the milky white suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and the product solution was decanted and filtered using a 0.2 pm Supor membrane syringe filter. The product was purified by dialysis against pH 3 water using a 3 kDa MWCO Amicon centrifugal filtration unit. The product solution was frozen and lyophilized for at least 72 hours to yield purified (NPBA)2-Lys-PEGm (2NPBA-Lys-PEGjooo-COOMe, 87 mg, 80 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.NMR (500 MHz, D2O): 8.41, 8.38, 8.29, 8.15, 8.06, 4.57, 3.6-3.3 (PEG), 3.22, 1.92, 1.85, 1.64,1.57, 1.49.Lys-PEGm (NPBA)2-Lys-PEGm

[0134] For (NPBA)2-Lys-PEGm synthesized by this method, s is about 114. Accordingly, the PEG component (NH2-PEG5000IT1) has a number average molecular weight of about 5,000 Da. This value will change if a different starting material is used (e.g., s is about 80 for NH2-PEG35oom or about 227 for NH2-PEG10000m).Example 6: Synthesis of (NPBA)i-Lys-PEG-COOH

[0135] A reactor was charged with amine PEG acetic acid (NH2-PEG5000-COOH, 403 mg, 0.081 mmol) and bis-Boc-L-lysine-N-hydroxysuccinimide ester (Boc-Lys(Boc)-OSu, 100 mg, 0.23 mmol). The reactor was purged with argon, and DCM was added to achieve an amine PEG acetic acid concentration of 0.2 g / mL. DIPEA (47 pL, 0.24 mmol) was added, and the reaction mixture was stirred at 19-25 °C for 16-22 hours, then dried under vacuum. The solids were dissolved in DMSO to obtain a homogenous solution and concentration of about 0.05 g starting amine PEG acetic acid / mL. The product was purified by dialysis using a pre-washed 2 kDa MWCO Spectra Por membrane. Dialysis was performed three times against DMSO and threetimes against Milli-Q water (each exchange is into about 125x of the product solution volume). The product solution was frozen and lyophilized for at least 72 hours to yield purified Boc- Lys(Boc)-PEG-COOH (Boc-Lys(Boc)-PEGsooo-COOH, 377 mg, 88 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.

[0136] A reactor was charged with Boc-Lys(Boc)-PEG-COOH (186 mg, 0.42 mmol), purged with argon, and vented with a needle. TIPS, DCM, and TFA were added at a volume ratio of 5:72:23, with TFA added dropwise, to achieve a Boc-Lys(Boc)-PEG-COOH concentration of 0.08 g / mL total volume TIPS:DCM:TFA. The reaction was stirred under vent at 19-25 °C for 16- 22 hours. DCM and TFA were removed under vacuum. The solids were dissolved in a minimum amount of DCM and added dropwise to methyl tert-butyl ether (MTBE, about 60x the DCM volume), and the suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and supernatant liquid was decanted. These steps (dissolving in DCM, adding MTBE, stirring, centrifuging and decanting) were repeated, and the isolated solid was dried under vacuum to remove excess solvent. Optionally, the solid was dissolved in a minimum amount of Milli-Q water and lyophilized for at least 72 hours to yield purified Lys- PEG-COOH (Lys-PEGsooo-COOH, 102 mg, 57 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon.

[0137] A reactor was charged with Lys-PEG-COOH (300 mg, 0.059 mmol), and was purged with argon. DCM was added to achieve a Lys-PEG-COOH concentration of about 0.1 g / mb. DIPEA (31 pL, 0.18 mmol) was added, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 10 minutes). A separate reactor was charged with PyAOP (87 mg, 0.17 mmol) and NPBA (176 mg, 0.84 mmol). The reactor was purged with argon, and DCM was added to achieve an NPBA concentration of 0.08 g NPBA / mL. To this suspension, DMF was added dropwise, and the mixture was stirred at 19-25 °C until a clear, homogenous solution was achieved (ca. 0.18x DCM volume and 15 minutes). DIPEA (291 pL, 1.67 mmol) was added dropwise resulting in a change in color of the solution to yellow. The Lys-PEG-COOH was transferred dropwise to the NPBA solution, and the reaction was stirred at 19-25 °C for 16- 22 hours, then dried under vacuum. 0.5 N HC1 was added to achieve a NPBA concentration of about 0.03 g NPBA / mL, and the milky white suspension was stirred at 19-25 °C for approximately 1 hour. The resulting suspension was centrifuged, and the product solution was decanted and filtered using a 0.2 pm Supor membrane syringe filter. The product was purified by dialysisagainst pH 3 water using a 3 kDa MWCO Amicon centrifugal fdtration unit. The product solution was frozen and lyophilized for at least 72 hours to yield purified (NPBA)2-Lys-PEG-COOH ((NPBA)2-Lys-PEG5ooo-COOH, 258 mg, 79 % yield) as a solid. The solid was stored at < -20 °C in glass vials under argon. 'H N.MR (500 MHz, D2O): 8.41, 8.37, 8.28, 8.15, 8.06, 4.57, 4.09, 3.6- 3.3 (PEG), 1.91, 1.85, 1.65, 1.58, 1.49.

[0138] For (NPBA)2-Lys-PEG-COOH synthesized by this method, s is about 114.Accordingly, the PEG component (NH2-PEG5000-COOH) has a number average molecular weight of about 5,000 Da. This value will change if a different starting material is used (e.g., s is about 80 for NH2-PEG3500-COOH or about 227 for NH2-PEG10000-COOH).Example 7: Synthesis of (NPBA)2-Lys-PEG-COO-PFP

[0139] A reactor was charged with (NPBA)2-Lys-PEG-COOH (102 mg, 0.018 mmol), and was purged with argon. DCM (2 mL) was added to achieve a 2NPBA-Lys-PEG-COOH concentration of 0.05 g / mL. N-methylmorpholine (NMM, 6 pL, 0.054 mmol) and bis(pentafluorophenyl) carbonate ((PFPO)2O, 30 mg, 0.076 mmol) were added, and the reaction mixture was stirred at 19-25 °C for 2-4 hours. Progress of the reaction was monitored by HPLC. After reaction completion, the reaction mixture was added dropwise into MTBE (about lOx the DCM volume), and the suspension was stirred at 19-25 °C for approximately 15 minutes underargon. The resulting suspension was centrifuged, and supernatant liquid was decanted. Heptane (about lOx the DCM volume) was added, and the suspension was stirred at 19-25 °C for approximately 15 minutes under argon. The resulting suspension was centrifuged, and supernatant liquid was decanted. The isolated solid was dried under vacuum to remove excess solvent to yield purified (NPBA)2-Lys-PEG-COO-PFP ((NPBA)2-Lys-PEG5ooo-COO-PFP, 94 mg, 90 % yield).The solid was stored at < -20 °C in glass vials under argon.1. DCM

[0140] As stated in Example 6, s is about 114 for (NPBA)2-Lys-PEG-COO-PFP synthesized by this method. This value will change if a different starting material is used.Example 8: Preparation of (NPBA)i-Lys-PEG-AbSynthesis of Crude Mixture

[0141] A 15 mL 30 kDa MWCO centrifugal filtration unit was pre-washed with 50 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) pH 7.8 buffer and centrifuged at 3,200 x g for 5 minutes. 100 mg (0.0069 mmol) of anti-CD8 antibody at 25 mg / mL was added and centrifuged at 3,200 x g for 15 minutes to allow buffer exchange into 50 mM HEPES pH 7.8 buffer. The flow-through was then replaced with 50 mM HEPES buffer and centrifugation was repeated until < 0.01 % of the initial solution remained. The concentration of antibody was determined with an Unchained Labs Little Lunatic UV / Vis absorbance spectrometer. The antibody concentration was then adjusted to 10 mg / mL in 50 mM HEPES pH 7.8 buffer. Following solubilization, 10.21 mg (0.00187 mmol) of (NPBA)2-Lys-PEG-COO-PFP in 0.093 mL DMSO was added slowly to the antibody solution and mixed well. The reaction solution was stirred at 19-25 °C and sampled periodically to determine progress of the reaction. Reaction progress was analyzed on a Cytiva AKTA Go fast protein liquid chromatography (FPLC) systemattached to a 1 mL HiTrap Phenyl HP HIC column with a mobile phase A of 1.5 M Ammonium Sulfate, 20 mM Sodium Phosphate, pH 7.4 and a mobile phase B of 20 % isopropyl alcohol (IP A), 10 mM Sodium Phosphate, pH 7.4 at a flow rate of 1 mL / min. The gradient was run from 40 % B to 100 % B over 25 column volumes. The column was connected to an absorbance detector set to 280 nm. FPLC analysis indicated that the reaction was complete after two hours, at which time the (NPBA)2-Lys-PEG-Ab crude mixture was added to a pre-washed 30 kDa MWCO Amicon centrifugal filtration unit with 50 mM HEPES pH 7.8 buffer and centrifuged at 3,200 x g in 15—20 minutes intervals with intermediate mixing to achieve a concentration of 30-35 mg / mL of Ab in the crude mixture.Purification of (NPBAf-Lys-PEG-Ab (Linker-to-Ab Ratio (LAR) I)

[0142] 2 mL of (NPBA)2-Lys-PEG-Ab crude mixture, at approximately 30 mg / mL in 50 mM HEPES, pH 7.8, was added to a reaction vessel. 3 mL of mobile phase A (1.5 M Ammonium Sulfate, 20 mM Sodium Phosphate pH 7.4) was added to achieve a final solution of 0.9 M ammonium sulfate, 12 mM sodium phosphate, pH 7.4. After mixing, a 2.5 mL sample was loaded onto a 5 mL HiTrap Phenyl HP HIC column attached to an AKTA Go FPLC system using a 5 mL sample loop. Mobile phase B (20 % IP A, 10 mM Sodium Phosphate pH 7.4) was used as the eluent. The monoPEGylated (NPBA)2-Lys-PEG-Ab (LAR 1) was isolated using a continuous gradient of elution buffer 40 % to 100 % B over 25 column volumes at a flow rate of 5 mL / min. The fractions containing monoPEGylated 2NPBA-Lys-PEG-Ab (LAR 1) were combined and transferred to a pre-washed 15 mL 30 kDa MWCO Amicon centrifugal filtration unit with lx PBS, pH 7.4. The fractions were concentrated using the 15 mL 30 kDa MWCO Amicon centrifugal filtration unit and centrifuged at 3,200 x g for 15 minutes. The flow-through was replaced with lx PBS pH 7.4 buffer and centrifugation was repeated. This process was repeated until < 0.01 % of the initial solution remained. The concentration of the purified monoPEGylated (NPBA)2-Lys- PEG-Ab (LAR 1) was adjusted to 30-40 mg / mL and the purified (NPBA)2-Lys-PEG-Ab material was stored at -80 °C.

[0143] Substitution, in the method described above, of anti-CD8 antibody with any other protein-, peptide-, or aptamer- based targeting agent yields the respective targeting agentcontaining (NPBA)2-Lys-PEG polymer crude mixtures and purified components.Example 9: Formation of nanoparticles

[0144] mRNA-containing nanoparticles, not containing a targeting agent, were formulated by first mixing Im-cMAP-Im with (NPBA)2-Lys-PEGm in 10 mM phosphate buffer, pH 7.4 at a 1 : 1 molar ratio of Im-cMAP-Im vicinal diols to (NPBA)2-Lys-PEGm. The mixtures were briefly vortexed, centrifuged down, and allowed to sit for 15 min at room temperature. mRNA at a concentration of up to 1 mg / mL, in a volume of RNAse-free water equivalent to the volume of the centrifuged polymer conjugate, was added to the centrifuged polymer conjugate at a charge ratio (+ / -) of cationic polymer to mRNA varying from 3:1 to 1 : 1.

[0145] Nanoparticles containing a targeting agent were made in a similar fashion, except Im-cMAP-Im was first mixed with (NPBA)2-Lys-PEGm and (NPBA)2-Lys-PEG-Ab, in 10 mM phosphate buffer, pH 7.4, at molar ratios of Im-cMAP-Im:(NPBA)2-Lys-PEGm:(NPBA)2-Lys- PEG-Ab that varied from 1 :0.95:0.05 to 1 :0.995:0.005.

[0146] Substitution, in the method described above, of (NPBA)2-Lys-PEG-Ab with any other 2NPBA-Lys-PEG-conjugated targeting agent yields the respective differently targeted nanoparticles.

[0147] For formulations without (NPBA)2-Lys-PEGm or (NPBA)2-Lys-PEG-Ab (i.e., Im- cMAP-Im and RNA), equal volumes of the Im-cMAP-Im polymer (in 10 mM phosphate buffer, pH 7.4) and mRNA (in RNAse-free water) were simply mixed at an appropriate charge ratio.

[0148] Any RNA (e.g., mRNA, siRNA, miRNA, antisense RNA, tracr RNA, guide RNA) of choice can be used in this method. Exemplary RNAs include, for example, CD 19 CAR mRNA, anti-EGFR siRNA (siEGFR), or anti-BRAF siRNA (siBRAF).Example 10: Characterization of nanoparticles

[0149] The ability of the Im-cMAP-Im polymers and polymer conjugates to encapsulate mRNA was analyzed using two methods: a gel retardation assay and a RiboGreen assay. For the gel retardation assay, increasing concentrations of polymer or polymer drug conjugate in 14 pL water were mixed with 1 pL of 1 mg / mL mRNA in water at charge ratios (+ / -) of 0, 0.5, 1, 1.5, 2, 2.5, 3, and 5 for a total volume of 15 pL. The mixtures were briefly vortexed, centrifuged down, and allowed to sit for 15 min at room temperature. 3 pL of 6x DNA loading dye was added to each mixture, and the samples were loaded onto a 1 % agarose gel (in TBE) and run in 0.5x TBE buffer. The gel was visualized on a gel imaging system.

[0150] The RiboGreen assay was performed in a manner similar to the gel retardation assay, except that it used increasing concentrations of polymer or polymer conjugate in 99 pL water and 1 pL of 0.1 mg / mL mRNA in water for a total volume of 100 pL in a 96-well plate. To each mixture, 100 pL of RiboGreen RNA reagent working solution (prepared according to the kit’s protocol) was added. The plate was briefly shaken, incubated in the dark for 5 min at room temperature, and the fluorescence intensity measured on a Thermo Fisher Scientific Varioskan LUX plate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The results of at least 3 measurements were averaged.

[0151] Nanoparticle size was determined using dynamic light scattering (DLS). DLS was performed on a Malvern Zetasizer Nano ZS instrument. The particles were diluted to a concentration of 0.2 mg / mL mRNA or less, depending on the formulation. The results of at least 10 measurements were averaged.

[0152] Surface charge, or zeta potential, of nanoparticles was measured using a Malvern Zetasizer Nano ZS instrument. 10 pL of nanoparticle formulation was mixed with 1.5 mL of either 10 mM phosphate buffer, pH 7.4 or 1 mM potassium chloride, pH 5.5. The results of at least 10 measurements were averaged.Incorporation by Reference

[0153] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.Equivalents

[0154] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMSWhat is claimed is:

1. A cationic polymer described by the structure of Formula (II):wherein:End Groups A and B independently comprise an amine-containing group with a pKa between 4 and 7 inclusive; and cMAP iswherein: m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is independently at each occurrence 0, 1, 2, 3, 4, or 5; and p is sufficient to provide a number average molecular weight for the subunit comprising cMAP in a range from about 500 Da to about 50,000 Da.

2. The cationic polymer of claim 1, wherein m is 4, 5, or 6.

3. The cationic polymer of claim 1, wherein the amine-containing group is an imidazole group.

4. The cationic polymer of claim 3, wherein:End Group A is:End Group B is:wherein:Li and L2 are linking groups; and k is independently at each occurrence 0, 1, 2, 3, 4, or 5.

5. The cationic polymer of claim 4, wherein Li and L2 are independently at each occurrence:-(Co-6alkylene)-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-,-(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.

6. The cationic polymer of claim 4, wherein Li is -C-C(=O)- and L2 is -C(=O)-C-.

7. The cationic polymer of claim 4, wherein: m is 5; n is 1; p is 4-40; k is 1;Li is -C-C(=O)- ; and L2 is -C(=O)-C-.

8. The cationic polymer of claim 7, further comprising a biological agent.

9. The cationic polymer comprising a biological agent of claim 8, wherein the biological agent is a nucleic acid.

10. The cationic polymer comprising a biological agent of claim 9, wherein the nucleic acid is a RNA molecule.

11. The cationic polymer comprising a biological agent of claim 10, wherein the RNA molecule is a messenger RNA (mRNA) molecule or a siRNA molecule.

12. The cationic polymer comprising a biological agent of claim 11, wherein the mRNA molecule encodes a chimeric antigen receptor (CAR).

13. The cationic polymer comprising a biological agent of claim 12, wherein the chimeric antigen receptor is targeted to CD 19 or B-cell maturation antigen (BCMA).

14. The cationic polymer comprising a biological agent of claim 9, wherein the nucleic acid is bound to the polymer by electrostatic interactions between the backbone of the polynucleotide and amidine groups of the cMAP.

15. The cationic polymer comprising a biological agent of claim 8, wherein the biological agent is covalently bound to the cationic polymer.

16. The cationic polymer comprising a biological agent of claim 15, wherein the biological agent is bound through a ether, ester, amide or carbamate linkage to the cMAP.

17. A polymer conjugate comprising:(a) the cationic polymer comprising a biological agent of any of claims 8-16; and(b) one or more boronic acid-containing polymers comprising a structure of Formula (III):wherein: the cationic polymer and the boronic acid-containing polymer are reversibly linked to one another by one or more borate condensation linkages between the one or more boronic acid moiety or moieties of Formula (III) and one or more pairs of vicinal diols of the cMAP portion of the cationic polymer of Formula (II); the spacer is selected from any linear or branched organic group;L3 is a linking group, or linking groups, between the one or more boronic acid moieties and the spacer;L4 is a linking group, or linking groups, between the spacer and the one or more polyethylene oxide linkages; q is independently at each occurrence 0, 1, 2, 3, or 4; r is 1, 2, or 3; s is independently at each occurrence 20-1200; t is 1, 2, or 3; andXi is(a) -Ci-6 alkyl, optionally substituted with -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH2, -NH(alkyl), or N(alkyl)2; or(b) a salt or protected analog of any of the foregoing.

18. The polymer conjugate of claim 17, wherein q is 1.

19. The polymer conjugate of claim 17, wherein r is 2.

20. The polymer conjugate of claim 17, wherein s is 50-300.

21. The polymer conjugate of claim 17, wherein t is 1.

22. The polymer conjugate of claim 17, wherein L3 and L4 are independently at each occurrence:-(Co-6alkylene)-NH-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-NH-(Co-6alkylene)-,-(Co-6alkylene)-0-C(=0)-(Co-6alkylene)-,-(Co-6alkylene)-C(=0)-0-(Co-6alkylene)-, -(Co-6alkylene)-NH-C(=0)-0-(Co-6alkylene)-, or -(Co-6alkylene)-0-C(=0)-NH-(Co-6alkylene)-.

23. The polymer conjugate of claim 17, wherein the spacer is an organic group bearing at least one terminal amine and at least one terminal carboxylic acid.

24. The polymer conjugate of claim 23, wherein the spacer has the formula -NH-CH-Ri-C(=O)-, wherein Ri is an amino acid functional group.

25. The polymer conjugate of claim 24, wherein:(a) Ri is the functional group of lysine;(b) L3 is -C(=O)-NH-; and(c) L4is -C(=O)-NH-.

26. The polymer conjugate of claim 17, wherein: the spacer is a lysyl group; q is 1; r is 2; s is 50-300; t is 1; for the first instance of r, L3 is -C(=O)-NH-; for the second instance of r, L3 is -C(=O)-NH-;L4is -C(=O)-NH-; andXi is -CH3or -C-C(=O)-OH.

27. The polymer conjugate of claim 26, further comprising one or more targeting molecules.

28. The polymer conjugate of claim 27, wherein the targeting molecule is selected from the group consisting of a protein, a peptide, an aptamer, and a ligand for a cellular receptor.

29. The polymer conjugate of claim 28, wherein the protein is an antibody or an antibody fragment.

30. The polymer conjugate of claim 29, wherein the antibody or antibody fragment is directed to CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR).

31. The polymer conjugate of claim 28, wherein the cellular receptor is CD3, CD4, CD8, the epidermal growth factor receptor (EGFR) or the transferrin receptor (TfR).

32. The polymer conjugate of claim 28, wherein the ligand for a cellular receptor is transferrin.

33. The polymer conjugate of claim 27, wherein the targeting molecule is a covalent conjugate of Xi, such that Xi has the structure -C-C(=O)-NH-targeting molecule.

34. A nanoparticle comprising the cationic polymer comprising a biological agent of claim 11.

35. A nanoparticle comprising the cationic polymer comprising a biological agent of claim 13.

36. A nanoparticle comprising the polymer conjugate of claim 31.

37. The polymer conjugate of claim 17, the conjugate comprising a plurality of molecules of Formula III, wherein:(a) in at least one of the molecules of Formula III, Xi is -CH3; and(b) in at least one of the molecules of Formula III, Xi comprises a targeting agent.

38. The polymer conjugate of claim 37, wherein the targeting agent is an anti-CD8 antibody.

39. The polymer conjugate of claim 38, wherein the biological agent is a mRNA encoding a chimeric antigen receptor that is targeted to CD 19 or B-cell maturation antigen (BCMA).

40. A nanoparticle comprising the polymer conjugate of claim 39.

41. A pharmaceutical composition comprising the nanoparticle of any of claims 34-36 or 40 and a pharmaceutically acceptable carrier or excipient.

42. A method for the treatment of a disease or disorder in a subject, the method comprising administering the pharmaceutical composition of claim 41 to the subject.

43. The method of claim 42, wherein the disease or disorder is a malignancy.

44. The method of claim 43, wherein the malignancy is selected from the group consisting of a hematologic malignancy and a solid tumor.

45. The method of claim 44, wherein the hematologic malignancy is a B-cell malignancy.

46. The method of claim 45, wherein the B-cell malignancy is a leukemia or a lymphoma.

47. The method of claim 44, wherein the solid tumor is a breast tumor, a lung tumor, an ovarian tumor, a gastric tumor, a colorectal tumor, a pancreatic tumor, a primary brain tumor or a brain metastasis.

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