In VIVO expression of a polymer polynucleotide complex

Abstract

The present invention is directed to cationic polymer delivery systems for DNA-mediated antibody expression. The present invention provides cationic polymers electrostatically complexed with gene-encoding nucleic acids including DNA, pDNA, ncDNA, npDNA, and mRNA for delivery to cells to induce the production of antibodies from the polymer polynucleotide complex in vivo. The present invention further relates to a polymer pDNA complex that includes a backbone, any associated pendant groups, ions, or salts thereof. The present invention also provides pharmaceutical compositions, the use of the pharmaceutical compositions as well as kits.

Description

IN VIVO EXPRESSION OF A POLYMER POLYNUCLEOTIDE COMPLEXField of the Disclosure

[0001] The present disclosure relates generally to the fields of biology, medicine, and immunology. More particularly, the disclosure relates to the use of polymeric nanoparticles to deliver pDNA to a target cell of interest, resulting in a biological outcome and neutralization of a disease, virus, or cancer.BACKGROUND

[0002] Delivery of biological agents, such as nucleic acids, peptides, proteins, or small molecules, to cells both in vitro and in vivo has been performed using various recombinant viral vectors, lipid delivery systems, and electroporation. Such techniques have sought to treat various diseases and disorders by reducing or inhibiting gene expression, providing genetic constructs for gene therapy or to study various biological systems. Despite the vast curative potential of biological agents, widespread clinical deployment faces an uncertain outlook due to difficulties in delivery of the agent to the desired tissue.

[0003] Monoclonal antibodies (mAbs) are synthetic proteins that are able to bind to and neutralize various viruses, bacteria, and other pathogens. Since their first clinical approval in the 1980’s, mAbs have become a highly successful pharmaceutical class that are broadly used to treat various ailments including cancers and infectious, autoimmune, and inflammatory diseases. However, widespread global access to mAb therapies is hindered by high costs associated with both development and manufacturing, mAb storage conditions, and the need for repeated administrations. In order to combat these associated challenges, researchers have developed a strategy called antibody gene transfer, in which genes encoding for the necessary protein, in the form of DNA or mRNA, are introduced to host cells in order to trigger the production of the therapeutic mAb of interest. These proteins are then secreted for a prolonged period of time either locally or systemically depending on the site of production. The sustained and in situ production of mAbs can help reduce costs associated with manufacturing a storage, eliminate the necessity for or reduce the frequency of repeated dosing, and broaden the prophylactic applications of mAb therapies.

[0004] While gene transfer strategies have the potential to transform the landscape of antibody therapeutics, delivery of the genetic encoding information to the host cell presents a significant challenge. Up to this point, the majority of pre-clinical and clinical gene therapies designed for the production of mAbs have utilized viral vectors, naked genetic cargo, or lipid nanoparticles (LNPs). Viral vectors (including adenoviral vectors, adeno-associated viral vectors, and oncolytic viruses) are evolutionarily designed to successfully deliver genetic information to cells, but high production and purification costs combined with a variety of issues regarding immunogenicity have limited the accessibility and impact of these vectors. Issues associated with using viral vectors for gene transfer have been avoided by eliminating the vector and delivering naked genetic cargo in the form of plasmid DNA (pDNA), minicircle DNA (mcDNA), or nanoplasmidDNA (npDNA). Compared to mAbs or viral vectors, DNA is more straightforward to produce and store at a lower cost and is considered to have low immunogenicity. However, without a vector to deliver the genetic information to the nucleus, physical methods of transfection such as electroporation or hydrodynamic injection are necessary to coax the host cells into accepting foreign DNA with both intravenous and intramuscular injections. While injecting naked DNA has resulted in expression of the encoded proteins in a relatively safe and cost-effective manner, transgene expression is significantly limited when compared to viral vectors.

[0005] Genome editing, for example, based on clustered regularly interspersed palindromic repeats (CRISPR) technology has transformed the therapeutic landscape for diseases wherein the deletion, insertion or repair of genetic sequences can restore healthy cellular states. Clinical trials of investigational gene therapeutics for P-thalassemia and sickle cell disease suggest that safe and efficacious treatment is possible using CRISPR-based genome editing technology.Additional clinical trials are underway to develop CRISPR-based therapeutics for debilitating conditions such as Duchenne’s muscular dystrophy (DMD), Leber congenital amaurosis (LCA) and for chimeric antigen receptor T-cell (CAR-T) therapies for cancer.

[0006] Despite the vast curative potential of CRISPR, widespread clinical deployment faces an uncertain outlook due to reliance on engineered viral vectors, which can be used to deliver therapeutic biomacromolecule payloads such as messenger RNA (mRNA), plasmid DNA (pDNA) and small interfering RNA (siRNA). However, the high costs, lengthy time requirements, and regulatory challenges involved in manufacturing clinical grade viruses at scalefor large patient populations have imposed severe logistical bottlenecks. In addition to manufacturing and regulatory delays, the cargo capacity of viral vectors is limited, and this size restriction is particularly problematic in the context of bulky multi-component CRISPR cargoes.

[0007] Although advances in virus manufacturing have minimized occurrences of carcinogenic mutations, genomic integration and fatal systemic inflammatory responses, these risks are amplified when repeated dosing or large dosages are involved. For CRISPR therapeutics to become safe, scalable, and affordable, there is a need to identify synthetic substitutes for viral carriers.

[0008] Polymeric delivery vehicles have been used in clinical therapies due to their versatility, relative low production cost, and low immunogenicity. Synthetic polymers have been used to deliver biomacromolecule payloads such as, for example, pDNA, ribonucleoproteins (RNP), and the like, due to their versatility, low toxicity, and the ability to encapsulate large payloads. Some recent examples indicate that synthetic polymer-based systems achieved biomacromolecule based gene delivery and gene editing both in vitro and in vivo.

[0009] For example, in aqueous physiological solutions, cationic polymers can spontaneously bind with negatively charged pDNA and form interpolyelectrolyte complexes. These complexes are predominately internalized by various endocytic routes, followed by cargo release from these vesicles inside the cells via different proposed mechanisms, and subsequent entry into the cell nucleus to promote gene expression. Compared to viral vehicles, polymeric delivery systems typically have lower delivery efficiency, and various optimization strategies can be used to improve this parameter such as changing the cationic moieties on polymers, adding targeting ligands, and installing responsive monomers, which can improve uptake efficiency and help to balance transfection efficiency and cytotoxicity. However, their utility in genome editing is relatively underexplored.

[0010] In the sera of human immunodeficiency virus type 1 (HIV-1) infected patients, anti-virus antibodies can be detected over a certain period after infection without any clinical manifestations of the acquired immunodeficiency syndrome (AIDS). At this state of active immune response, high numbers of antigen-specific B-cells are expected in the circulation. These B-cells are used as fusion partners for the generation of human monoclonal anti-HIV antibodies. One major drawback to finding a vaccine composition suitable for more reliable prevention of human individuals from HIV-1 infection and / or for more successful therapeutic treatment ofinfected patients is the ability of the HIV-1 virus to escape antibody capture by genetic variation, which very often renders the remarkable efforts of the researchers almost useless. Such escape mutants may be characterized by a change of only one or several of the amino acids within one of the targeted antigenic determinants and may occur, for example, as a result of spontaneous or induced mutation. In addition to genetic variation, certain other properties of the HIV-1 envelope glycoprotein make it difficult to elicit neutralizing antibodies making generation of undesirable non-neutralizing antibodies a major concern (see, Phogat SK and Wyatt RT, Curr Pharm Design 2007;13(2):213-227).

[0011] HIV-1 is among the most genetically diverse viral pathogens. Of the three main branches of the HIV-1 phylogenetic tree, the M (main), N (new), and 0 (outlier) groups, group M viruses are the most widespread, accounting for over 99% of global infections. This group is presently divided into nine distinct genetic subtypes, or clades (A through K), based on full length sequences. Env is the most variable HIV-1 gene, with up to 35% sequence diversity between clades, 20% sequence diversity within clades, and up to 10% sequence diversity in a single infected person (Shankarappa, R. et al. 1999. J. Virol. 73:10489-10502). Clade B is dominant in Europe, the Americas, and Australia. Clade C is common in southern Africa, China, and India and presently infects more people worldwide than any other clade (McCutchan, FE. 2000.Understanding the genetic diversity of HIV-1. AIDS 14(Suppl. 3): S31-S44). Clades A and D are prominent in central and eastern Africa.

[0012] Accordingly, what is needed, therefore, is a novel and efficient polymer-based delivery vehicle to deliver nucleic acids to produce antibodies against, e.g., HIV.SUMMARY

[0013] Herein, we present a new strategy to deliver gene encoding DNA to host cells using cationic polymer nanoparticles (PNPs). As a consequence of their versatility, large encapsulation efficiency, low production cost, and low toxicity, polymers have been widely explored as delivery vehicles in various genetic therapies for both PNP gene delivery and editing, but have not yet been used for DNA-mediated antibody gene transfer. In this approach, anionic DNA is electrostatically complexed with cationic polymer chains to produce interpolyelectrolyte nanoparticles. These particles can be internalized into the cell via endocytic mechanisms and release cargo into the nucleus to promote sustained transgene expression and antibody transfer.

[0014] Disclosed herein is a pharmaceutical composition comprisinga. a first polymer comprising:i. a polymer comprising a backbone and one or more charged pendant groups,ii. a polymer comprising a backbone and one or more charged pendant groups, and one or more uncharged pendant groups, iii. a polymer comprising a backbone derived from one or more than one first monomer, which is a ionizable N-alkyl or N, N-dialkyl substituted (alkyl) methacrylamide, methacrylate, styrenic, acrylamide, or acrylate, iv. a polymer comprising a backbone derived from one or more than one first monomer, which is a ionizable N-alkyl or N, N-dialkyl substituted (alkyl) methacrylamide, methacrylate, styrenic, acrylamide, or acrylate, and one or more than one second monomer comprising non-ionizable structural units,v. a polymer having a backbone comprising a plurality of structural units derived from one or more than one first monomer, which is an N-alkyl or N, N-dialkyl substituted (alkyl)acrylamide monomer, and a plurality of hydrophilic structural units derived from one or more than one second monomer, which is an ethylenically unsaturated monomer,vi. a polymer comprising a backbone and one or more pendant groups selected from aminoalkyl group or amino aryl group, a dialkyl group, a neutral substituted alkyl group, or an aryl group or a neutral heteroalkyl group,vii. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, orviii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. a heterologous first nucleic acid encoding one or more proteins.

[0015] Disclosed herein is a modified cell comprising:a. a first polymer comprising;i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5-95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. an expression cassette comprising a heterologous polynucleotide sequence encoding one or more therapeutic proteins and a regulatory sequence operably linked to the therapeutic protein.

[0016] Further disclosed herein is a method for administering a polymer polynucleotide complex into a cell in a subject in need thereof comprising:a. introducing into the cell a polymer polynucleotide complex comprising:i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. recombinantly expressing a polynucleotide sequence encoding a therapeutic protein and a regulatory sequence operably linked to the therapeutic protein.

[0017] In additional embodiments, disclosed herein is a method of treating a patient in need thereof by administering a polymer nucleic acid complex comprising:a. a means for transfecting a cell with the polymer nucleic acid complex comprising:i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5-95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains;b. a means for encoding a nucleic acid to one or more proteins; andc. a means for binding a target of interest with the one or more proteins.

[0018] Disclosed herein is a method of treating, preventing, or ameliorating an immune response to an antigen comprising administering to a subject in need thereof a pharmaceutical composition comprisinga. a polymer nucleic acid complex comprising:i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5-95% of the total number of first and second pendant groups and the first and second pendant groups are different, or a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. a polynucleotide encoding an antibody.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] FIG. 1A shows non-PEGylated polymers (APMAm 15% / MAETMA 10% / PhHPMA 75%) assembled into complexes with the biological agent, pDNA, which were evaluated for efficient delivery of pDNA.

[0021] FIG. IB shows non-PEGylated polymers (APMAm 15% / MAETMA 10% / PhHPMA 75%) assembled into complexes with the biological agent, mRNA, which were evaluated for efficient delivery of mRNA.

[0022] FIG. 2A shows PEGylated polymers (APMAm 15% / MAETMA 10% / PhHPMA 75%) assembled into complexes with the biological agent, pDNA, which were evaluated for efficient delivery of pDNA.

[0023] FIG. 2B shows PEGylated polymers (APMAm 15% / MAETMA 10% / PhHPMA 75%) assembled into complexes with the biological agent, mRNA, which were evaluated for efficient delivery of mRNA.

[0024] FIG. 3 A shows polymers (APMAm 65% / MAETMA 10% / PhHPMA 25%) assembled into complexes with the biological agent, pDNA, which were evaluated for efficient delivery of pDNA.

[0025] FIG. 3B shows polymers( APMAm 65% / MAETMA 10% / PhHPMA 25%) assembled into complexes with the biological agent, mRNA, which were evaluated for efficient delivery of mRNA.

[0026] FIG. 4A shows polymers (APMAm 40% / MAETMA 10% / PhHPMA 50%) assembled into complexes with the biological agent, pDNA, which were evaluated for efficient delivery of pDNA.

[0027] FIG. 4B shows polymers (APMAm 40% / MAETMA 10% / PhHPMA 50%) assembled into complexes with the biological agent, mRNA, which were evaluated for efficient delivery of mRNA.

[0028] FIG. 5A shows the biodistribution of polymers from A-2, wherein n = 4 and including one multiplex, across liver, lung, spleen, heart, and kidney.

[0029] FIG. 5B shows the real quantitation of biodistribution of polymers from A-2, wherein n = 4 and including one multiplex, across liver, lung, spleen, heart, and kidney.

[0030] FIG. 5C shows the biodistribution of polymers from A-2, wherein n = 2 and including one multiplex, across liver, lung, spleen, heart, and kidney.

[0031] FIG. 5D shows the real quantitation of biodistribution of polymers from A-2, wherein n = 2 and including one multiplex, across liver, lung, spleen, heart, and kidney.

[0032] FIG. 5E shows the biodistribution of polymers from A-2, wherein n = 3 with all as singleplex, across liver, lung, spleen, heart, and kidney.

[0033] FIG. 5F shows the real quantitation of biodistribution of polymers from A-2, wherein n = 3 with all as singleplex, across liver, lung, spleen, heart, and kidney.

[0034] FIG. 5G shows a spider plot of the biodistribution of polymers from the three A-2 complexes.

[0035] FIG. 5H shows a spider plot of the real quantitation of the biodistribution of polymers from the three A-2 complexes.

[0036] FIG. 6 depicts Nanite’s PGT121 polymer optimization using the SAYER platform for a design across a 1060space to predict novel polymers to target PGT121 serum expression.

[0037] FIG. 7 shows the human IgG concentration in a HEK293T supernatant 6 days posttransfection, using 250 ng of Elipovimab (P2A) GenCircle / Well. These results outperform the results in the scientific literature (Wise MC, et al., (2020). J Clin Invest, 130(2):827-837).

[0038] FIG. 8 shows the IgG concentration in mouse serum levels, with a low in vivo hlgG protein levels (5-30 ng / ml) relative to target level (>10,000 ng / ml).

[0039] FIG. 9A / B shows the Polymer nanoparticle tissue biodistribution with pDNA and the polymer nanoparticle tissue transfection levels with RNA for the candidates.

[0040] FIG. 10A / B shows the top three candidate polymers for in vivo pElipovimab (P2A) injections, and tissue biodistribution and RNA generation.

[0041] FIG. 11 shows the PGT121 GenCircle (Catalent Sequence) delivered in vitro using various polymers.

[0042] FIG. 12 confirms the delivery of lead polymer candidates in vitro.

[0043] FIG. 13 shows the dosing and in vivo jetPEI tissue transfection, and GenCircle dose resulting in hlgG concentration.

[0044] FIG. 14A / B shows the in vitro hit polymers transfect PGT121 in vivo.

[0045] FIG. 15 is an in vivo confirmation of the hit polymers using different antimals, showing hlgG signal in serum using ELISA.

[0046] FIG. 16 shows an in vivo increase of serum levels in mice, providing a strong baseline for a Rag2 - / - intravenous study. These results are due in part to optimization of both the formulation and ELISA.

[0047] FIG. 17 shows the promising initial data from the first IM injections in the right gastrocnemius. These results depict little to no localization in tissues other than the target tissue, as there is a significantly higher signal in target tissues than non-target tissues.

[0048] FIG. 18A shows the relative binding affinity of polymer 1 with pDNA.

[0049] FIG. 18B shows the relative binding affinity of polymer 1 with mRNA.

[0050] FIG. 19 shows transfection of Jurkat cells with mRNA by polymer 1 polyplex with (left) and without (right) calcein red signal.

[0051] FIG. 20 shows transfection of Jurkat cells with mRNA by benchmark LNP (top row) and polymer 1 polyplex with a 100:1 antibody conjugate (bottom row). The polymer 1 complex had both higher average transfection efficiency and viability.

[0052] FIG. 21 shows the delivery of DNA-encoded PGT121 with IM injection in mice. Error bars = SEM.

[0053] FIG. 22 depicts the in vitro transfection of PNPs. FIG. 22A depicts the representative images of HEK293T cells transfected with PNPs carrying a ZsGreen reporter plasmid across different N / P ratios. ZsGreen expression and cell nuclei marker are shown in green and blue, respectively. FIG. 22B represents the heat map of the average effective transfection efficiency (product of transfection efficiency and viability) from experiments in FIG. 22A. FIG. 22C represents the HEK293T supernatant PGT121 levels 1 week after in vitro transfection ofPGT121 GenCircle delivered with PNPs. n=2-5, error bars=SEM for data in FIG. 22B and FIG.22C.

[0054] FIG. 23 depicts the PNP Characterization of PGT121 DNA Cargo by Dynamic Light Scattering. FIG. 23 A depicts the representative intensity-weighted distributions of PNPs approximately 45 minutes after PNP formulation. FIG. 23B depicts the hydrodynamic radius of PNPs across a range of N / P ratios from experiments in Fig. 23 A. FIG. 23C depicts the poly dispersity of PNPs across a range of N / P ratios from experiments in Fig. 23 A. n=3, error bars=SEM for data in FIG. 23B and FIG. 23C.

[0055] FIG. 24 represents the serum antibody levels following intravenous delivery of PNPs. FIG. 24A depicts the mouse serum PCT121 protein expression after i.v. administration of PNPs with PGT121 GenCircle at 1.8 mg / kg. FIG. 24B depicts the peak serum PGT121 expression levels following intravenous administration from the experiments in FIG. 24A. n=2, error bars=SEM.

[0056] FIG. 25 represents the serum antibody levels following i.m. delivery of PNPs. FIG. 25A represents the ELISA readout of serum PGT121 antibody levels after i.m. PNP delivery of PGT121 GenCircle in RAG2- / - mice on Day 0. polymer, N / P ratio, and dose of GenCircle are denoted on the right for each condition. FIG. 25B represents the peak serum PGT121 expression levels following i.m. administration from the experiments in FIG. 25A. FIG. 25C depicts the focus on the time course of PGT121 serum levels from PNP D5588 shown in FIG. 25A. FIG.25D depicts the focus on the time course of PGT121 serum levels from PNP D3503 shown in FIG. 25A. FIG. 25E depicts the focus on the time course of PGT121 serum levels from PNP D5586 shown in FIG. 25A. FIG. 25F represents a single redosing of PNP D3503 at Day 8 (dashed line) shows higher and more durable PGT121 serum levels when compared to a single dose of PNP 3503 (straight line). n=2, Error bars=SEM.

[0057] FIG. 26 represents the in vitro transfection of DNA-encoded osteocalcin peptide using polymers. HEK293T supernatant osteocalcin peptide levels were measured on anti-osteocalcin MSD assay 2 days post-transfection. FLAG-tagged osteocalcin plasmid cargos with different polymers shown. Fc=human IgG Fc region, ABD=human albumin binding domain. n=3, error bars=SEM.

[0058] FIG. 27 represents the in vivo transfection of DNA-encoded GLP-1 peptide using polymer D9302. This figure depicts the plasma GLP-1 levels following intramuscular delivery of polymer D9302 complexed with a GLP-1 -encoding plasmid. n=l.

[0059] FIG. 28 represents the in vivo transfection of DNA-encoded FGF-21 protein using polymer D35O3. The plasma FGF-21 levels are depicted following intramuscular delivery of polymer D35O3 complexed with an FGF-21 -encoding plasmid. N / P ratios and total dose of FGF-21 -encoding plasmid are shown. n=4 technical replicates for a single mouse, error bars=SEM.

[0060] FIG. 29 represents the in vivo transfection of DNA-encoded FST protein using polymers. Serum follistatin (FST) levels are shown following intramuscular delivery of polymers complexed with an FST-encoding plasmid. n=3, error bars=SEM.

[0061] FIG. 30 represents the subcutaneous delivery of db-DNA by polymer DI 1042 transfects BALB / c mouse cells in vivo. FIG. 30A left panel depicts an example IVIS images from bilateral subcutaneous delivery of polymer DI 1042 carrying db-DNA-fLuc cargo showing both a photograph of mice and a luminescence signal overlaid. FIG. 30A right panel depicts the ROI for analysis (white box) and luminescence signal from the same animals in FIG. 30A, left panel. FIG. 30B depicts the quantification of IVIS transfection signal within ROIs shown in FIG. 30A. n=3, error bars=SEM.

[0062] FIG. 31 represents the subcutaneous delivery of db-DNA by polymers durably transfects in vivo. FIG. 31 A depicts the mouse IVIS transfection signal after bilateral subcutaneous administration of db-DNA-fLuc complexed with polymers. FIG. 3 IB depicts the mouse body weights after bilateral subcutaneous administration of db-DNA-fLuc complexed with polymers, same animals as in FIG. 31 A. FIG. 31C depicts the day 28 ex vivo db-DNA tissue biodistribution after bilateral subcutaneous administration of db-DNA-fLuc complexed with polymer DI 1042 measured via DNA qPCR. The animals are the same in all panels. n=2, error bars=SEM.

[0063] FIG. 32 represents the subcutaneous delivery of db-DNA with polymer DI 1042 maintains a strong transfection signal two months post-injection. FIG. 32A depicts the mouse IVIS transfection signal after bilateral subcutaneous administration of db-DNA-fLuc complexed with polymer DI 1042. FIG. 32B depicts the mouse body weights after bilateral subcutaneous administration of db-DNA-fLuc complexed with polymer DI 1042, with the same animals as in FIG. 32A. n=3, error bars=SEM.

[0064] FIG. 33 represents the subcutaneous delivery of polymer DI 1042 transfects across a wide range of DNA cargo dosages and is well tolerated. FIG. 33A left panel depicts the example IVIS images from bilateral subcutaneous delivery of polymer DI 1042 carrying 0.25 mpk of db-DNA-CMV-fLuc cargo, showing both a photograph of mice and a luminescence signal overlaid. FIG.33A right panel depicts the ROI for analysis (white box) and luminescence signal from the same animals in FIG. 33 A, left panel. FIG. 33B represents the quantification of IVIS transfection signal across a range of db-DNA-CMV-fLuc dosages. FIG. 33C depicts the cytokines from the cGAS-STING pathway were analyzed via MSD assay using blood plasma isolated from the 0.5 mpk group in FIG. 33B at ~24 hours pre-PNP administration (dark gray bars) and 4 hours post-PNP administration (light gray bars). mpk=mg of cargo per kg of mouse weight, n=3, error bars=SEM for all panels.

[0065] FIG. 34 represents the polymer Ml 1163 specifically delivers and expresses db-DNA-fLuc in iWAT via subcutaneous delivery. FIG. 34A depicts the example IVIS images from bilateral subcutaneous delivery of polymer Ml 1163 carrying 0.25 mpk of db-DNA-CAG-fLuc cargo, showing a photograph of mice and a luminescence signal overlaid (left) and a luminescence signal (right). One ROI for each injection are shown in each image (white ovals). FIG. 34B depicts the quantification of in vivo IVIS transfection signal from Ml 1163 PNP animal shown in FIG. 34A. FIG. 34C depicts the day 28 ex vivo IVIS signal from animal shown in FIG.34A. Note the specific transfection signal, shown as luminescence, in both iWATs that is absent in the liver. FIG. 34D depicts the day 28 ex vivo db-DNA-CAG-fLuc tissue biodistribution, measured via DNA qPCR. n=l animal. In FIG. 34B, n=2 iWAT ROIs (which are shown in A) for a single animal, error bars=SEM.

[0066] FIG. 35 shows the transfection of 16HBE-G542X cells across a N / P ratio of 1.5 to 48 for a benchmark commercial polymer, negative control, and DI 56 with dual reporter cargo (pDNA and mRNA).

[0067] FIG. 36 shows the transfection of primary human basal cells across a N / P ratio of 1.5 to 48 for DI 56 with dual reporter cargo (pDNA and mRNA).

[0068] FIG. 37 represents the plasmid map of pDNA-Osteocalcin-FLAG-Fc, used in FIG. 26.

[0069] FIG. 38 represents the plasmid map of pDNA-Osteocalcin-FLAG-ABD, used in FIG. 26.

[0070] FIG. 39 represents the plasmid map of pDNA-GLP-1, used in FIG. 27.

[0071] FIG. 40 represents the plasmid map of pDNA-FGF-21, used in FIG. 28

[0072] FIG. 41 represents the plasmid map of pDNA-FST, used in FIG. 29.

[0073] FIG. 42 represents the plasmid map of db-DNA-CMV-ILuc, used in FIGs. 30-34.

[0074] FIG. 43 represents the plasmid map of db-DNA-CAG-fLuc, used in FIGs. 31, 32, and 34.DESCRIPTIONS OF THE EMBODIMENTS

[0075] The disclosure provides polymeric delivery vehicles for the delivery of biological agents. The polymers described herein may be advantageous in their ability to deliver biological agents to a desired tissue.

[0076] In one aspect, copolymers are synthesized from two monomers, a cyclic anhydride and an epoxide. Ring-opening copolymerization (ROCOP) produces alternating copolymers such that the backbone of the polymer scaffold alternates between the residues of the cyclic anhydride and epoxide monomers to form a polyester. The monomers may contain functionalities that remain intact during the polymerization, which allows for subsequent functionalization after the synthesis of the polymer backbone. In the present invention, copolymer intermediates contain at least one degree of unsaturation that can be utilized in a subsequent conjugation reaction, which allows for the incorporation of additional side chain functionalities. One such conjugation reaction is the thiol-ene reaction. The choice in thiol can be leveraged to produce polymers that are cationic or poly ampholytes. Another conjugation reaction used to produce the present invention is azide-alkyne cycloaddition, e.g., click chemistry. The choice in azide can be leveraged to produce polymers with different 1,2,3-triazolylene side chains. The polymerizations can be initiated with a monovalent initiator, which may contain either a hydroxyl group or carboxylic acid. The initiator becomes one terminus of the polymer and may be used to introduce chain end functionality that can be further used for conjugation with small molecule end-groups, biological agents (e.g., peptides), or coupling with other polymers (e.g., PEG). The use of a monovalent initiator results in a linear polymer. Alternatively, the polymerizations can be initiated with a multivalent initiator, which would result in star polymers.

[0077] In general, the present disclosure is directed to polymers that may be associated with at least one biological agent payload such as pDNA, ribonucleoproteins (RNP), mRNA, and the like. The complexes may be internalized by a cell via various endocytic routes, the biological agent may be released inside the cell, and it subsequently may enter the cell nucleus to alter gene expression. The polymers disclosed herein thus provide a polymeric scaffold that provides awell-defined host configured to bind with biological macromolecular agents and facilitate intracellular delivery thereof. The polymers have physiochemical properties such as, for example, composition, molecular weight, (^-potential, pKa, complex diameter, nucleic acid condensation, and combinations thereof selected for efficient nucleic acid payload delivery using, for example, a CRISPR / Cas9 delivery process. The polymers and related complexes may also have good gene editing efficiency, cellular internalization, cytotoxicity, and combinations thereof.

[0078] The present polymeric delivery systems harness the use of the polymers as delivery systems to take advantage of biological agents having a negative charge, e.g., nucleic acids due to the phosphate groups along each nucleotide. The present polymeric delivery vehicles may condense biological agents, such as CRISPR payloads (for example, mRNA, pDNA or RNP), which can vary widely in their lengths, topologies, physical characteristics, and biological mechanisms, into discrete nanosized polyelectrolyte complexes. Upon administration, the complexes may navigate both extracellular barriers such as serum DNAases (or RNAases) and reticuloendothelial system clearance, as well as intracellular barriers such as endosomal interrogation and lysosomal degradation. Finally, the biological agent may be released within the spatiotemporal window that is optimal for payload translocation to the nucleus, where the biological agent can undergo further processing and realization of targeted edits. In addition to meeting high standards for safety, efficiency and cost-effectiveness, synthetic delivery systems may minimize immune activation and cellular toxicity. In the presence of water, the present polymers undergo hydrolytic degradation into byproducts that are either resorbable, or benign and small enough for renal clearance. Thus, in a clinical setting, treatments using these polymers could be given to patients multiple times without concerns over toxic polymer buildup within the body.DEFINITIONS

[0079] To facilitate the understanding of this invention, a number of terms are defined below and throughout the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0080] Terms such as "a", "an," and "the" are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration.

[0081] The term "about," is afforded a general understanding, known or knowable by one skilled in the art. And may depend upon a particular situation. In various embodiments, about, refers to a value that is within 30%, 20% or 10% above or below the value being described. In various embodiments, about pH 7 includes, but is not limited to, physiological pH.

[0082] The term "antibody" or "immunoglobulin (Ig)" is used in the broadest sense and includes monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, chimeric antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), and antibody fragments (as described in greater detail herein). An antibody typically comprises both "light chains" and "heavy chains." The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, s, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0083] " Antibody fragments" or "fragments" comprise only a portion of an intact antibody. The portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments (e.g., single-chain variable fragments (scFv)); diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigenbinding fragments, called " Fab" fragments, each with a single antigen-binding site, and a residual " Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibodyfragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function, ADCVI function, and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise an antigen-binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[0084] The term "antibody-dependent cellular cytotoxicity" or " ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet (Annu. Rev. Immunol. 9:457-92, 1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U. S. Patent No. 5,500,362 or 5,821,337 or Presta U. S. Patent No. 6,737,056 may be performed.

[0085] By "broadly neutralizing antibody" or "bnAb," with respect to HIV (e.g., HIV-1), is meant an antibody that recognizes a specific antigen (e g., gpl20 of HIV) and inhibits the effect(s) of the antigen of at least 2, 3, 4, 5, 6, 7, 8, 9 or more different strains of HIV, the strains belonging to the same or different clades, in the host subject (e.g., human). As used herein, the antibody can be a single antibody or a plurality of antibodies.

[0086] The term "conjugated moiety" as used herein, refers to a residue of a conjugation reaction.

[0087] The term "conjugation reaction" as used herein, refers to a chemical reaction in which a functional group containing at least one degree of unsaturation is utilized to link atoms together.

[0088] The term "effective amount," as used herein, refers to the amount that is necessary to result in a physiological change in the cell, organism, or tissue to which it is administered.

[0089] An "enzyme replacement therapy enzyme" or " ERT enzyme" refers to an enzyme that is deficient in a lysosomal storage disorder. An " ERT enzyme variant" refers to a functionalvariant, including allelic and splice variants, of a wild-type ERT enzyme or a fragment thereof, where the ERT enzyme variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or fragment thereof, e.g., when assayed under identical conditions. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, where the catalytically active fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ERT enzyme or variant thereof, e.g., when assayed under identical conditions.

[0090] The term "individual" or "subject" is an animal, such as a mammal, bird, amphibian, or reptile.

[0091] Mammals, as used herein, include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Particularly, the individual or subject is a human.

[0092] As used herein, by " PGT family antibody" is meant an antibody, or antibody fragment thereof, including PGT121, and PGT121 derivatives and clonal relatives thereof (e.g., antibody 10-1074), such as those disclosed in WO 2012 / 030904; WO 2013 / 055908; Walker et al. Nature.477: 466-470, 2011; Mouquet et al. Proc. Natl. Acad. Sci. 109(47): E3268-E3277, 2012; Julien et al., PLoS Pathog. 9: el003342, 2013; and Kong et al., Nat. Struc. Mol. Biol. 20: 796-803, 2013.

[0093] The term "pharmaceutical composition," as used herein, refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0094] The term "pharmaceutically acceptable carrier," as used herein, refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0095] By "proviral DNA" is meant viral (e.g., retroviral, e.g., HIV, e.g., HIV-1) genomic DNA that is integrated into the DNA of a host cell, such as a tissue cell (e.g., a lymph node, gastrointestinal, or peripheral blood tissue cell).

[0096] The term "therapeutically effective amount," as used herein, e.g., of a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes, reduces, or prevents adverse effects of a disease.

[0097] The term "individual" or "subject" is an animal, such as a mammal, bird, amphibian, or reptile. Mammals, as used herein, include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.

[0098] By "specifically binds" is meant the preferential association of an antibody, or fragment thereof, to a target molecule (e.g., a viral protein, e.g., gpl20, e.g., the N332 glycan of gpl20) in a sample (e.g., a biological sample) or in vivo or ex vivo. It is recognized that a certain degree of non-specific interaction may occur between an antibody and a non-target molecule.Nevertheless, specific binding may be distinguished as mediated through specific recognition of the target molecule. Specific binding results in a stronger association between the antibody, or fragment thereof, and, e.g., an antigen (e.g., gpl20, e.g., the N332 glycan of gpl20) than between the antibody and, e.g., a non-target molecule (e.g., non-viral polypeptide). In one example, the antibody may specifically bind to the N332 glycan of envelope glycoprotein gpl20 of HIV. In another example, the antibody may specifically bind to the CD4 binding site (CD4bs) of envelope glycoprotein gp!20 of HIV. The antibody (e.g., PGT121) may have, e.g., at least 2-fold greater affinity (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 102-, 103-, 104-, 105-, 106-, 107-, 108-, 109-, or 1010-fold greater affinity) to the gpl20 protein than to other viral or non-viral polypeptides (e.g., PGT121 has at least 2-fold greater affinity to gpl20 than a comparable IgG antibody).

[0099] The term "therapeutically effective amount," as used herein, e.g., of a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of anagent for example eliminates, decreases, delays, minimizes, reduces, or prevents adverse effects of a disease.

[0100] The term "alkenyl," as used herein, refers to an acyclic straight or branched chain monovalent hydrocarbon group containing one or more double bonds, no triple bonds, and from 2 to 12 (e.g., 2 to 6) carbons, unless otherwise specified. Alkenyl groups may be substituted or unsubstituted. Exemplary substituents include alkoxy, alkylthio, alkynyl, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, epoxy, ester, halo, heterocyclyl, hydroxyl, oxo, phospho, sulfa, and thiol.

[0101] The term "alkyl," as used herein, refers to an acyclic straight or branched chain, saturated, monovalent hydrocarbon group having from 1 to 12 carbons (e.g., 1 to 6), unless otherwise specified. Alkyl groups may be substituted or unsubstituted. Exemplary substituents include alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, epoxy, ester, halo, heterocyclyl, hydroxyl, oxo, phospho, sulfa, and thiol. An alkyl may be substituted with an oxo to form an aldehyde or ketone.

[0102] The term "alkynyl," as used herein, refers a straight or branched monovalent hydrocarbon group containing one or more triple bonds and from 2 to 12 (e.g., 2 to 6) carbons, unless otherwise specified. Alkynyl groups may be unsubstituted or substituted as alkenyl groups.Exemplary substituents include alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, epoxy, ester, halo, heterocyclyl, hydroxyl, oxo, phospho, sulfa, and thiol.

[0103] The term "alkoxy," as used herein, refers to a group of the formula RO-, wherein R is an alkyl group as defined herein. Alkoxy groups may be unsubstituted or substituted as alkyl groups. An alkoxy may be substituted with an oxo group to form an ester. Three alkoxy groups may be bound to the same carbon to form an orthoester.

[0104] The term "alkylthio," as used herein, refers to a group of the formula RS-, wherein R is an alkyl group as defined herein. Alkylthio groups may be unsubstituted or substituted as alkyl groups.

[0105] The term "amido," as used herein, refers to a group of the formula -NRGC(=O)RHor -C(=O)NRGRH, where each of RG and RH are independently H, alkyl, alkenyl, alkynyl, carbocyclyl, heteroalkyl, heterocyclyl, or aryl.

[0106] The term "amino," as used herein, refers to a group of formula -NRGRHor-NR^nR1, where each of RG, RH, and Rxis independently H, alkyl, alkenyl, alkynyl, carbocyclyl, heteroalkyl, heterocyclyl, or aryl.

[0107] The term "aminoalkyl," as used herein, refers to an alkyl group substituted with an amino group. Aminoalkyl groups may be primary, secondary, tertiary, or quaternary.

[0108] The term "aryl," as used herein, refers to any monocyclic or fused ring bicyclic or multicyclic system containing only carbon atoms in the ring(s), which has the characteristics of aromaticity in terms of electron distribution throughout the ring system, e.g., phenyl, naphthyl, or phenanthryl. An aryl group may have, e.g., six to sixteen carbons (e.g., six carbons, ten carbons, thirteen carbons, fourteen carbons, or sixteen 5 carbons). Aryl groups may be unsubstituted or substituted. Exemplary substituents include alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, ester, halo, heteroalkyl, heterocyclyl, hydroxyl, phospho, sulfa, and thiol.

[0109] The term "acyl," as used herein, refers to a group having the general formula -C(=O)RJ, wherein RJis hydrogen, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl.

[0110] The term "azido," as used herein, refers to -N3.

[0111] The term "carbonate," as used herein, refers to a group of the formula -OC(=O)OR, wherein R is H, alkyl, heteroalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, or aryl.

[0112] The term "carboxyl," as used herein, refers to a group of the formula -(C=O)OH.

[0113] The term "cyano," as used herein, refers to -C=N.

[0114] The term "cyanato," as used herein, refers to -O-C=N.

[0115] The term "carbocyclyl," as used herein, refers to a cyclic, saturated or unsaturated, monovalent hydrocarbon group having from 3 to 12 carbons (e.g., 3 to 6), unless otherwise specified. Carbocyclyl groups may be substituted or unsubstituted. Exemplary substituents include alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, epoxy, ester, halo, heterocyclyl, hydroxyl, oxo, phospho, sulfa, and thiol.

[0116] The term "cycloalkyl," as used herein, refers to a cyclic, saturated, monovalent hydrocarbon group having from 3 to 12 carbons (e.g., 3 to 6), unless otherwise specified.Cycloalkyl groups may be substituted or unsubstituted. Exemplary substituents include alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amido, amino, carbonate, carboxyl, cyano, cycloalkyl, epoxy, halo, heterocyclyl, hydroxyl, oxo, and thiol.

[0117] The suffix "-ene," as used herein, refers to a divalent radical of the group. For example, alkylene is a divalent alkyl group, and heterocyclylene is a divalent heterocyclyl group.

[0118] The term "epoxy," as used herein, refers to >0, where the oxygen is bound to adjacent carbon atoms.

[0119] The term "ester," as used herein, refers to a group having the general formula -C(=O)ORJor -OC(=O)RJ, wherein RJis alkyl, heteroalkyl, alkenyl, alkynyl, heterocyclyl, or aryl.

[0120] The term "formed from," as used herein, refers to the formal structure of the product and does not imply that the stated moiety was necessarily employed in the direct synthesis. For example, thiols with protecting groups may be employed, that, after deprotection, result in the named thiol. Other reactions may also be employed to result in a final product.

[0121] The term "halide," as used herein, refers to a F, Cl, Br, or I anion.

[0122] The term "halo," as used herein, refers to a F, Cl, Br, or I radical.

[0123] The term "heteroalkenyl," as used herein, refers to an alkenyl group in which one or more of the carbon atoms not forming a double bond has been replaced by a heteroatom selected from S, 0, and N. A neutral heteroalkenyl group includes only S or O as heteroatoms replacing carbon atoms. Heteroalkenyl groups may be substituted or unsubstituted as alkenyl groups.

[0124] The term "heteroalkyl," as used herein, refers to an acyclic straight or branched chain, saturated, monovalent hydrocarbon group having from 1 to 12 carbons (e.g., 1 to 6), unless otherwise specified, wherein one or more of the carbon atoms has been replaced by a heteroatom selected from S, 0, and N. A neutral heteroalkyl group includes only S or O as heteroatoms replacing carbon atoms. Heteroalkyl groups may be substituted or unsubstituted. Exemplary substituents include alkenyl, alkynyl, alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, epoxy, ester, halo, heterocyclyl, hydroxyl, oxo, phospho, sulfa, and thiol.

[0125] The term "heteroalkynyl," as used herein, refers to an alkynyl group in which one or more of the carbon atoms not forming a triple bond has been replaced by a heteroatom selected from S, O, and N. A neutral heteroalkynyl group includes only S or O as heteroatoms replacing carbon atoms. Heteroalkynyl groups may be substituted or unsubstituted as alkynyl groups.

[0126] The term "heteroaryl," as used herein, refers to an aromatic heterocyclyl group. For example, a single-ring heteroaryl group includes pyridyl; fused-ring heteroaryl groups include benzimidazolyl, quinolinyl, acridinyl; and a non-fused bi-heteroaryl group includes bipyridinyl.Further examples of heteroaryls include, but are not limited to, furanyl, thienyl, oxazolyl, acridinyl, phenazinyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzothiophenyl, benzoxadiazolyl, benzotri azolyl, imidazolyl, indolyl, isoxazolyl, isoquinolinyl, indolizinyl, isothiazolyl, isoindolyloxadiazolyl, indazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazinyl, pyrrolyl, pyrazinyl, pyrazolyl, purinyl, phthalazinyl, pteridinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazolyl, tetrazolyl, thiazolyl, triazinyl, thiadiazolyl and the like. Heteroaryl groups may be substituted or unsubstituted. Exemplary substituents include alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, ester, halo, heterocyclyl, hydroxyl, phospho, sulfa, and thiol.

[0127] The term "heterocyclyl," as used herein, represents a monovalent, monocyclic or fused ring bicyclic or multicyclic system having at least one heteroatom as a ring atom. For example, a heterocyclyl group may have, e.g., one to fifteen carbon ring atoms (e.g., a C1-C2, C1-C3, Cl-C4, C1-C5, C1-C6, C1-C7, C1-C8, C1-C9, C1-C10, Cl-Cll, C1-C12, C1-C13, C1-C14, or Cl-C15 heterocyclyl) and one or more (e.g., one, two, three, four, or five) ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyl groups may or may not include a ring that is aromatic (e.g., heteroaiyl). Heterocyclyl groups may be unsubstituted or substituted. In preferred embodiments of the invention, a heterocyclyl group is a 3- to 8-membered ring, a 3- to 6-membered ring, a 4- to 6-membered ring, a 5-membered ring, or a 6-membered ring. Exemplary 5-membered heterocyclyl groups may have zero to two double bonds, and exemplary 6-membered heterocyclyl groups may have zero to three double bonds.

[0128] The term "basic nitrogen-containing heterocycle," as used herein, refers to a heterocyclyl group having at least one ring nitrogen (e.g., 1 to 4 nitrogen atoms, e.g., 1 or 2) that can accept a proton from solution, e g., imidazolyl or benzimidazolyl. Heterocyclyl groups may be substituted or unsubstituted. Exemplary substituents include alkyl, alkenyl, alkynyl, alkoxy, alkylthio, amido, amino, aryl, azido, carbonate, carboxyl, cyano, carbocyclyl, epoxy, ester, halo, heteroalkyl, heterocyclyl, hydroxyl, oxo, phospho, sulfa, and thiol.

[0129] The term “guanidinium,” as used herein, refers to a positively charged or protonated guanidine or guanidinyl group. As a non-liming example, a guanidinium group is a protonated guanidinyl group at physiological pH. Guanidinium groups may include any of its resonance forms.

[0130] The term "hydrazide," as used herein, refers to: -NH-NH2.

[0131] The term "imino," as used herein refers to =N-RK, where each RKis H, alkyl, or heteroalkyl.

[0132] The term "isocyanato," as used herein, refers to -N=C=O.

[0133] The term "isothiocyanate," as used herein, refers to -N=C=S.

[0134] The term "oxo," as used herein, refers to =0.

[0135] The term "nitro," as used herein, refers to -NO2.

[0136] The term "nitroso," as used herein, refers to -N=O.

[0137] The term "nitrosooxy," as used herein, refers to -O-N=O.

[0138] The term "phosphino," as used herein, refers to -PRk2, where each RKis H, alkyl, or heteroalkyl.

[0139] The term "phospho," as used herein refers to -P(=O)(QRK)2, wherein each RKis hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, aryl, or heterocyclyl, e.g., heteroaryl.

[0140] The term "silyl," as used herein, refers to -SiRL3, wherein each RLis independently H, alkyl, or heteroalkyl.

[0141] The term "sulfide," as used herein, refers to -SRM, wherein RMis alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, aryl, or heterocyclyl, e.g., heteroaryl.

[0142] The term "sulfa," as used herein refers to -S(=O)2O-RM, wherein RMis H, alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, aryl, or heterocyclyl, e.g., heteroaryl.

[0143] The term "sulfinyl," as used herein refers to -S(=O)-RM, wherein RMis alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, or heterocyclyl, e.g., heteroaryl.

[0144] The term "sulfonyl," as used herein refers to -S(=O)2-RM, wherein RMis alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, or heterocyclyl, e.g., heteroaryl.

[0145] The term "sulfo," as used herein refers to -S(=O)2O-RM, wherein RMis alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, or heterocyclyl, e.g., heteroaryl.

[0146] The term "thiol," as used herein, refers to -SH.POLYMERS

[0147] The polymer may include formula (I) or formula (II):(II),or a salt or ion thereof,wherein

[0148] each X is independentlywherein R1is -NRARBor OR4, wherein R4and RBare each independently H, aminoalkyl, or aminoheteroalkyl, provided that at least one of RAand RBis aminoalkyl or aminoheteroalkyl, R1is amino, aminoalkyl, or aminoheteroalkyl, and u is 1 to 5, and wherein R2is H or CH3;

[0149] each Y is independentlywherein R3is -NRCRDor -ORC, wherein Rc and RD are each independently H or quaternary aminoalkyl or quaternary aminoheteroalkyl, provided that at least one Rcand RDis quaternary aminoalkyl or quaternary aminoheteroalkyl, R3is quaternary amino, quaternary aminoalkyl, or quaternary aminoheteroalkyl, and v is 1 to 5, and wherein R4is H or CH3;

[0150] each Z is independently, wherein R5is -NRERFor -ORE, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFis neutral substituted alkyl or neutral heteroalkyl, R5is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3;

[0152] each W is independently, wherein n is from 1 to 8 and each R7is independently H, alkyl, alkenyl, alkynyl, or heteroalkyl;

[0153] each Q is independently■6,-ORE, -CH2SO3, -CH2CH2SO3, or -CH2CH2CH2SO3, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFis neutral substituted alkyl or neutral heteroalkyl, R5is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3;

[0155] m is from 10 to 100;

[0156] wherein each a, b, c, d, and e represents a fraction of m, and the sum of a, b, c, d, and e is 1, provided that at least two of a, b, c, d and e are not 0.

[0157] In various embodiments, a is 0 - 0.3, b is 0 - 0.15, c is 0 - 0.9, d is 0 - 0.15, and e is 0 -0.3. In various embodiments, a is 0 - 0.3, b is 0 - 0.15, c is 0 - 0.9, and d is 0 - 0.15 and each represents a fraction of m, and the sum of a, b, c, and d is 1, provided that at least two of a, b, c, and d are not 0; or an ion or salt thereof.

[0158] In some embodiments, X is, S, wherein R8is aminoalkyl, and q is 1 to 4, or an ion or salt thereof.

[0160] In some embodiments, Y iss\R8'

[0161] , wherein each R8is independently alkyl, each R8is independently quaternary aminoalkyl, and r is 1 to 4, or a salt thereof.

[0162] In some embodiments, Z is

[0164] wherein each R8is independently neutral substituted alkyl or neutral heteroalkyl, each R9is independently H, OH, or SH, and R10is aryl or heteroaryl, wherein s is 2 to 5.

[0165] In some embodiments, the monomer employed resulting in the first pendant group or X is 3 -aminopropyl methacrylamide. In some embodiments, the monomer resulting in the second pendant group or Y is 3 -trimethylaminoethyl methacrylate.

[0166] In some embodiments, the monomer employed resulting in the third pendant group or Z is selected from PhHPMA (2-hydroxy-3 -phenoxy propyl methacrylate), HEMA (hydroxy ethylmethacrylate), PEGMA (poly (ethylene glycol) methacrylate), e.g., PEG300 or PEG500, EGPhEMA (ethylene glycol phenyl ether methacrylate), THFMA (tetrahydrofurfuryl methacrylate), GlyMA (glycidyl methacrylate), MPC (2-methacryloyloxyethyl phosphorylcholine), DMMAm (N, N-dimethylmethacrylamide), and HPMAm (2-hydroxypropyl methacrylate).

[0167] In some embodiments, each W is independentlywherein n is from 1 to 8, e.g., 1 to 6, e.g., 3, and each R7is independently H, alkyl, alkenyl, alkynyl, or heteroalkyl, e.g., substituted with amino, imino, amido, azido, cyano, cyanato, isocyanato, isothiocyanto, hydrazide, nitro, nitroso, nitrosooxy, hydroxyl, alkoxy, carboxyl, ester, acyl, halo, phosphino, phospho, sulfide, thiol, sulfonyl, sulfo, sulfinyl, or silyl, e.g., as described in WO2023 / 122807..In some embodiments, the monomer employed resulting in group W is lipoic acid, which can be further modified via esterification or amidation.

[0168] The polymers described herein may be synthesized be any suitable method, e.g., controlled polymerization mechanisms such atom transfer radical polymerization (ATRP), stable free radical polymerization (SFRP), or reversible addition-fragmentation chain transfer (RAFT) polymerization. The present invention may employ RAFT polymerization or ATRP. In particular embodiments, the end groups of formula (II), formula (III), or formula (IV) may be from any RAFT or ATRP agent or a chemical derivative thereof. Specific examples include, but are not limited to, RAFT agents that incorporate dithiobenzoate or trithiocarbonate, such as 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2-cyano-2-propyl dodecyl trithiocarbonate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, and 2-cyano-2-propylbenzodithioate. Specific examples include, but are not limited to, ATRP agents that incorporate halogens, such as methyl-2-bromopropionate, methyl-2-chloropropionate, ethyl-2-bromoisobutyrate, and 2-hydroxyethyl 2-bromo-2-methylpropanoate. End groups may also include modifications made to the reacted RAFT or ATRP agent, e.g. displacement or thioesterification.O

[0169] In some embodiments, the polymer is terminated at one end with, wherein U is H or alkyl, and V is alkylene e.g.,

[0170] In some embodiments, the polymer is terminated at one end withembodiments, the polymer is terminated withIn some embodiments, the polymer is terminated with H, thiol, alkyl,wherein o is 1 to 20.

[0171] ATRP and RAFT agents also include macromolecular agents, such as poly(ethylene)glycol. These also include branched structures that are formed from branched to dendrimer RAFT reagents:CH39 CH-,(CH2'!0CH2S^, S_3X K C i ( oYM / TVn WWH» S <0H>CCH3S 4— "OA^S.^SCH2(CH2>„; CH3H-’CCH-, S

[0172] Other groups may also be included in the polymers. Such groups include polyethylene glycol, a polynucleic acid (e.g., sequence specific, such as an aptamer), and / or a polypeptide (e.g., sequence specific such as an antibody). Such polymers may be attached by standard conjugation methods known in the art.

[0173] In some embodiments, the polymer includes formula (III):wherein each R8is independently CH3 or C2H5, R9is OH or SH, R10is aryl or heteroaryl,, or a salt thereof. In some embodiments, R8is methyl. In some embodiments, R9is OH. In some embodiments, R10is phenyl.

[0174] In particular embodiments, the compound of formula (III) is selected from:

[0175] wherein m is from 10 to 1000; a is 0.5 - 0.3, b is 0.5 - 0.15, and c is 0.55 - 0.9; and each represents a fraction of m, and the sum of a, b, and c is 1; or an ion or salt thereof.

[0176] In some embodiments, the polymer including formula (IV):(IV), wherein n is 1 to 6, each R7is independently H or alkyl, each R8is independently CH3 or C2H5, R9is OH or SH, R10is aryl or heteroaryl, or a salt thereof.OH

[0177] In some embodiments, n is 3. In some embodiments, R7is H oror a C1-C3 ester or di-Co-Cs amide thereof, e.g.,

[0178] where one and only one R7is ® or a C1-C3 ester or di-Co-Cs amide thereof. In some embodiments, R8is methyl. In some embodiments, R9is OH. In some embodiments, R10is phenyl.

[0179] In particular embodiments, the compound of formula (IV) is selected from:(IVa),

[0180] wherein m is from 10 to 1000; a is 0.4 - 0.3, b is 0.4 - 0.15, c is 0.4 - 0.9, and d is 0 -0.15; and each represents a fraction of m, and the sum of a, b, c, and d is 1; or an ion or salt thereof.

[0181] In some embodiments, the polymer includes formula (IVc):wherein m is from 10 to 1000; a is 0.4 - 0.3, b is 0.4 - 0.15, c is 0.4 - 0.9, and d is 0 - 0.15 and e is 0 - 0.3; and each represents a fraction of m, and the sum of a, b, c, d and e is 1; or an ion or salt thereof.

[0183] In some embodiments, the polymer includes formula (V):

[0184] In formulas (I), (II), (III), (Illa), (Illb), (IV), (IVa), (IVb), (IVc), LA, ILA, IILA, IV-A, V-A, VLA, VILA, VIILA, VIX-A, VX-A, VXLA, VXILA, VXIILA, D156, D5588, D5586, and D5503, m may be from about 10 to about 1000, e.g., from 10 to 20, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 75, 10 to 80, 10 to 90, 10 to 100, 10 to 125, 10 to 150, 10 to 175, 10 to 200, 10 to 250, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 15 to 45, 15 to 50, 15 to 55, 15 to 60, 15 to 70, 15 to 75, 15 to 80, 15 to 90, 15 to 100, 16 to 24, 17 to 23, 18 to 22, 19 to 21, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 75, 20 to 80, 20 to 90, 20 to 100, 25 to 30, 25 to 35, 25 to 40, 25 to 50, 25 to 75, 25 to 100, 25 to 125, 26 to 34, 27 to 33, 28 to 32, 29 to 31, 30 to 35, 30 to 40, 30 to 45, 30 to 50, 30 to 60, 30 to 70, 30 to 80, 30 to 90, 30 to 100, 35 to 65, 40 to 50, 40 to 60, 40 to 70, 40 to 80, 40 to 90, 40 to 100, 50 to 60, 50 to 70, 50 to 80, 50 to 90, 50 to 100, 50 to 125, 50 to 150, 50 to 175, 50 to 200, 50 to 250, 50 to 300, 50 to 350, 50 to 400, 50 to 450, 50 to 500, 50 to 550, 50 to 600, 50 to 700, 50 to 800, 50 to 900, 50 to 1000, 60 to 100, 60 to 140, 70 to 100, 70 to 130, 80 to 100, 80 to 120, 90 to 110, 100 to 125, 100 to 150, 100 to 200, 100 to 300, 100 to 500, 100 to 750, 100 to 1000, 150 to 200, 150 to 250, 200 to 300, 200 to 500, 200 to 700, 200 to 1000, 250 to 500, 250 to 750, 250 to 1000, 300 to 500, 300 to 700, 400 to 500, 400 to 600, 500 to 600, 500 to 750, 500 to 800, 500 to 1000, 750 to 1000, 800 to 900, 800 to 1000, or 900 to 1000, e.g., is about 10, 25, 50, 75, 90, 100, 125, 150, 190, 200, 225, 250, 300, 350, 400, 450, or 500.

[0185] In formulas (I), (II), (III), (Illa), (Illb), (IV), (IVa), (IVb), (IVc), LA, ILA, IILA, IV-A, V-A, VLA, VILA, VIILA, VIX-A, VX-A, VXLA, VXILA, VXIILA, D156, D5588, D5586, and D5503a may be from about 0.04 to about 0.3, e.g., from 0.04 to 0.05, 0.04 to 0.1, 0.04 to 0.15, 0.04 to 0.2, 0.04 to 0.25, 0.04 to 0.3, 0.1 to 0.3, 0.15 to 0.3, e.g., is about 0.04, 0.1, 0.15, 0.2, 0.25, or 0.3.

[0186] In formulas (I), (II), (III), (Illa), (Illb), (IV), (IVa), (IVb), (IVc), LA, ILA, IILA, IV-A, V-A, VLA, VILA, VIILA, VIX-A, VX-A, VXLA, VXILA, VXIILA, D156, D5588, D5586,and D5503) b may be from about 0.04 to about 0.15, e.g., from 0.04 to 0.05, 0.04 to 0.1, 0.04 to 0.15, 0.05 to 0.1, 0.05 to 0.15, 0.1 to 0.15, e.g., is about 0.04, 0.1, 0.15.

[0187] In formulas (I), (II), (III), (Illa), (Illb), (IV), (IVa), (IVb), (IVc), LA, ILA, IILA, IV-A, V-A, VLA, VILA, VIILA, VIX-A, VX-A, VXLA, VXILA, VXIILA, D156, D5588, D5586, and D55O3, c may be from about 0.4 to about 0.9, e.g., from 0.4 to 0.45, 0.4 to 0.5, 0.4 to 0.9, 0.55 to 0.6, 0.55 to 0.65, 0.55 to 0.9, 0.6 to 0.9, 0.65 to 0.9, 0.7 to 0.9, 0.75 to 0.8, 0.75 to 0.9, 0.8 to 0.9, 0.85 to 0.9, e.g., is about 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.75, 0.8, 0.85, or 0.9.

[0188] In formulas (1), (11), (111), (Illa), (Illb), (IV), (IVa), and (IVb), (IVc), d may be from about 0 to about 0.15, e.g., from 0 to 0.05, 0 to 0.1, 0 to 0.15, 0.05 to 0.1, 0.05 to 0.15, 0.1 to 0.15 e.g., is about 0, 0.05, 0.1, 0.15.

[0189] Polyester copolymers

[0190] In various embodiments, the polymer comprises a polyester copolymer comprising a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains.

[0191] The present polyester copolymer scaffolds may be synthesized from alternating cyclic anhydride and epoxide monomers. In some embodiments, the cyclic anhydride is succinic anhydride or a derivative thereof, such as maleic anhydride, diglycolic anhydride, glutaric anhydride, allylsuccinic anhydride, or phthalic anhydride. When cyclic anhydrides including a double bond like maleic anhydride may be employed, the resulting polymer may be isomerized around the double bond after the initial polymerization to the fumaric orientation. In some embodiments, the epoxide is allyl glycidyl ether (AGE). In some embodiments, the epoxide is 4-vinyLl -cyclohexene 1,2-epoxide (vCHO). In some embodiments, the epoxide is propylene oxide. In some embodiments, the epoxide is propargyl glycidyl ether (PGE).

[0192] An intermediate polyester copolymer unit contains at least one degree of unsaturation that may be a part of the epoxide monomer, cyclic anhydride monomer, or both. The unsaturated moieties on the polyester copolymer intermediates may be utilized in conjugation reactions to produce polyester copolymers containing additional side chains functionalities. Another such conjugation is a thioLyne reaction. Conjugated moieties include -S-, aminocarbonyl, e g., -NC(=O)- or -C(=O)N-, oxy carbonyl, e.g., -C(=O)O- or -OC(=O)-, carbocyclylene, and heterocyclylene, e.g., 1,2, 3 -tri azole. Exemplary conjugation reactions include the thiol-ene reaction between a thiol and an alkene, thiol-yne reaction between a thiol and an alkyne, click reaction between an azide and an alkyne (e.g., copper-catalyzed or copper-free), amide bond formation between carboxylic acid or activated derivative and an amine, esterification between carboxylic acid or activated derivative and an alcohol, and the Diels Alder reaction between a diene and a dienophile.

[0193] In one embodiment polyester copolymers may include formula (I- A):R1R2R3R4(I-A),

[0194] wherein n is from 2-80, R1 and R2 are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, or ionizable side chain, or R1 and R2, together with the atoms to which they are attached, combine to form a 5-7 membered ring, R3 and R4 are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, or ionizable side chain or R3 and R4, together with the atoms to which they are attached, combine to form a 5-7 membered ring, and the dashed bond is either absent or a bond of either E or Z orientation, provided at least one Rl, R2, R3, or R4 or combination thereof, on at least one monomer, comprises an ionizable side chain. In some embodiments, the side chain is -S-. In some embodiments, the side chain comprises an amido moiety, e.g., -NC(=O)- or -C(=O)N-. In some embodiments, the side chain comprises an oxycarbonyl moiety, e.g., -C(=O)O- or -OC(=O)-. In some embodiments, the side chain comprises -S-, -NC(=O)-, -C(=O)N-, -C(=O)O-, -OC(=O)-, carbocyclylene, or heterocyclylene. In some embodiments, the side chain comprises a carbocyclylene or heterocyclylene, e.g., 1,2,3-triazole. In some embodiments, the side chain comprises a 1,2,3-triazole.

[0195] In various embodiments, the polyester copolymers comprise Formula (A):R2(A),wherein:n is a targeted degree of polymerization. In various embodiments, n is about 1 to about 1000. In various, n is about 1 to about 100. In various, n is about 2 to about 80.m is 0 or 1,X is O, CH or C,R1, R2R3, R4and R5, are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, or an ionizable side chain,R1and R2, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising a pendant group,R3and R4, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain, and

[0196] wherein at least one monomer unit comprises at least one of Rl, R2 R3, R4 and R5 comprises an ionizable side chain,

[0197] or an ion or salt thereof.

[0198] In some embodiments, the ionizable side chain comprises an ionizable base. In some embodiments, the ionizable side chain comprises an ionizable acid.

[0199] In some embodiments, the ionizable base comprises a nitrogenous moiety. In some embodiments, the ionizable base comprises an amine, imidazole, or guanidine

[0200] In some embodiments, the ionizable acid comprises a carboxylic acid or sulfonic acid.

[0201] In some embodiments, the amine comprises -NH2, -N(CH3)2, -N(ethyl)2, -N(propyl)2 morpholine, pyrrolidine, or piperidine.

[0202] In some embodiments, 0-80% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, the ionizable acids can be negatively charged at about pH 7. In some embodiments, 0-70% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, the ionizable acids can be negatively charged at about pH 7. In some embodiments, 0-60% of the side chains in thepolyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, the ionizable acids can be negatively charged at about pH 7. In some embodiments, 0-50% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, the ionizable acids can be negatively charged at about pH 7.

[0203] In some embodiments, 20-100% of the side chains in the polyester copolymer are ionizable bases. In some embodiments, 30-100% of the side chains in the polyester copolymer are ionizable bases. In some embodiments, 40-100% of the side chains in the polyester copolymer are ionizable bases In some embodiments, 50-100% of the side chains in the polyester copolymer are ionizable bases. In some embodiments, the ionizable bases can be positively charged at about pH 7.

[0204] In some embodiments, 0-50% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 50-100% of the side chains in the polyester copolymer are ionizable bases which can be positively charged at pH 7.

[0205] In some embodiments, 0-50% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids and 50-100% of the side chains in the polyester copolymer are ionizable bases.

[0206] In some embodiments, 0-30% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 30-100% of the side chains in the polyester copolymer are ionizable bases which can be positively charged at pH 7. In some embodiments, 30-50% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 30-50% of the side chains in the polyester copolymer are ionizable bases which can be positively charged at pH 7. In some embodiments, 50-80% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 50-80% of the side chains in the polyester copolymer are ionizable bases which can be positively charged at pH 7.

[0207] In some embodiments, 0-30% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 0-40% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 0-50% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 0-60% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 0-70% of the side chains in the polyester copolymer areneutral, zwitterionic, or ionizable acids. In some embodiments, 0-80% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids. In some embodiments, 30-100% of the side chains in the polyester copolymer are ionizable bases which can be positively charged at about pH 7. In some embodiments, 0-30% of the side chains in the polyester copolymer are neutral, zwitterionic, or ionizable acids which are negatively charged at about pH 7. In some embodiments, 30-100% of the side chains in the polyester copolymer are positively charged at about pH 7.

[0208] In some embodiments, 30-100% of the side chains in the polyester copolymer are positively charged at a pH ranging from about pH 4 to about pH 8. In some embodiments, 50-100% of the side chains in the polyester copolymer are positively charged at a pH ranging from about pH 4 to about pH 8. In some embodiments, 75-100% of the side chains in the polyester copolymer are positively charged at a pH ranging from about pH 4 to about pH 8.

[0209] In some embodiments, at least about 10% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 20% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 30% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 40% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 50% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 60% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 70% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 80% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, at least about 90% of the side chains in the polyester copolymer are positively charged at about pH 7.

[0210] In some embodiments, about 50% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, about 60% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, about 70% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, about 80% of the side chains in the polyester copolymer are positively charged at about pH 7. In some embodiments, about 90% of the side chains in the polyester copolymer arepositively charged at about pH 7. In some embodiments, about 100% of the side chains in the polyester copolymer are positively charged at about pH 7.

[0211] In some embodiments, at least about 50% of the side chains in the polyester copolymer are positively charged at about physiological pH. In some embodiments, at least about 60% of the side chains in the polyester copolymer are positively charged at about physiological pH. In some embodiments, at least about 70% of the side chains in the polyester copolymer are positively charged at about physiological pH. In some embodiments, at least about 80% of the side chains in the polyester copolymer are positively charged at about physiological pH. In some embodiments, at least about 90% of the side chains in the polyester copolymer are positively charged at about physiological pH.

[0212] In some embodiments, about 50% of the side chains in the polyester copolymer are positively charged at physiological pH. In some embodiments, about 60% of the side chains in the polyester copolymer are positively charged at physiological pH. In some embodiments, about 70% of the side chains in the polyester copolymer are positively charged at physiological pH. In some embodiments, about 80% of the side chains in the polyester copolymer are positively charged at physiological pH. In some embodiments, about 90% of the side chains in the polyester copolymer are positively charged at physiological pH. In some embodiments, about 100% of the side chains in the polyester copolymer are positively charged at physiological pH.

[0213] In some embodiments, the polyester copolymer comprises 30-100% ionizable bases and 0-70% neutral, zwitterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises 40-100% ionizable bases and 0-60% neutral, zwitterionic, or ionizable acids.

[0214] In some embodiments, the polyester copolymer comprises about 20% ionizable bases and about 80% neutral, zwitterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises about 30% ionizable bases and about 70% neutral, zwitterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises about 40% ionizable bases and about 60% neutral, zwitterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises about 50% ionizable bases and about 50% neutral, zwitterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises about 60% ionizable bases and about 40% neutral, zwitterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises about 70% ionizable bases and about 30% neutral,zwiterionic, or ionizable acids. In some embodiments, the polyester copolymer comprises about 80% ionizable bases and about 20% neutral, zwitterionic, or ionizable acids

[0215] In particular embodiments, the side chain comprises a sulfide. In particular embodiments, the side chain comprises a triazole.

[0216] In particular embodiments, the side chain is a formed from a thiol. In particular embodiments, the side chain is a formed from an azide. In particular embodiments, the side chain is a formed from reaction between a thiol and an alkene. In particular embodiments, the side chain is a formed from reaction between a thiol and an alkyne. In particular embodiments, the side chain is a formed from a reaction between an azide and an alkene.

[0217] For example, a neutral, zwitterionic, or ionizable acid may be formed from 1-propanethiol, 1 -butanethiol, 1 -pentanethiol, 1 -hexanethiol, 1 -octanethiol, 1 -decanethiol, 1-undecanethiol, 1 -hexadecanethiol, 2-ethylhexanethiol, cyclopentanethiol, cyclohexanethiol, 2-mercaptoethanol, 3 -mercapto- 1 -propanol, 6-mercapto-l -hexanol, mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptohexanoic acid, 8-mercaptoctanoic acid, 12-mercaptododecanoic acid, 16-mercaptohexadecanoic acid, 11-mercaptoundecanoic acid, benzimidazole ethanethiol, 2-sulfanylethanesulfonic acid, 1 -thioglycerol, 7-mercapto-4-methylcoumarin, L-glutathione, or (4-nitrobenzyl)mercaptan.

[0218] In various embodiments, a zwitterionic moiety comprises an amino acid. In various embodiments, a zwiterionic moiety comprises L-cysteine (LCYS), D-cysteine, DL-cysteine, L-homocysteine, D-homocysteine, or DL -homocysteine.

[0219] In particular embodiments, an ionizable base side chain is formed from the amine salts, e.g., amine hydrochloride, of cysteamine, 2-dimethylaminoethanethiol, 2-diethylaminoethanethiol, (2-butylamino)ethanethiol, L-cysteine, D-cysteine, DL-cysteine, L-cysteine methyl ester, L-cysteine ethyl ester, L-homocysteine, D-homocysteine, or DL-homocysteine. In particular embodiments, an ionizable base is formed from the guanidine salts, e.g., guanidinium hydrochloride of 2-mercaptoethyl guanidinium or 3 -mercaptopropyl guanidinium.

[0220] In various embodiments, an ionizable base side chain is formed from an ionizable acids. In particular embodiments, the side chain is formed form the acids, mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptohexanoic acid, 8-mercaptoctanoic acid, 12-mercaptododecanoic acid, 16-mercaptohexadecanoic acid, 11-mercaptoundecanoic acid, or sulfanylethanesulfonic acid.

[0221] In some embodiments, the side chain comprises a 1,2,3-triazolylene. In particular embodiments, the 1,2,3-triazolylene is substituted with an alkyl, heteroalkyl, aryl, or heteroaryl substituent. In particular embodiments, the 1,2,3-triazolylene comprises an ionizable acid substituent, or an ionizable base substituent. In particular embodiments, the 1,2,3-triazolylene is substituted with an alkyl, heteroalkyl, aryl, or heteroaryl substituent, wherein each alkyl, heteroalkyl, aryl, or heteroaryl substituent independently comprise a neutral substituent, an ionizable acid substituent, or an ionizable base substituent.

[0222] In particular embodiments, the polyester copolymer contains negatively charged and positively charged side chains resulting in a polyampholyte.

[0223] In particular embodiments, the polyester copolymer comprises an ionizable acid and an ionizable base side chains resulting in a polyampholyte at about pH 7.

[0224] In particular embodiments, the polyester copolymer comprises an ionizable acid and an ionizable base side chains resulting in a zwitterion at about pH 7.

[0225] In particular embodiments, the polyester copolymer comprises an ionizable acid and an ionizable base side chains resulting in a net positive charge at about pH 7.

[0226] In some embodiments, the polyester copolymer contains a terminus resulting from an initiator. The initiator can be monovalent or multivalent. The initiator can be an alcohol. In some embodiments, the initiator is propargyl alcohol. In particular embodiments, the initiator is selected from 2-hydroxymethyl-l,3-propanediol, pentaerythritol, or meso-erythritol. In some embodiments, the use of a multivalent initiator results in the formation of a star polymer.

[0227] In some embodiments, one terminus of the polyester copolymer is conjugated to poly (ethyleneglycol) (PEG).

[0228] In some embodiments, one terminus of the polyester copolymer is conjugated to a moiety capable of interacting selectively with biomolecules or cells, such as peptides, antibodies, antibody fragments, monosaccharides, polysaccharides, or aptamers.

[0229] In another aspect, the invention provides a composition including a complex as described herein and a liquid carrier.

[0230] In another aspect, the invention provides a method including contacting a cell with the complex, wherein the biological agent is delivered into the cell.

[0231] In another aspect, the invention provides a composition comprising a product by process. In various embodiments, the composition comprises a product by process comprising the steps of:a. reacting one or more cyclic anhydride monomers and one or more epoxide monomers to form a polyester polymer backbone, wherein and at least one monomer comprises a reactive group; b. reacting one or more reactive groups with a side chain reactive molecule; wherein the side chain reactive molecule comprises an ionizable base, a neutral, a zwitterionic, or an ionizable acid moiety.

[0232] In another aspect, the invention provides a method of synthesizing a polyester copolymer. In various embodiments, the method comprisesa. reacting one or more cyclic anhydride monomers and one or more epoxide monomers to form a polyester polymer backbone, wherein and at least one monomer comprises a reactive group; andb. reacting one or more reactive groups with a side chain reactive molecule; wherein the side chain reactive molecule comprises an ionizable base, a neutral, a zwitterionic, or an ionizable acid moiety.

[0233] In various embodiments, the reactive group of the polyester backbone comprises an alkene, alkyne, or azide.

[0234] In various embodiments, the side chain reactive molecule comprises a thiol, amino, hydroxy, carboxy, activated carboxy, azido, alkyne, diene, alkene, linear alkene, or cyclic alkene. In particular embodiments, the side chain reactive molecule comprises a thiol. In particular embodiments, the side chain reactive molecule comprises an azide.

[0235] In various embodiments, the side chain reactive molecule comprises an ionizable moiety. In various embodiments, the ionizable moiety comprises an ionizable acid. In various embodiments, the ionizable moiety comprises an ionizable base.

[0236] In various embodiments, the side chain reactive molecule comprises i. a thiol, amino, hydroxy, carboxy, activated carboxy, azido, alkyne, diene, alkene, linear alkene, or cyclic alkene, and ii. an ionizable moiety.

[0237] In particular embodiments, the side chain reactive molecule comprises i. a thiol and an ionizable moiety. In particular embodiments, the side chain reactive molecule comprises i. a thioland ii. an ionizable base. In particular embodiments, the side chain reactive molecule comprises i. a thiol and ii. an ionizable acid.

[0238] In particular embodiments, the side chain reactive molecule comprises i. an azide and ii. an ionizable moiety. In particular embodiments, the side chain reactive molecule comprises i. an azide and ii. an ionizable base. In particular embodiments, the side chain reactive molecule comprises i. an azide and ii. an ionizable acid.

[0239] In particular embodiments, the side chain reactive molecule comprises 1 -pentyne, 1-hexyne; 4-methyl-l -pentyne; 4-octyne; cyclopropylacetylene; 4-pentyn-l-ol; 1 -dodecyne; methyl-5-hexynoate, propargyl amine; N-methylpropargylamine; amino-PEG3-alkyne; 3, dimethylamino- 1 -propyne; 10-undecynoic acid; or 4-pentynoic acid.

[0240] In another aspect, the invention features a complex comprising of any one of the polyester copolymers described and a negatively charged biological agent.

[0241] In various embodiments, each side chain is independently neutral, negatively charged or positively charged at about physiological pH. In various embodiments, each pendant group is neutral or positively charged at about physiological pH.

[0242] In various embodiments, the polymer comprises a mixture of charged and / or neutral side chains.

[0243] In various embodiments, each side chain is independently neutral, negatively charged or positively charged at pH 7. In various embodiments, each side chain is neutral or positively charged at about pH 7.

[0244] In various embodiments, the polymer comprises a mixture of ionizable and / or neutral side chains.

[0245] In various embodiments, the ionizable base side chain comprises an amino group. In various embodiments, the amino group comprises an alkylamino group.

[0246] In various embodiments, the ionizable base side chain comprises a guanidine group. In various embodiments, an ionizable base side chain comprises a guanidinium group.

[0247] In various embodiments, the side chain comprises a cysteaminyl, 2-dimethylaminoethanethiolyl, 2-diethylaminoethanethiolyl, (2-butylamino)ethanethioylyl, L-cysteinyl, D-cysteinyl, DL-cysteinyl, L-cysteinyl methyl ester, L-cysteinyl ethyl ester, L-homocysteinyl, D-homocysteinyl, or DL-homocysteinyl, 2-mercaptoethyl guanidinyl or 3-mercaptopropyl guanidinyl group.

[0248] In various embodiments, the ionizable side chain comprising an amino group is formed from a compound selected from the group consisting of cysteamine, 2-dimethylaminoethanethiol, 2-diethylaminoethanethiol, (2-butylamino)ethanethiol, L-cysteine, D-cysteine, DL-cysteine, L-cysteine methyl ester, L-cysteine ethyl ester, L-homocysteine, D-homocysteine, or DL-homocysteine, 2-mercaptoethyl guanidine or 3 -mercaptopropyl guanidine.

[0249] In various examples, the present polymers include a backbone and first and second pendant groups. The first pendant group includes an aminoalkyl group or amino aryl group (e.g., primary, secondary, or tertiary), and the second pendant group includes an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group an aryl group or a neutral heteroalkyl group, e.g., being hydrophilic or hydrophilic and lipophilic. The polymer may include the first pendant group and the second pendant group, where each is 5-95% of the total number of first and second pendant groups and the first and second pendant groups are different. Exemplary backbones include styrene, acrylate / acrylamide, methacrylate / methacrylamide, and mixtures thereof. The backbone may also include disulfide groups, e.g., separated by alkylene, polyetheneglycol, polynucleic acids, and / or polypeptides. In some embodiments, the polymer includes a third pendant group or an ion or salt thereof, wherein the first pendant group includes an aminoalkyl group, the second pendant group includes a quaternary aminoalkyl group, and the third pendant group includes a dialkyl group, a neutral substituted alkyl group, or a neutral heteroalkyl group, wherein the first pendant group is 5-30%, the second pendant group is 5-15%, and the third pendant group is 55-90% of the total number of first, second, and third pendant groups.

[0250] Use of disulfide monomers in the synthesis of the polymers may result in the incorporation of sulfur atoms and disulfide bonds in the backbone. Incorporation of sulfur atoms and disulfide bonds in the backbone introduces sites for cleavage or reduction, e.g., by thiols or antioxidants in vivo. Reduction of disulfides may be pH dependent and, e.g., allow for delivery of the intact polymer inside a cell prior to degradation. Degradation of the polymer results in smaller fragments that may be more able to be excreted or eliminated.

[0251] One terminus of the polyester copolymer scaffolds may result from an initiator. The initiator may contain either a hydroxyl group or carboxylic acid. In some embodiments, the initiator polymer terminus may be used to introduce chain end functionality for further conjugation with small molecule end-groups, biological agents (e.g., peptides), or coupling with other polymers (e.g., PEG, e.g., 500 to 3000 Da or 2000 Da). A monovalent initiator may beused to form a linear scaffold. In some embodiments, the initiator is propargyl alcohol. In particular embodiments, the alkyne terminus resulting from the propargyl alcohol initiator is used to attach PEG or peptides to the chain terminus.

[0252] In various embodiments, a terminus comprises a hydrogen, alkyl, alkenyl, alkynyl, or benzyl, -O-alkyl, -O-alkenyl, -O-alkynyl, or -O-benzyl. -O-CH2-alkenyl, -O-CH₂-alkynyl. In various embodiments, the terminus comprises alkynyl or benzyl. In various embodiments, the terminus comprises -O-CH2CCH. In various embodiments, the terminus comprises -O-benzyl.

[0253] The polyester copolymers may also include formula (A-I):

[0254] The polyester copolymers may also include formula (A-II):0 R20 R3R R R (A-II), wherein: n, R1, R2, R3, R4, and R5are as described herein.

[0255] The polyester copolymers may also include formula (A-III):(A-III), wherein: n, R1, R3, R4, and R5are as described herein.

[0256] In various embodiments, the polyester copolymer may comprise Formula (A-IV):(A-IV), wherein: a, b, c, d, e, f, g, m, q, R1, R2, R3, R4, and R5are as described herein.

[0257] In various embodiments, the polyester copolymer may comprise Formula (II-A):R1R2R3R4R1R2(I-A), wherein: a, b, c, q, R1, R2, R3, and R4are as described herein.

[0258] In various embodiments, the polyester copolymer may comprise Formula (I-B):R1R2R3R4R1R2R3R4R1R2R3R4R1R2R3R4R1R2R3R4R1R2R3R4R1R2R3R4(I-B), wherein: a, b, c, d, e, f, g, q, R1, R2, R3, and R4are as described herein.

[0259] In various embodiments, the polyester copolymer may comprise Formula (A- VI):(A- VI), wherein: a, b, c, d, e, f, g, m, q, R1, R2, R3, R4, and R5are as described herein.

[0260] In various embodiments, the polyester copolymer may comprise Formula (A- VII):(A-VIII), wherein: a, b, c, d, e, f, g, m, q, R1, R3, R4, and R3are as described herein.

[0261] In various embodiments, the polyester copolymer may comprise Formula (B-I):(B-I).

[0262] In various embodiments, the polyester copolymer may comprise Formula (B-II):(B-II)or

[0263] In another embodiment, polyester copolymers may also include formula (B-III):

[0264] In another embodiment, polyester copolymers may also include formula (B-IV):(B-IV).

[0265] In another embodiment, polyester copolymers may also include formula (B-V):

[0266] In another embodiment, polyester copolymers may also include formula (B-VI):

[0267] In another embodiment, polyester copolymers may also include formula (B-VII):

[0269] In another embodiment, polyester copolymers may also include formula (B-IX):

[0270] In another embodiment, polyester copolymers may also include formula (B-X):

[0273] In another embodiment, polyester copolymers may also include formula (B-XIII):

[0274] In various embodiments the polyester copolymers may also include formula (B-XIII): wherein a is 0.70, b is0.30, and q is 48.

[0275] In another embodiment, polyester copolymers may also include formula (B-XIV):

[0276] In various embodiments the polyester copolymers may also include formula B-XIV wherein a is 0.60, b is0.20, c is 0.20, and q is 28.

[0277] In another embodiment, polyester copolymers may also include formula (B-XV):(B-XV).

[0278] In various embodiments the polyester copolymers may also include formula B-XV wherein a is 0.70, b is0.10, c is0.20, and q is 28.

[0279] In another embodiment, polyester copolymers may also include D3503

[0280] In various embodiments the polyester copolymers may also include formula (B-VI): wherein a is 0.40, b is 0.60, and q is 48.

[0281] In various embodiments, a is 0.3, b is 0.7, and q is 8. In various embodiments, the a is 0.3, b is 0.7, q is 8, RT1is alkynyl, and RT2is H. In various embodiments, RT1is benzyl.

[0282] Variables a, b, c, d, e, f and g represent fractions of m. The sum of a, b, c, d, e, f and g add up to 1.0. Variables a, b, c, d, e, f, and g, are each independently 0 to 1. In various embodiments, a, b, c, d, e, f, and g, are each independently zero or about 0.1 to about 0.9. In various embodiments, a, b, c, d, e, f, and g, are each independently 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.

[0283] In various embodiments, at least one of a, b, c, d, e, f and g is not 0. In various embodiments, at least two of a, b, c, d, e, f and g are not 0. In various embodiments, at least three of a, b, c, d, e, f and g are not 0. In various embodiments, at least four of a, b, c, d, e, f and g are not 0. In various embodiments, at least five of a, b, c, d, e, f and g are not 0. In various embodiments, at least six of a, b, c, d, e, f and g are not 0.

[0284] In various embodiments q is from 1 to about 1,000. In various embodiments q is from about 10 to about 1,000. In various embodiments m is from about 2 to about 100. In various embodiments, m is from about 10 to about 100. In various embodiments, m is from about 2 to about 80.

[0285] In various embodiments, q is from 10 to 100; a is 0 - 0.3, b is 0 - 0.15, c is 0 - 0.9, and d is 0 - 0.15 and each represents a fraction of q, and the sum of a, b, c, and d is 1, provided that at least two of a, b, c, and d, are not 0.

[0286] Rla, R2a, R3a, R4a, Rlc, R2c, R3c, R4c, Rld, R2d, R3d, and R4d, are each independently an ionizable acid, and ionizable base, a neutral group, an anionic group, or a cationic group. In certain embodiments, Rla, R2a, R3a, R4a, Rlc, R2c, R3c, R4c, Rld, R2d, R3d, and R4d, are each independently H, amino, quaternary amino, guanidine, hydroxyl, sulfonyl, or carboxyl. In certain embodiments, Rla, R2a, R3a, R4a, Rlc, R2c, R3c, R4c, Rld, R2d, R3d, and R4d, are each independently alkyl, alkenyl, alkynyl, carbocyclyl, heteroalkyl, heterocyclyl, or aryl.

[0287] Each of u, v, w, x, y, z, uu, vv, ww, xx, yy, zz, are independently 0 to 20. In some embodiments, each of u, v, w, x, y, z, uu, vv, ww, xx, yy, zz, are independently 0 to 10. In some embodiments, each of u, v, w, x, y, z, uu, vv, ww, xx, yy, zz, are independently 0 to 6.

[0288] RT1and RT2each independently is H, a terminal group, or a side chain. In various embodiments, the terminal group comprising a hydrogen, alkyl, alkenyl, alkynyl, or benzyl, -O-alkyl, -O-alkenyl, -O-alkynyl, or -O-benzyl. -O-CH₂-alkenyl, -O-CH2-alkynyl.

[0289] In various embodiments, RT1is alkynyl or benzyl. In various embodiments, RT1is -O-CH2CCH. In various embodiments, RT1is -O-benzyl.

[0290] In various embodiments, RT2is H

[0291]

[0292] Exemplary copolymer intermediates are shown in Table 1.Table 1: Polyester copolymer reactive backbonesPoly(maleate-co-AGE)s1) Poly(phthalic anhydride-co- f'AGE)M.,0, 0 00TOjH Poly(succinic anhydride-co-0AGE) 0 L_Poly(glutaric anhydride-co- o..AGE) 'Y,0 / „x~,, O, \H If0 o Poly(diglycolic anhydride-co- 0.AGE) ■:Y 0 / x-^ x'.' P Y ° „ 0 01o0 / x. J\ X-, O - Poly (allyl succinic anhydride- Y X T * co-AGE) 1 'Polypropylene fumarate) b oCH, Polypropylene maleate)fl If0 Y 0 / X-S JI x~. Oi Polypropylene allysuccinate) A T ° T #0ISPoly(succinic anhydride-co- vCHO)y o HCH; o>o= / Poly(fumarate-co-vCHO)O \-- \ o / Poly(maleate-co-vCHO) / Poly(glutarate-co-vCHO)4 -- o / — X / A / ,o, Qi Poly(diglycolate-co-vCHO) vr0¥? \ r o o \,) o. <o H. h ' Poly(allylsuccinate-co-vCHO. / \S. \\ / -’ Poly(phthalic anhydride-co- vCHO) ' ¥"0; W°4 0 0 \ / \. oPoly(succinic anhydride-co- PGE)« N3„ 0Poly(succinic anhydride-co- ^.,-A o. A. AH glycidyl azide) or p(SA-co- Q\ -•' Y 07 4zH Gaz) 0

[0293] In addition to linear reactive backbones, the polyester copolymer could be nonlinear, e.g., by initiating the reaction from a multivalent initiator. For example, scaffolds of arbitrary size can be formed, e.g., 3-, 4-, 5-, or 6-armed stars. Examples of multivalent initiators are shown in Table 2.Table 2: Multivalent initiatorsName Structure # of arms 2-hydroxymethyl- 1,3- '"" OH3 propanediol'"" OHpentaerythritol 4HO—' OHOHmeso-erythritol A.-OHHO Y 4OH

[0294] Neutral, negatively charged, positively charged, non-ionizable, ionizable base, or ionizable acid side chains may be formed. In some embodiments, this is accomplished via conjugation reactions with a side chain reactive group on the polyester polymer backbone and at least one side chain reactive molecule. In some embodiments, a side chain is formed via conjugation reactions with various thiols as shown in Tables 3 and 4. Neutral and negatively charged side chains may be 0-50% of the side chains present, and positively charged side chains may be 50-100% of the side chains present. For other conjugation reactions, the thiol in Tables 3 and 4 may be replaced with another group, e.g., amino, hydroxy, carboxy (or activated derivative), azido, alkyne (linear or cyclic), diene, or alkene.

[0295] In various embodiments, the side chain reactive molecule comprises an alkyne. In various embodiments, the alkyne is a neutral alkyne. In various embodiments, the neutral alkyne is propargyl alcohol; 1 -pentyne, 1 -hexyne; 4-methyl-l -pentyne; 4-octyne; cyclopropylacetylene; 4-pentyn-l-ol; 1-dodecyne; or methyl-5-hexynoate. In various embodiments, the alkyne is an ionizable base alkyne. In various embodiments, the ionizable base alkyne is propargyl amine; N-methylpropargylamine; amino-PEG3 -alkyne; or 3-dimethylamino-l-propyne. In variousembodiments, the alkyne is an ionizable acid alkyne. In various embodiments, the ionizable acid alkyne is 10-undecynoic acid; or 4-pentynoic acid.

[0296] Table 3: Neutral and negatively charged thiols.1 -propanethiol1 -butanethiol HS 1 -pentanethiol1 -hexanethiol (1HT)x''41 -octanethiol (1OT)1 -decanethiol HS '1 -undecanethiolf s 1 -hexadecanethiol' 142-ethylhexanethiolcyclopentanethiolcyclohexanethiol?H2-mercaptoethanol(2ME)SH3 -mercapto- 1 -propanol(3M1P) 1OHSH 6-mercapto- 1 -hexanol(MHO)GH SH8-mercapto- l -octanol (MOO)-Tu' OH SH9-mercapto- 1 -nonanol (9M1N)7'0H SH11 -mercapto- 1 -undecanol (MUD)^" OH SH16-mercapto- 1 -hexadecanol (MHDol)mercaptopropionic acid(MPA)11-mercaptoundecanoic acid(MUA)benzimidazole ethanethiol(BET) _benzimidazole octanethiol (BETC8)benzimidazole undecathiol (BETCI 1)SHsodium 2-sulfanyl ethanesulfonates (MESA)ON a SHS odium 3 -m ercaptopropanesul fonate0 (S3MPS)nQ®'ONa SH1 -thioglycerol(1TG)SH7-mercapto-4-methylcoumarin(MMC)c H 34-mercaptobutyric acid SHV OH6-mercaptohexanoic acid (MHA) SHOH8-mercaptoctanoic acid (MO A) SHjn' oOH12-mercaptododecanoic acid (MDDA) SH' TaOH16-mercaptohexadecanoic acid (MHDA) SHOHL-glutathione _SH0C * f H? A N A HO’'' N' Y " OH RH2n0(4-nitrobenzyl)mercaptan 0 n +1 " V 0 HS

[0297] Table 4: ionizable base thiolsSHcysteamine hydrochloride(CYS) sNH2HGI2-dimethylaminoethanethiol hydrochloride(CAP)HJC:2-diethylaminoethanethiol hydrochloride(DiE)HS(2-butylamino)ethanethiol 'N- H SHL-cysteine hydrochloride,. NH?HCI (LCYS)'"'OHD-cysteine hydrochlorideSH DL-cysteine hydrochloride NH2HCI O'' ''" OH HS,.L-homocysteine hydrochloride LX,. NH?HCI 0 " OH HS.XD-homocysteine hydrochloride -NH, HCI TC 'A'OHHS„DL-homocysteine hydrochlorideNF.. HCI (DLHCYS) 0 ’ ’OHSHL-cysteine methyl ester hydrochlorideX HCI(LCMEH) O ' OSHL-cysteine ethyl ester hydrochloride L.,, NH21 HCi(LCEEH) O' O2-mercaptoethyl guanidine L n,<o-;■ " A >i.NH HCIH3 -mercaptopropyl guanidine (MPGdm) HS. N., NH?NH HCI

[0298] Pendant groups may be present in the monomers during polymerization or may be added after polymerization. For example, groups substituted with thiols may be reacted with pendant haloalkyl or alkenyl groups on the polymer. Exemplary thiolated groups are shown in Table 1. Neutral and positively charged side chains may be formed. In some embodiments, this is accomplished via conjugation reactions with various thiols as shown in Tables 1 and 2. For other conjugation reactions, the thiol in Tables 5 and 6 may be replaced with another group, e.g., amino, hydroxy, carboxy (or activated derivative), azido, alkyne (linear or cyclic), diene, or alkene.Table 5: Neutral thiols2-mercaptoethanol(2ME)3 -mercapto- 1 -propanol (3M1P)6-mercapto- 1 -hexanol (MHO)benzimidazole ethanethiol (BET)1 -thioglycerol(1TG)7-mercapto-4-methylcoumarin (MMC)Table 6: Positively charged thiolscysteamine hydrochloride (CYS)^ 2-dimethylaminoethanethiol hydrochloride X (CAP)Jk2-di ethylaminoethanethiol hydrochloride(DiE)o L-cysteine hydrochloride(LCYS)L-cysteine methyl ester hydrochloride(LCMEH) XL-cysteine ethyl ester hydrochloride(LCEEH)

[0299] Exemplary polymers are shown in Table 9.TABLE 9.first Pendant Apcmp {a} Second P’eihJ-sfitGroiip (hj Third Pendant Greap (c) % la.b,cj m PEG BJockJ _1fi, l[T '.. j.,... c x,...lv...^Y Y ""' '"i... iT.. 1.. JO xT ' I ’ ’[4. j^^jQ... 1...., OT XS... AI i. i.... OT' r. i.,, O % A _.<»T I£ - I..1,. X % A,.iST " I.. i,... O~T "" I' "='„ ii „.. ^u... 1.... jO Tr. ' - I '"' I ’■ L. a x.,- Tv■'" T -. ^VyjO1a _ n a. T Ia^jx^,v xL x.....1.. i..' J ' " '». JO., D T ' X ’ '.. 1 -.. i... O T 'r.. Xax X '' I" '". 1... »,.., i...,nTT" cvT * r '■- "■'.. i,.....i T;' ' UUyUO.,i y =.......,- jrn...... L.-.,... O X ’ L '" ' ^T JTx.-. XX), i... x ':i T ' X

[0300] Exemplary polyester polymers are shown in Table 10.TABLE 10.Thir SixthReactiv First Second d Fourt Fifth sidee side side side h side side chai %Polymer backbo lengt chain chain chai chain chai n (a,b,c,d,e, PEG # ne h (q) (a) (b) n (c) (d) n (e) (f) f) block SA-co- MOD2592 AGE 48 A Cys 30,70SA-co- MHD42 AGE 48 O Cys 30,70SA-co- D126 AGE 28 BET Cys 30,70SA-co- MUD51 AGE 48 A Cys 30,70SA-co- MH PEG2 D164 AGE 48 O Cys 10,90 k SA-co- MHD2397 AGE 48 DA Cys 30,70SA-co- MUD2386 AGE 28 A Cys 30,70SA-co- MUD1167 AGE 8 A Cys 23,77SA-co- MUD4172 AGE 8 A Cys 30,70SA-co- MHD1169 AGE 8 0 Cys 31,69SA-co- MUD5588 AGE 28 A MHO Cys 20,20,60SA-co- D3859 AGE 8 1OT Cys 40,60SA-co- MUD3576 AGE 28 D Cys 40,60SA-co- MDD4764 AGE 8 DA Cys 10,90SA-co- MHD4894 AGE 28 Doi Cys 10,90SA-co- MH LCMEDI 462 AGE 8 0 H 20,80SA-co- MHD4175 AGE 28 DA Cys 30,70MA-co- MUD156 AGE 12 D Cys 40,60Thir SixthReactiv First Second d Fourt Fifth sidee side side side h side side chai %Polymer backbo lengt chain chain chai chain chai n (a,b,c,d,e, PEG # ne h (q) (a) (b) n (c) (d) n (e) (f) f) block SA-co- MUD4800 AGE 48 A Cys 30,70SA-co- MUD5580 AGE 28 A MHO Cys 30,10,60SA-co- MUD1163 AGE 8 A Cys 15,85SA-co- MUD188 AGE 28 A Cys 30,70SA-co- D178 AGE 28 BET Cys 30,70SA-co- D3560 AGE 28 1OT Cys 40,60SA-co- MUD3503 AGE 48 D Cys 40,60SA-co- MOD2598 AGE 48 A Cys 40,60SA-co- MHD5386 AGE 28 Doi Cys 20,80SA-co- MUD4895 AGE 28 D Cys 40,60SA-co- MUD5584 AGE 28 A MHO Cys 10,20,70SA-co- MUD5585 AGE 28 A MHO Cap 10,20,70SA-co- MUD5586 AGE 28 A MHO DiE 10,20,70LC SA-co- MU MED5587 AGE 28 A MHO H 10,20,70SA-co- MUD5592 AGE 28 A MHO Cys 10,30,60SA-co- MUD5366 AGE 48 A MHO Cys 20,10,70SA-co- S3M MUD11883 AGE 28 PS 1HDT D BET Cap 10,20,20,10,40LC SA-co- MH DLHC ME 30,10,20, DI 1344 AGE 28 0 YS H Cys 40MA-co- MHD6014 PO 6 0 Cys 30,70Thir SixthReactiv First Second d Fourt Fifth sidee side side side h side side chai %Polymer backbo lengt chain chain chai chain chai n (a,b,c,d,e, PEG # ne h (q) (a) (b) n (c) (d) n (e) (f) f) _ block SA-co- MH 9M1D11910 AGE 48 A MHO N BET Cys Cap 10,10,15,15,10,40SA-co- MH MES 10,20,10, D11491 AGE 48 O MUD A Cys 60SA-co- MU MPGdD11273 AGE 28 D m DiE 30,10,60SA-co- MUD6352 AGE 48 A Cys 30,70SA-co- MU MU 10,10,20, DI 1052 AGE 13 A MHO D Cys 60SA-co- MH 9M1DI 1042 AGE 48 A MHO N BET Cys 10,10,15,15,50 SA-co- S3M MUD11867 AGE 28 PS 1HDT D BET Cap 20,20,20,10,30

[0301] In various embodiments, the polymer comprises:(Ml 1163),HCIO^OH OH N NH OH NH2o > o > o > o (DI 1042), HCI O. OH OH N NH OH NH9< Y <(DI 1052), lNJ O OH OH J HCI X9, X4Y 4( Y -VY T(D5586)UP, O. OH OHHCINH2Y <4"(D5588)>«Cl<.0HoU / l9(D3503), or(D6352).ANTIBODIES

[0302] The invention provides a number of isolated human monoclonal antibodies, wherein each said monoclonal antibody binds to HIV-1 infected or transfected cells; and binds to HIV-1 virus. A neutralizing antibody having potency in neutralizing HIV-1, or a fragment thereof is provided. In some embodiments, a neutralizing antibody of the invention exhibits a higher neutralization index and / or a higher affinity for binding to the envelope proteins gpl20, or gp41 than anti -HIV mAbs known in the art, such as the mAb bl2. (Burton DR et al., Science Vol. 266. no. 5187, pp.1024 - 1027). Exemplary monoclonal antibodies 1496_C09 (PG9), 1443_C16 (PG16), 1456P20 (PG20), 1460 G14 (PGG14), and 1495C14 (PGC14) exhibit binding to the envelope glycoprotein gpl20, but not gp41, in an ELISA assay, however gpl20 binding does not always correlate with neutralization activity against specific strains of HIV- 1. In some embodiments, monoclonal antibodies display none or weak gpl20 binding activity against a particular strain but bind to HIV-1 trimer on transfected or infected cell surface and / or virion and exhibit broad and potent neutralization activity against that strain of HIV- 1.

[0303] The present invention features an anti -HIV gpl20 antibody for use methods of curing subjects (e.g., humans) infected with HIV (e.g., HIV Type 1 (HIV-1)) by administration of an N332 glycan-dependent antibody (e g., PGT121), alone, in combination with other HIV-specific antibodies (e.g., other broadly neutralizing antibodies (bnAbs)), and / or in combination with antiretroviral therapies, which are described herein below.

[0304] In one aspect the antibody is a monoclonal antibody which may comprise one or more polypeptides selected from the group consisting of 1443C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480108 (PG16), 1456P20 (PG20), 1460G14 (PGG14), 1495_C14 (PGC14),1496_C09 (PG9) (TCN-109), 4838 L06 (PGT-121),4873_E03 (PGT-121),4877_D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125), 5141_B17 (PGT-126), 5145_B14 (PGT-127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136_HO1 (PGT-131), 5343_B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT-136), 5366P21 (PGT-136), 4964 G22 (PGT-141), 5345101 (PGT-137), 4993 K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138G07 (PGT-138), 5120_N10 (PGT-139), 6831A21 (PGT-151), 6889 117 (PGT-152), 6891F06 (PGT-153), 6843 G20 (PGT-154), 6892D19 (PGT-155), 6808_B09 (PGT-156), 6892C23 (PGT-157), and 6881_N05 (PGT-158); which may comprise a heavy chain selected from the group consisting of the heavy chain of 1443 C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN 117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480_108 (PG16), 1456_P20 (PG20), 1460G14 (PGG14), 1495C14 (PGC14), 1496_C09 (PG9) (TCN-109), 4838_L06 (PGT-121), 4873_E03 (PGT-121), 4877D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125), 5141_B17 (PGT-126), 5145B14 (PGT-127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136 HO (PGT-131), 5343B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT-136), 5366_P21 (PGT-136), 4964G22 (PGT-141), 5345101 (PGT-137), 4993_K13 (PGT-141), 4995_E20 (PGT-142), 4980N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138 G07 (PGT-138), 5120_N 10 (PGT-139), 6831_A21 (PGT-151), 6889 117 (PGT-152), 6891 F06 (PGT-153), 6843 G20 (PGT-154), 6892D19 (PGT-155), 6808 B09 (PGT-156), 6892C23 (PGT-157), and 6881_NO5 (PGT-158); which may comprise a heavy chain which may comprise a CDR selected from the group consisting of the CDRs of the heavy chain of 1443_C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480108 (PG16), 1456_P20 (PG20), 1460G14 (PGG14), 1495C14 (PGC14), 1496_C09 (PG9) (TCN-109), 4838_L06 (PGT-121), 4873_E03 (PGT-121), 4877D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125),5141 B17 (PGT-126), 5145B14 (PGT-127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136 HO (PGT-131), 5343B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT-136), 5366_P21 (PGT-136), 4964G22 (PGT-141), 5345101 (PGT-137), 4993_K13 (PGT-141), 4995_E20 (PGT-142), 4980N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138_G07 (PGT-138), 5120_N10 (PGT-139), 6831_A21 (PGT-151), 6889 117 (PGT-152), 6891 F06 (PGT-153), 6843 G20 (PGT-154), 6892D19 (PGT-155), 6808 B09 (PGT-156), 6892C23 (PGT-157), and 6881_NO5 (PGT-158); which may comprise a light chain selected from the group consisting of the light chain of 1443_C16 (PG16) (TCN 116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN 118), 1489 113 (PG16) (TCN-120), 1480_108 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), 1495_C14 (PGC14),1496_C09 (PG9) (TCN-109),4838_L06 (PGT-121),4873_E03 (PGT-121), 4877_D15 (PGT-122), 4858_P08 (PGT-123), 6123A06 (PGT-125), 5141B17 (PGT-126), 5145_B14 (PGT-127), 5114_A19 (PGT-128), 5147N06 (PGT-130), 5136_HO1 (PGT-131), 5343_B08 (PGT-135), 5344 E16 (PGT-135), 5329C19 (PGT-136), 5366P21 (PGT-136), 4964 G22 (PGT-141), 5345101 (PGT-137), 4993 K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970_K22 (PGT-144), 4995P16 (PGT-145), 4835F12 (PGT-124), 4869-K15 (PGT-133), 4876_M06 (PGT-134), 5131A17 (PGT-132), 5138G07 (PGT-138), 5120_N10 (PGT-139), 6831 A21 (PGT-151), 6889117 (PGT-152), 6891 F06 (PGT-153), 6843 G20 (PGT-154), 6892 D19 (PGT-155),6808_B09 (PGT-156),6892_C23 (PGT-157), and 6881 NO5 (PGT-158); which may comprise a light chain which may comprise a CDR selected from the group consisting of the CDRs of the light chain of 1443 C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480 108 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), 1495_C14 (PGC14),1496_C09 (PG9) (TCN-109),4838_L06 (PGT-121),4873_E03 (PGT-121), 4877_D15 (PGT-122), 4858P08 (PGT-123), 6123 A06 (PGT-125), 5141B17 (PGT-126), 5145 B14 (PGT-127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136_HO1 (PGT-131), 5343_B08 (PGT-135), 5344E16 (PGT-135), 5329_C19 (PGT-136), 5366P21 (PGT-136), 4964_G22 (PGT-141), 5345101 (PGT-137), 4993_K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970_K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-K5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138G07 (PGT-138), 5120_N10 (PGT-139),6831_A21 (PGT-151), 6889 117 (PGT-152), 6891_F06 (PGT-153), elipovimab, 6843_G20 (PGT-154), 6892D19 (PGT-155), 6808 B09 (PGT-156), 6892C23 (PGT-157), and 6881 N5 (PGT-158) as described in US10376583 and US20240132578.

[0305] The invention relates to an antibody or a fragment thereof, such as Fab, Fab', F(ab')2 and Fv fragments that binds to an epitope or immunogenic polypeptide capable of binding to an antibody selected from 1443C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480_108 (PG16), 1456P20 (PG20), 1460_G14 (PGG14), 1495C14 (PGC14), 1496C09 (PG9) (TCN 109), 4838_L06 (PGT-121), 4873E03 (PGT-121), 4877_D15 (PGT-122), 4858P08 (PGT 123), 6123_A06 (PGT-125), 5141B17 (PGT-126), 5145_B14 (PGT-127), 5114A19 (PGT 128), 5147 N06 (PGT-130), 5136 HO1 (PGT-131), 5343 B08 (PGT-135), 5344E16 (PGT135),5329_C19 (PGT-136),5366_P21 (PGT-136), 4964G22 (PGT-141), 5345101 (PGT-137), 4993 K13 (PGT-141), 4995_E20 (PGT-142), 4980N08 (PGT-143), 4970K22 (PGT-144), 4995 P16 (PGT-145), 4835F12 (PGT-124), 4869-K15 (PGT-133), 4876M06 (PGT-134), 5131 A17 (PGT-132), 5138 GO7 (PGT-138), 5120 N10 (PGT-139), 6831A21 (PGT-151), 6889 117 (PGT-152), 6891F06 (PGT-153), 6843 G20 (PGT-154), elipovimab, 6892D19 (PGT-155), 6808 B09 (PGT-156), 6892C23 (PGT-157), and 6881_N05 (PGT-158).

[0306] The invention also relates to immunogenic polypeptides encoding such epitopes.

[0307] Nucleic acid molecules encoding such antibodies, and vectors and cells carrying such nucleic acids are also provided.

[0308] The invention relates to a pharmaceutical composition which may comprise at least one antibody or fragment as recited herein, together with a pharmaceutically acceptable carrier.

[0309] The invention relates to a method of immunizing, preventing or inhibiting HIV infection or an HIV-related disease which may comprise the steps of identifying a patient in need of such treatment and administering to said patient a therapeutically effective amount of at least one monoclonal antibody as recited herein.

[0310] In a further aspect the HIV antibodies according to the invention are linked to a therapeutic agent or a detectable label.

[0311] Additionally, the invention provides methods for stimulating an immune response, treating, preventing or alleviating a symptom of an HIV viral infection by administering an HIV antibody to a subject.

[0312] In another aspect, the invention provides methods of administering the HIV antibody of the invention to a subject prior to, and / or after exposure to an HIV virus. For example, the HIV antibody of the invention is used to treat or prevent HIV infection. The HIV antibody is administered at a dose sufficient to promote viral clearance or eliminate HIV-infected cells.

[0313] Also included in the invention is a method for determining the presence of an HIV virus infection in a patient, by contacting a biological sample obtained from the patient with an HIV antibody; detecting an amount of the antibody that binds to the biological sample; and comparing the amount of antibody that binds to the biological sample to a control value.

[0314] The invention relates to a broadly neutralizing antibody (bNAb) wherein the antibody neutralizes at least one member of each clade with a potency greater than that known in the art of the bNAbs bl2, 2G12, 2F5 and 4E10 respectively.

[0315] The invention relates to a broadly neutralizing antibody (bNAb) wherein the antibody binds or does not bind monomeric gp!20 or gp41 proteins of the HIV-1 env gene. The antibody binds with higher affinity to trimeric forms of the HIV-1 Env expressed on a cell surface than to the monomeric gpl20 or artificially trimerized gpl40. In some aspects, the antibody binds with high affinity to uncleaved HIV-1 gpl60 trimers on a cell surface.

[0316] The invention relates to a broadly neutralizing antibody (bNAb) wherein the antibody binds an epitope within the variable loop of gpl20, wherein the epitope may comprise the conserved regions of V2 and V3 loops of gpl20, wherein the epitope may comprise N glycosylation site at residue Asn-160 within the V2 loop of gpl20, wherein the antibody binds an epitope presented by a trimeric spike of gpl20 on a cell surface, wherein the epitope is not presented when gpl20 is artificially trimerized. In some embodiments, the antibody does not neutralize the HIV-1 in the absence of N-glycosylation site at residue Asn-160 within the V2 loop of gp!20.

[0317] The invention relates to a broadly neutralizing antibody (bNAb) selected from the group consisting of PG16 and PG9. Moreover, the invention relates to a broadly neutralizing antibody (bNAb) selected from the group consisting of 1443_C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480108 (PG16), 1456P20 (PG20), 1460G14 (PGG14), 1495_C14 (PGC14),1496_C09 (PG9) (TCN-109), 4838_L06 (PGT-121),4873_E03 (PGT-121),4877_D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125), 5141_B17 (PGT-126), 5145_B14(PGT-127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136_HO1 (PGT-131), 5343_B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT-136), 5366P21 (PGT-136), 4964 G22 (PGT-141), 5345101 (PGT-137), 4993 K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138G07 (PGT-138), 5120_N10 (PGT-139), 6831A21 (PGT-151), 6889 117 (PGT-152), 6891_F06 (PGT-153), 6843_G20 (PGT-154), 6892D19 (PGT-155), 6808 B09 (PGT-156), 6892C23 (PGT-157), and 6881 NO5 (PGT-158).

[0318] The invention relates to an antigen or an immunogenic polypeptide, or a vaccine which may comprise such antigen or immunogenic polypeptide, for producing a broadly neutralizing antibody (bNAb) by an immune response, the antigen which may comprise an epitope within the variable loop of gpl20 according to the invention.

[0319] The invention relates to method for passive or active immunization of an individual against a plurality of HIV-1 species across one or more clades, the method which may comprise: providing a broadly neutralizing antibody (bNAb) wherein the bNAb neutralizes HIV-1 species belonging to two or more clades, and further wherein the potency of neutralization of at least one member of each clade is determined by an IC50 value of less than 0.005 Pg / mL. In some embodiments, the antibody is selected from the group consisting of PGT121 and elipovimab. Alternatively, or in addition, the antibody is selected from the group consisting of 1443_C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480_108 (PG16), 1456_P20 (PG20), 1460_G14 (PGG14), 1495C14 (PGC14), 1496C09 (PG9) (TCN-109), 4838L06 (PGT-121), 4873_E03 (PGT-121), 4877_D15 (PGT-122), 4858P08 (PGT-123), 6123A06 (PGT-125), 5141_B17 (PGT-126), 5145B14 (PGT-127), 5114A19 (PGT-128), 5147N06 (PGT-130), 5136 HO (PGT-131), 5343 B08 (PGT-135), 5344E16 (PGT-135), 5329C19 (PGT-136), 5366_P21 (PGT-136), 4964_G22 (PGT-141), 5345101 (PGT-137), 4993K13 (PGT-141), 4995_E20 (PGT-142), 4980_N08 (PGT-143), 4970K22 (PGT-144), 4995P16 (PGT-145), 4835_F12 (PGT-124), 4869-KI5 (PGT-133), 4876_M06 (PGT-134), 5131A17 (PGT-132), 5138_G07 (PGT-138), 5120_N 10 (PGT-139), 6831A21 (PGT-151), 6889117 (PGT-152), 6891 F06 (PGT-153), 6843 G20 (PGT-154), 6892D19 (PGT-155), 6808 B09 (PGT-156), 6892_C23 (PGT-157), and 6881_NO5 (PGT-158).

[0320] In some embodiments, the antibody is produced by active immunization with an antigen which may comprise an epitope within the variable loop of gpl20, wherein the epitope may comprise the conserved regions of V2 and V3 loops of gp!20 or, wherein the epitope may comprise an N-glycosylation site at residue Asn-160 within the V2 loop of gp!20. In some aspects, the epitope is presented by a trimeric spike of gp!20 on a cell surface, and the epitope is not presented when gpl20 is monomeric or artificially trimerized.

[0321] In some embodiments, the antibodies are specific for dengue virus, and is selected from a number of isolated antibodies with serological specificity for dengue virus, including monoclonal antibody 12-4.1 (specific for DENV 1), monoclonal antibody 33-7.1 (specific for DENV 2), monoclonal antibody 43-1.3 (specific for DENV 3), and monoclonal antibody 22-1.5 (specific for DENV 4). These anti-dengue virus antibodies were found to be able to recognize one specific serotype of dengue virus without cross-reacting with other serotypes. More particularly, these anti-dengue virus antibodies are specific for different serotypes of dengue virus NS1 protein; that is, monoclonal antibody 12-4.1 is specific to the NS1 polypeptide derived from DENV1, monoclonal antibody 33-7.1 is specific to the NS1 polypeptide derived from DENV2, monoclonal antibody 43-1.3 is specific to the NS1 polypeptide derived from DENV3, and monoclonal antibody 22-1.5 is specific to the NS1 polypeptide derived from DENV4. These anti-dengue virus antibodies have been found to be effective and feasible in various forms of immunoassays for detecting dengue virus in a sample with excellent sensitivity and specificity. The invention is also based on the identification of dengue virus-specific cross-reactive antibodies (monoclonal antibody 82-1.1) that cross-react with various serotypes of dengue virus, including DENV1, DENV2, DENV3, and DENV4, and can be paired with any of the serospecific antibodies described herein for use in immunoassays to detect dengue virus in a sample.

[0322] In some embodiments, the antibodies are specific for the Ebola virus, and the Ebola virus therapeutic is selected from the group consisting of ribavirin, palivizumab, motavizumab, RSV-IGIV ( RespiGam® ), MEDI-557, A-60444, MDT -637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola convalescent plasma (ECP), TKM-100201, BCX4430 ((2S,3S, 4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), method Pilavir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (l-N,7-N-bisphosphate) [3-(dimethylamino)propyl]-3,9-dimethylquinoline[8,7-h]quinolinone-l,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2, VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, brincidofovir, Ebola vaccine based on Vaxart adenovirus vector 5, Ad26-ZEBOV, FiloVax vaccine, GOVX-E301, GOVX-E302, Ebola virus entry inhibitor (NPC1 inhibitor), amd rVSV-EBOV. In some embodiments, the Ebola virus therapeutic is selected from ZMapp, mABl 14, and REGEN-EB3.

[0323] In some embodiments, the antibodies are specific for the coronavirus, a severe acute respiratory syndrome (Severe Acute Respiratory Syndrome; SARS) related coronavirus. In some embodiments, the coronavirus is Middle-East Respiratory Syndrome (MERS) related coronavirus. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the SARS-CoV-2 (COVID- 19) therapeutic is an RNA polymerase inhibitor (eg, remdesivir, galidesivir). In some embodiments, the SARS-CoV-2 (COVID- 19) therapeutic is remdesivir (GS-5734). In some embodiments, the SARS-CoV-2 (COVID- 19) therapeutic is an anti-SARS-CoV-2 hyperimmune globulin therapy (plasma from convalescent COVID-19 patients, e.g., processed into hyperimmune globulin) ( For example, TAK-888). In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is selected from the group consisting of COVID-19 vaccines (eg, BN162, Ad5-nCoV, INO-4800, mRNA-1273); anti-IL6 receptor antibodies (eg, torcilla tocilizumab, sarilumab, TZLS-501); anti-IL6 antibodies (eg, siltuximab); RNA-dependent RNA polymerase (RdRp) inhibitors (eg, Favipravir (favipravir, remdesivir); anti-CCR5 antibodies (eg, lelonimab (PRO 140)); broadly neutralizing antibodies (eg, anti-ACE2 receptor antibodies, SAB-185, COVID-HIG, COVID-EIG); ACE2 (angiotensin converting enzyme 2)-Fc fusion protein (COVIDTRAP) or recombinant human ACE2 protein (APN1); designed to bind to coronaviruses including SARS-CoV-2 and SARS-CoV ACE-MAB ™ bispecific fusion proteins of spike proteins (eg, STI-4920, CMAB020); Janus kinase (JAK1 / JAK2) inhibitors (eg, ruxolitinib, baricitinib); siRNA (eg, targeting angiotensinconverting enzyme-2 (ACE2) or transmembrane protease, serine 2 (TMPRSS2)); HIV-1 protease inhibitors (eg, lopinavir / ritonavir; alone or dalunavir in combination with cobicistat); complement inhibitors (eg, eculizumab); HCV protease inhibitors (eg, danoprevir); stem cells Therapies (eg, MultiStem®, Remestemcel-L, CYNK-001); NK cell therapy (NKG2D-ACE2 CAR-NK cells); Neutralizing antibodies against human granulosa macrophage colony stimulating factor (GM-CSF) (eg, IZN-101, gimsilumab); vasoconstrictors (eg, angiotensin II); selective nuclear export inhibitors (SINEs), such as XPO1 inhibitors (eg, selinexor) NSAIDs, including COX inhibitors (eg, ibuprofen, aspirin, diclofenac, naxopren), and selective COX2inhibitors (eg, celecoxib ), rofecoxib, etoricoxib, lumiracoxib, valecoxib coxib)); and other antiviral agents (eg, ENU200, lopinavir / ritonavir combination). In some embodiments, the COVID- 19 vaccine is an mRNA vaccine (eg, BN162), including a lipid complex (eg, lipid-nanoparticle (LNP)) encapsulated vaccine (eg, mRNA1273). In some embodiments, the COVID-19 vaccine is a DNA vaccine (eg, INO-4800). In some embodiments, the COVID- 19 vaccine encodes a prefusion stable form of the spine (S) protein (eg, mRNA1273). In some embodiments, the COVID- 19 vaccine is a recombinant protein-based vaccine consisting of the receptor binding domain (RBD) of the coronavirus spike protein. In some embodiments, the COVID- 19 vaccine uses a ligand epitope presentation system (LEAPS) peptide comprising conserved regions of coronavirus proteins to stimulate protective cell-mediated T cell responses and reduce viral load. In some embodiments, the COVID-19 vaccine is a microneedle array (MNA) delivered vaccine. In some embodiments, the vaccine is based on an influenza vector expressing the surface antigen of SARS-CoV-2. In some embodiments, the COVID-19 vaccine is an intranasal vaccine (eg, AdCOVID). In some embodiments, the COVID- 19 vaccine is NVX-CoV2373, IN04800, orBNT-162. In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is selected from the group consisting of PIKfyve kinase inhibitors (eg, apiimod), immunomodulators (eg, ritalide), T cell immunotherapy, Recombinant sialidase (eg, DAS 181), CRAC channel inhibitors (eg, CM-4620-IE), cardiac cell therapy using allogeneic cardiosphere-derived cells (eg, CAP-1002), cardioprotective drugs (eg, aspirin, plavix, lipitor, opremazole), SIP receptor antagonists (eg, fingolimod), cyclooxygenase-2 (COX- 2) Inhibitors (eg, celecoxib), phosphodiesterase-5 (PDE5) inhibitors (eg, sildenafil citrate), serine protease TMPRSS2 inhibitors (methanesulfonic acid) Camostat mesylate), anti-human complement 5a antibodies (eg, IFX-1), macrophage migration inhibitory factor (MIF) inhibitors, phosphodiesterase (PDE)-4 and PDE-10 inhibitors (eg, ibudilast), eEFl A2 inhibitors (eg, plitidepsin), sphingosine kinase 2 (SK2) inhibitors (eg, ABC294640, RHB-107), galactose Lectin inhibitors (eg, BXT-10), membrane fusion inhibitors (eg, Eugene), anti-PDl antibodies, thymosin, antimalarial drugs (eg, chloroquine, hydroxychloroquine), and other antiviral therapeutics (for example, HTCC (N-(2-hydroxypropyl)-3 -trimethylammonium 47 chitosan chloride, 0YA1) as described in US11124582.

[0324] In some embodiments, the antibodies are specific for respiratory synctyial virus (RSV), and are selected from D25, MPE8, palivizumab, motavizumab, nirsevimab, 12C6, 3D3, 2D10, 5C4, RBI, MPE8, RV01, RV10, RV11, RV15, AM-14, AM-16, AM-22, and AM-23.

[0325] Additionally, therapeutic agents such as Factor IX, Factor VII, Factor VIII, glycoproteinoses, anti-TNF, GLP-1, anti-VEGF, anti-C5, vectorized PROTACs, vectorized siRNA and / or miRNA + / - genes for combined therapeutic strategies can be used with the current invention described herein.

[0326] In some embodiments, the therapeutic proteins described herein are selected from, but not limited to, metabolic, bone, muscle, immunomodulatory, hematology, longevity / regenerative, antibodies, rare disease, cardio-renal, anti-infective, and / or endocrine proteins.

[0327] In some embodiments, the therapeutic proteins described herein are selected from, but not limited to, GLP-1 receptor agonists, GIP receptor agonists, glucagon analogs, dual incretin agonists, triple / tri-agonists (GLP-1 / GIP / glucagon), FGF21 analogs, FGF19 analogs, amylin analogs, adiponectin mimetics, leptin and leptin analogs, insulin, insulin analogs, insulin fusion proteins, Follistatin, myostatin inhibitors, activin inhibitors, GDF-8 inhibitors, osteocalcin, osteoprotegerin (OPG), sclerostin inhibitors, parathyroid hormone (PTH), PTHrP analogs, BMP2, BMP4, BMP6, BMP7, Wnt ligands, sFRP inhibitors, R-spondin proteins, irisin, myonectin, myokines, IL- 10, IL-22, IL-2 muteins, IL-1 receptor antagonist (IL-IRa), IL-6 receptor blockers (e.g., tocilizumab), TNF inhibitors (e.g., etanercept, adalimumab), TGF-P inhibitors, GM-CSF, G-CSF, M-CSF, erythropoietin (EPO), thrombopoietin (TPO), Factor VIII, Factor IX, von Willebrand factor (vWF), fibrinogen, protein C, protein S, antithrombin III, hepcidin, complement Cl -INH, complement C3 inhibitors, complement C5 inhibitors, HGF, VEGF, PDGF, FGF2, IGF-1, OSK / OSKM reprogramming factors, GDF-11, GDF-15, klotho, neuregulin-1, relaxin, natriuretic peptides (ANP, BNP, CNP), apelin, defensins, cathelicidins, interferons (IFN-a, IFN-P, IFN-y), antiviral antibodies (RSV mAbs, influenza mAbs, COVID-19 mAbs), broadly neutralizing antibodies (bnAbs), antibacterial antibodies, anti-toxin antibodies, alpha- 1 antitrypsin (AAT), lysosomal enzymes (IDUA, IDS, GALC, GAA, ASB, NAGLU, GLA, SGSH, ARSB, HEXA, HEXB), GALC (galactocerebrosidase), GAA (acid alphaglucosidase), Cl esterase inhibitor (Cl -INH), GH (growth hormone), prolactin, ACTH, TSH, thyroid hormone-binding proteins, AMH, inhibin, activin, GnRH, FSH, LH, hCG, oxytocin, vasopressin, somatostatin analogs, osteopontin inhibitors, anti-fibrotic proteins (LOXL2inhibitors, CTGF inhibitors), PCSK9 inhibitors, ANGPTL3 inhibitors, apoA-I mimetics, anti-IL-13 antibodies, anti-IL-17 antibodies, anti-IL-23 antibodies, neuregulin, KIM-1 antagonists, antimicrobial peptides, immune checkpoint ligand traps (e.g., PD-L1 traps), immunoglobulins, bispecific antibodies, fusion cytokines, enzyme replacement therapies, complement regulatory proteins, renal protective peptides, cardioprotective peptides, digestive enzymes (pancrelipase), transferrin, ceruloplasmin, hemojuvelin, fibrinolytic proteins (tPA, uPA), anti-plasmin inhibitors, osteoanabolic proteins, anti -resorptive proteins, metabolic regulators (e.g., glucagon receptor antagonists as proteins), chemokines, chemokine receptor ligands, antiviral interferons, anti-infective fusion proteins, and / or anti-inflammatory cytokines.

[0328] In preferred embodiments, the therapeutic proteins described herein are selected from, but not limited to, GLP-ls, tri-agonists, FGF21 / 19, amylin, adiponectin mimetics, leptin, insulin / insulin fusions, Follistatin, myostatin inhibitors, osteocalcin, OPG, sclerostin inhibitors, IL- 10, IL-22, IL-2 muteins, IL-IRa, HGF, relaxin, EPO, Factor VIII / IX, vWF, hepcidin, OSK / OSKM, GDF-11 / 15, klotho, PCSK9, IL-6R, TNF, RSV / flu / COVID, bnAbs, AAT, GALC, GAA, lysosomal enzymes, ANP / BNP / CNP, apelin, defensins, interferons, Cl -INH, GH, IGF-1, PTH, AMH, GnRH, and / or FSH / LH / hCG.

[0329] PROTACs function in a ternary complex composed of a protein of interest-targeting ligand, an E3 ligase ligand, and a linker. This complex allows for the specific specific degradation of various proteins and the recyclability of PROTACs, increasing their efficacy. PROTACs are able to target and degrade specific proteins within a cell using the cell’s natural protein degradation machinery, and are under development to enhance degradation efficacy and drug characteristics such as bioavailability (Zhong G, et al., (2024). Sig Transduct Target Ther, 9:308).

[0330] As described herein, the monoclonal antibody as used herein can be selected from abciximab, adalimumab, alemtuzumab, alirocumab, avibactam, basiliximab, benralizumab, bezlotoxumab, blinatumomab, brodalumab, burosumab, canakinumab, caplacizumab, certolizumab pegol, daclizumab, denosumab, dupilumab, eculizumab, emicizumab, erenumab, evolocumab, fremanezumab, galcanezumab, golimumab, guselkumab, ibalizumab, idarucizumab, infliximab, itolizumab, ixekizumab, lanadelumab, lokivetmab, mepolizumab, natalizumab, obiltoxaximab, ocrelizumab, omalizumab, palivizumab, ranibizumab, raxibacumab, reslizumab, rmab, rovelizumab, ruplizumab, sarilumab, secukinumab, tildrakizumab, thiomab,tocilizumab, ustekinumab, vedolizumab, abrilumab, actoxumab, aducanumab, afasevikumab, afelimomab, anifrolumab, anrukinzumab (IMA-638), aselizumab, atorolimumab, bapineuzumab, BCD- 100, bertilimumab, besilesomab, biciromab, bimagrumab, bimekizumab, birtamimab, bleselumab, blosozumab, bococizumab, brazikumab, briakinumab, brolucizumab, carlumab, carotuximab, cedelizumab, clazakizumab, clenoliximab, concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab, crotedumab, depatuxizumab, mafodotin, derlotuximab biotin, dezamizumab, diridavumab, domagrozumab, dusigitumab, ecromeximab, edobacomab, efalizumab, efungumab, eldelumab, elezanumab, enokizumab, eptinezumab, erlizumab, etrolizumab, evinacumab, exbivirumab, fanolesomab, faralimomab, faricimab, fasinumab, felvizumab, fezakinumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foravirumab, frovocimab, fulranumab, gantenerumab, gavilimomab, gevokizumab, gimsilumab, gomiliximab, gosuranemab, ianalumab, inclacumab, inolimomab, iomab-B, keliximab, lampalizumab, landogrozumab, larcaviximab, lebrikizumab, lenvervimab, lerdelimumab, letolizumab, libivirumab, ligelizumab, lodelcizumab, lulizumab pegol, marstacimab, mavrilimumab, metelimumab, mirikizumab, motavizumab, muromonab CD3, nebacumab, nemolizumab, NEODOOl, nirsevimab, odulimomab, olendalizumab, olokizumab, OMS721, opicinumab, orticumab, otelixizumab, otilimab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, panobacumab, pascolizumab, pateclizumab, PDR001, perakizumab, pexelizumab, placulumab, plozalizumab, ponezumab, porgaviximab, prasinezumab, priliximab, PRO 140, quilizumab, rafivirumab, ralpancizumab, ranevetmab, ravagalimab, ravulizumab, refanezumab, regavirumab, relatlimab, rinucumab, risankizumab, roledumab, romosozumab, rontalizumab, SA237, satralizumab, sevirumab, SHP647, sifalimumab, simtuzumab, siplizumab, sirukumab, solanezumab, sonepcizumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tadocizumab, talizumab, tamtuvetmab, tanezumab, tefibazumab, telimomab aritox, teneliximab, teplizumab, teprotumumab, tezepelumab, tibulizumab, toralizumab, tralokinumab, trevogrumab, tuvirumab, ulocuplumab, urtoxazumab, varisacumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, zolimomab aritox, trastuzumab, gemtuzumab, brentuximab, vorsetuzumab, lorvotuzumab, cantuzumab, bivatuzumabor inotuzumab, or vadastuximab.ANTIBODY TARGETING AND DELIVERY

[0331] In some embodiments, a positively charged polymer is mixed with a negatively charged pDNA construct and is condensed, while the polymeric nanoparticles self-assemble. The polymeric nanoparticle is injected through a single route of administration such as intravenously, intramuscularly, or subcutaneously. The polymeric nanoparticle is passively or actively delivered to target cells and endocytosed into the cell. Through a variety of mechanisms, the polymeric nanoparticle is able to evade endosomes, such as a pH-sensitive polymeric nanoparticle that accepts protons in the acidic endosome causing swelling and endosomal rupture, or through direct interaction with the endosomal membrane. In the cytoplasm, the polymeric nanoparticle disassembles and the pDNA construct enters the nucleus through the nuclear pore complex, where the promoter encourages the transcription of the pDNA to mRNA, which exits the nucleus and is translated to proteins in the cytoplasm. The translated antibody protein sequence has a signal peptide attached, which drives the antibody out of the cell and towards bloodstream secretion to target specific cellular receptors on target cells of interest.

[0332] In some embodiments, the cargo is delivered to preferred cells for targeting, limiting off-target effects.

[0333] In other embodiments, PGT121 target cells include liver hepatocytes, lung epithelial cells, and lung endothelial cells.

[0334] Experiments by Elliott et al. disclosed a pDNA-mAb anti-influenza A virus hemagglutinin (HA) mouse IgGl antibody with initial expression of approximately 1 pg / mL anti-HA antibody in mice. After the utilization of an early-generation electroporation (EP) device, they showed the benefit of delivery and hyaluronidase pre-treatment of the muscle to increase muscle uptake of DNA, resulting in serum levels of> 15 pg / mL in mice and expression for 50 days (Elliott STC, et al., (2017). NPJ Vaccines, 2:18). As discussed in Andrews et al., anti-HA DMAbs were expressed to levels of > 10 pg / mL against A / HA and > 30 pg / mL against B / HA, with expression for 70 days using a pDNA construct uptake in muscle cells (Andrews CD, et al., (2017). Mol Ther Methods Clin Dev, 7:74-82).

[0335] Regarding Lyme disease, Wang et al. demonstrated an initial expression of the wt-DMAb averaging 5.7 pg / mL, and sequence optimizations further increased expression to 6.7 pg / mL (Wang Y, et al., (2019). J Infect Dis., 217(7): 1146-1150).

[0336] For anti-ebolavirus pDNA-mabs, Andrews et al. encoded the ZMapp cocktail antibodies into their pDNA-mAb platform, showing delivery of each individual mouse IgG2a antibody to amaximum of 10 pg / mL and 30 pg / mL for combined delivery and 13 weeks of in vivo expression (Andrews CD, et al., (2017). Mol Ther Methods Clin Dev., 7:74-82).

[0337] Similarly, in experiments conducted by Wise et al., they demonstrated that using an optimized delivery strategy with a 6-mg dose, divided across six injection sites, anti -HIV DMAb delivery can achieve in vivo expression levels as high as 34.4 pg / mL. Wise also revealed that the delivery of DMAbs, delivered alone or in combination, demonstrated long expression for 300 days and neutralized against the global panel viruses in an in vitro neutralization assay (Wise MC, et al., (2019). J Clin Investig).

[0338] In some embodiments, the antibody expression titer is 1000ng / mL-500 ng / ml per cell.

[0339] In some embodiments, the antibody expression titer is 500ng / mL-100 ng / mL per cell.

[0340] In some embodiments, the antibody expression titer is 100ng / mL-10ng / mL per cell.

[0341] In some embodiments, the antibody expression titer is 10ng / mL-1ng / mL per cell.

[0342] In some embodiments, the antibody expression titer is Ipg / ml per cell.

[0343] In some embodiments, the antibody expression titer is 2 pg / ml per cell.

[0344] In some embodiments, the antibody expression titer is 3 pg / ml per cell.

[0345] In some embodiments, the antibody expression titer is 4 pg / ml per cell.

[0346] In some embodiments, the antibody expression titer is 5 pg / ml per cell.

[0347] In some embodiments, the antibody expression titer is 6 pg / ml per cell.

[0348] In some embodiments, the antibody expression titer is 7 pg / ml per cell.

[0349] In some embodiments, the antibody expression titer is 8 pg / ml per cell.

[0350] In some embodiments, the antibody expression titer is 9 pg / ml per cell.

[0351] In some embodiments, the antibody expression titer is 10 pg / ml per cell.

[0352] In some embodiments, the antibody expression titer is greater than 20 pg / ml per cell.

[0353] In some embodiments, the amount of antibodies expressed are at a rate of 1000ng / mL-500 ng / ml, 500ng / mL-100 ng / mL, 100ng / mL-10ng / mL, 10ng / mL-1ng / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 30 pg / mL to 85 pg / mL or greater at 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 3 months, 6 months, 9 months, 1 year, or greater.PROMOTERS

[0354] In some embodiments, the pDNA construct contains a constitutive promoter.

[0355] In some embodiments, the pDNA construct contains an inducible promoter.

[0356] In some embodiments, the pDNA construct contains a cell-specific promoter.

[0357] In some embodiments, a cell-specific promoter drives expression exclusively in target cells to minimize any off-target effects.

[0358] In some embodiments, an inducible promoter drives expression in target cells when a particular stimulus is present, such as specific signalling cascades.

[0359] In some embodiments, the promoter is a heterologous inducible promoter selected from the group consisting of cysteine synthase (cysK), ORF upstream of cysK (cysZ), ATP sulfurylase (cysN), sulfate adenylyltransferase (cysD), adenylyl sulfate kinase (cysC), Periplasmic sulfate-binding protein (sbp), phosphoenolpyruvate carboxylase (ppc), phosphoenolpyruvate synthase (pps), pyruvate carboxylase (pyc), acetyl-CoA synthetase (acs), homoserine O-transsuccinylase (metA), cystathionine gamma-synthase (metB), cystathionine beta-lyase (metC), 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (metE), 5,10-methylenetetrahydrofolate reductase (metF), B12-dependent homocysteine-N5-methyltetrahydrofolate transmethylase (metH), methionine adenosyltransferase (metK), aspartokinase II / homoserine dehydrogenase II (metL), aspartate-semialdehyde dehydrogenase (asd), aspartate aminotransferase (aspC), aspartokinase III (lysC), pyruvate kinase I (pykA), pyruvate kinase II (pykF) formyltetrahydrofolate deformylase (purU), the operons cysPUWAM (periplasmic sulphate binding protein, a component of sulphate ABC transporter, a membrane bound sulphate transport protein, a sulphate permease and an 0-acetyl serine sulfhydralase), cysJIH (alpha and beta subunits of a sulfite reductase and an adenylyl sulfate reductase) and gcvTHP (Tetrahydrofolate dependent aminomethyl transferase, a glycine cleavage, carrier of aminomethyl group and a glycine dehydrogenase), phosphoglycerate dehydrogenase (serA), phosphoserine phosphatase (serB), phosphoserine aminotransferase (serC), serine hydroxymethyl transferase (glyA), acetate kinase (ackA), phosphotransacetylase (pta), pyruvate dehydrogenase El (ace), pyruvate dehydrogenase E2 (aceF), lipoamide dehydrogenase (Tpd), succinyl-CoA synthetase beta subunit (sucC), succinyl-CoA synthetase alpha subunit (sucD), phosphoenolpyruvate carboxykinase (pck), malate dehydrogenase (maeB), pyruvate oxidase (poxB), acetohydroxy acid synthase I large subunit (ilvB), acetohydroxy acid synthase I small subunit (ilvN), acetohydroxy acid synthase II large subunit (ilvG), acetohydroxy acid synthase II small subunit (ilvM), acetohydroxy acid synthase III large subunit (ilvl), acetohydroxy acid synthase III small subunit (ilvH), DAHP synthetase (aroF), DAHP synthetase (aroG), DAHP synthetase (aroH), homoserine kinase (thrB), threonine synthase (thrC), serine deaminase (sdaA),serine deaminase (sdaB), S-Adenosylmethionine decarboxylase (speD), ornithine decarboxylase (speC), arginine succinyltransferase (astA), dihydrodipicolinate synthase (dapA), malate dehydrogenase (mdh), malate dehydrogenase FAD / NAD(P)-binding domain (mqo), and citrate synthase (gltA).

[0360] In some embodiments, the promoter is an inducible promoter selected from araBAD, prpBCDE, rhaSR, xlyA, araA, araB, prpB, prpD, rhaA, rhaB, rhaD, xylA, xylB, scpA / sbm, argK / ygfD, scpB / ygfG, scpC / ygfH, rmlA, rmlB, rmlC, T7, anhydrotetracycline-inducible promoter, IPTG-inducible promoter, rhamnose-inducible promoter, arabinose-inducible promoter, a tetracycline-inducible promoter, a light-inducible promoter, a heat-inducible promoter, a phage shock promoter (PSP), ADH1, GALI, CaMKIIa,rtTA, tTA, TRE, LacO, or rm ID.

[0361] In some embodiments, the promoter is a constitutive promoter selectedfrom constitutive cytomegalovirus (CMV) promoter, a constitutive RNA polymerase III promoter, SP6, SV40, EF-1 alpha, PGK1, UBI, human beta actin, CAG, Ac5, Polyhedrin, TEF1, GDS, CaMV35S, or a UBC promoter.

[0362] In some embodiments, the pDNA construct includes a P2A self-cleavable linker.

[0363] In other embodiments, the pDNA construct includes a cleavable linker.

[0364] In other embodiments, the pDNA construct includes a non-cleavable linker.pDNA Expression

[0365] In certain embodiments, the disclosure provides compositions comprising plasmid DNA (pDNA) encoding a therapeutic polypeptide, RNA, or regulatory element. The pDNA may be formulated for administration to a human subject in a manner that enables in vivo cellular uptake and sustained expression of the encoded molecule. Suitable pDNA constructs may comprise a eukaryotic promoter, enhancer sequences, polyadenylation signals, and optional regulatory elements that enhance transcriptional efficiency or improve transcript stability.

[0366] In some embodiments, the pDNA is taken up by host cells, such as, but not limited to, pancreatic (3-cells, pancreatic a-cells, hepatocytes, adipocytes (white and brown), skeletal muscle fibers, hypothalamic neurons (including pome and agrp / npy neurons), enteroendocrine 1-cells, intestinal epithelial cells, kupffer cells, osteoblasts, osteoclasts, osteocytes, chondrocytes, satellite cells (muscle stem cells), mesenchymal stromal cells (mses), T cells (cd4+, cd8+, tregs), macrophages (ml / m2), dendritic cells, nk cells, fibroblasts, endothelial cells, erythroidprogenitors (bfu-e, cfu-e), megakaryocytes, neural stem cells, renal tubular epithelial cells, hematopoietic stem cells (hscs), b cells, plasma cells, airway epithelial cells, alveolar macrophages, neurons, oligodendrocytes, microglia, cardiac muscle cells, cardiomyocytes, cardiac fibroblasts, vascular smooth muscle cells, podocytes, neutrophils, thyroid follicular cells, ovarian granulosa cells, leydig cells, sertoli cells, pituitary cells (somatotrophs, gonadotrophs), and / or adrenal cortical cells, where it remains episomal and drives transient or semi-durable expression of the encoded gene product. Expression is generally dose-dependent and may persist for days to months, depending on promoter strength, delivery modality, and tissue turnover. In some embodiments, pDNA expression results in secretion of a therapeutic protein into the systemic circulation; in other embodiments, expression is localized within the transfected cells to mediate intracellular or cell-surface activity.

[0367] In some embodiments, the pDNA may be delivered as a naked DNA formulation, complexed with polymers or lipids, or encapsulated within lipid nanoparticles (LNPs) or other non-viral vectors. Administration routes include, but are not limited to, intramuscular, intradermal, subcutaneous, or intravenous injection. In some embodiments, electroporation may be used to enhance cellular uptake and transgene expression.

[0368] In preferred embodiments, the pDNA is delivered through the complex of a polymer described herein.

[0369] In certain embodiments, the disclosure provides polymer-pDNA complexes designed to enable efficient in vivo delivery and transient expression of a therapeutic transgene in human subjects. The pDNA may encode a therapeutic protein, peptide, or regulatory RNA and may include a eukaryotic promoter, enhancer sequences, and a suitable polyadenylation signal. In some embodiments, the encoded polypeptide comprises an N-terminal secretion signal peptide to promote extracellular release of the therapeutic product.

[0370] In some embodiments, the pDNA is delivered using a biodegradable or bioreducible polymer, as described herein, that condenses the nucleic acid via electrostatic interactions and facilitates uptake into host cells. Suitable polymers include poly(P-amino esters), polyethylenimine derivatives, polysaccharide-based polymers, polypeptide polymers, dendrimers, or block copolymers with tunable charge density and hydrophobicity. The polymer may provide protection from nuclease degradation, enhance endosomal escape, and enable controlled intracellular release of the pDNA.

[0371] In certain embodiments, the polymer-pDNA complex lacks a nuclear localization signal (NLS), relying instead on passive nuclear entry during mitosis or low levels of spontaneous diffusion into the nucleus. This configuration may be advantageous for achieving transient, nonintegrating transgene expression, while reducing the risk of prolonged or supraphysiologic expression.

[0372] In some embodiments, a nuclear localization signal (NLS) is included in the pDNA delivery.

[0373] In preferred embodiments, an NLS is not required to be included in the pDNA delivery.

[0374] Following uptake into target cells, the pDNA remains episomal and promotes transient transcription of the encoded product. The absence of an NLS may limit nuclear import efficiency, resulting in lower but more controllable expression kinetics. In embodiments where a secretion signal is present, the encoded protein is co-translationally directed to the endoplasmic reticulum and subsequently secreted into circulation, enabling systemic pharmacological activity while minimizing intracellular accumulation.

[0375] Expression duration may range from several days to several weeks depending on polymer degradation rate, uptake efficiency, promoter strength, and tissue turnover. In certain embodiments, the polymer is optimized to degrade rapidly into non-toxic byproducts, promoting fast clearance of both polymer and released pDNA from the body.

[0376] An effective amount of pDNA is an amount sufficient to generate therapeutically relevant expression of the encoded product in a human subject. Typical dose ranges may be from 0.1 mg to 20 mg of pDNA per administration, with single or multiple dosing regimens. Expression levels may be quantified by detecting the encoded protein in serum, measuring target engagement, or evaluating downstream pharmacodynamic biomarkers.

[0377] In some embodiments, the polymer is engineered for rapid systemic clearance to minimize prolonged tissue exposure. For example, the polymer may exhibit enhanced hydrophilicity, reduced molecular weight, or cleavable linkers that enable rapid depolymerization in physiological fluids. Clearance pathways may include renal filtration, hepatobiliary elimination, or degradation into small, water-soluble fragments that are rapidly excreted.

[0378] The pDNA may also be formulated at doses and in polymer complexes designed to ensure transient exposure, such that expression wanes as the polymer is cleared. In certainembodiments, both the polymer and the pDNA demonstrate half-lives enabling substantially complete systemic clearance within hours to a few days, while still permitting sufficient intracellular delivery to elicit a therapeutic effect.

[0379] In certain embodiments, the pDNA does not integrate into the host genome, minimizing the risk of insertional mutagenesis. Episomal maintenance leads to gradual dilution of the plasmid during cell division, providing a controlled and reversible expression profile. Safety may also be enhanced through the inclusion of tissue-specific promoters, inducible expression cassettes, or suicide-gene elements if desired.

[0380] In some embodiments, the pDNA does not integrate into the host genome and remains episomal until it is diluted through cell division or degraded intracellularly. The use of polymers lacking NLS elements further limits long-term nuclear persistence. The polymer may be selected or engineered to minimize immunogenicity, complement activation, or off-target organ accumulation.

[0381] In additional embodiments, tissue-specific promoters or inducible regulatory elements may be included within the pDNA to further limit off-target expression, enhance safety, or permit reversible gene expression.

[0382] In various embodiments, the therapeutic composition is administered at a dose sufficient to achieve a therapeutically effective level of the active agent. The dose may vary depending on the route of administration, formulation, subject characteristics, and desired pharmacological effect.

[0383] In some embodiments, the dose of administered pDNA is expressed as an absolute amount, a weight-per-body-weight amount, or on a per-surface-area basis. Non-limiting examples of suitable dose ranges include from about O. OOlmg / kg to about 0.05mg / kg, from about 0.05mg / kg to about O.lmg / kg, from about O.lmg / kg to about 0.5mg / kg, from about 0.5mg / kg to about Img / kg, from about Img / kg to about 2mg / kg, from about 2mg / kg to about 3mg / kg, from about 3mg / kg to about 4mg / kg, from about 4mg / kg to about 5mg / kg, from about 5mg / kg to about 6mg / kg, from about 6mg / kg to about lOmg / kg, from about lOmg / kg to about 20mg / kg, from about 20mg / kg to about 30mg / kg, from about 30mg / kg to about 40mg / kg, or from about 40mg / kg to about 50mg / kg.

[0384] In preferred embodiments, a suitable dose ranges from about 0. Img / kg to about 6mg / kg.

[0385] In some embodiments, dose selection accounts for the stability, clearance, and biodistribution of the active agent. For polymer-delivered pDNA or expressed proteins, suitable doses may reflect factors such as expression kinetics, secreted protein half-life, and clearance from systemic circulation.

[0386] In various embodiments, the pDNA maintains measurable presence and / or expression for a duration selected from about 1 month, about 2 months, about 3 months, about 4 months, or longer.PROTEIN EXPRESSION

[0387] In one illustrative embodiment, the polymer nucleotide delivery composition further comprises a targeting component for targeting to cells. In one aspect, the targeting component can be a nucleotide that is an RNA that forms a ‘stem-and-loop’ structure. In this aspect, the polymer nucleotide complex delivery composition can be designed so that the polynucleotide strands fold into three-dimensional structures via a series of highly tuned ‘stem-and-loop’ configurations. In this embodiment, the polymer nucleotide complex delivery composition can have a high affinity for protein receptors expressed on specific cells resulting in targeting of the polymer nucleotide complex delivery composition and the payload to the specific cells. In this embodiment, the polynucleotide that binds to the target cell receptor can bind in conjunction with a peptide aptamer. In another aspect, the polymer nucleotide complex delivery composition can be folded so that, in the presence of certain biomarkers such as cell receptors, microRNA, DNA, RNA or an antigen, the self-base pairs are disrupted and the polymer nucleotide complex delivery composition can unfold, resulting in the triggered release of the payload only in the presence of the specific biomarker. For example, a lock-and-key mechanism for triggered opening of a polymer nucleotide complex delivery composition (e.g., a DNA origami construct) has been demonstrated previously (Andersen, et al., Nature, Vol. 459, pages 73-76(2009)), incorporated by reference herein. In these embodiments, the use of the polymer nucleotide complex delivery composition to create three-dimensional structures that target cells and tissues allows for more efficient delivery of payloads with fewer side effects, since the polymer nucleotide complex delivery composition can have low immunogenicity, and the payload will be released only in the presence of RNA or peptide biomarkers, for example, that exist in the cytosol of target cells and tissues.

[0388] In the embodiment where a polymer nucleotide complex delivery composition is used, computer aided design tools can predict the nucleotide sequence necessary to produce highly engineered polymer nucleotide complex delivery compositions. For gene delivery, these polymer nucleotide complex delivery compositions offer the advantages of encapsulation efficiency, as the size and shape of the structure can be tailored to fit the cargo. In another aspect, loading efficiency can be increased by incorporating nucleic acid payloads into the encapsulating polymer nucleotide complex delivery composition itself.

[0389] An expression cassette included herein can further include transposon insertion sequences recognized by a transposase operatively linked to the DNA sequence and the sequence terminus distal to the bidirectional promoter.

[0390] A second contemplated aspect of the DNA regulatory cassette comprises a polynucleotide sequence that includes: (i) a regulatory polynucleotide sequence that encodes an antibody; (ii) optionally a selection marker polynucleotide sequence that encodes a protein that confers resistance to an anti-bacterial agent; and (iii) an internal ribosome entry site (IRES) operatively linked; (iv) a promoter that confers expression of these collective sequences; and (v) optionally a transposase binding site operatively linked to the terminus of the promoter not operatively linked to the regulatory polynucleotide sequence and another transposase binding site operatively linked to the terminus of the selection marker polynucleotide sequence. The promoter is operatively linked to the regulatory polynucleotide sequence and promotes expression both of the regulatory and selection marker polynucleotide sequences.

[0391] Illustrative restriction endonuclease recognition sites includes one or more sites for enzymes selected from the group consisting of BamHI, Bglll, BspEI, Mfel, Mlul, Ncol, Pmel, PstI, SacI, Sall, Spel, and Xhol as described in US20180105833. Preferably, two or more restriction endonuclease recognition sites are present.

[0392] A contemplated DNA expression cassette in a commercial setting as a product for sale typically utilizes a recognition site for a restriction endonuclease as at least one of the first polynucleotide sequence and the second polynucleotide sequence.

[0393] A contemplated expression cassette can further include transposon insertion sequences recognized by a transposase operatively linked to the sequence at a polynucleotide sequence terminus distal to the bidirectional promoter. A cell comprising a beforedescribed expression cassette in its chromosomal DNA is also contemplated.

[0394] Thus, another aspect of this invention can comprise a DNA cassette that is present as a portion of a vector. One aspect of a vector embodiment of the invention comprises a vector operably linked to a DNA expression cassette at least one of whose DNA sequences is comprised of a multiple cloning site. Preferably, the DNA sequence contains multiple cloning sites. This type of vector is particularly useful in a commercial environment where it can be sold to others who insert protein or polypeptide sequences of their choice. The vector operably linked to the expression cassette can also have DNA sequences of that expression cassette that encode a protein or polypeptide. Preferably, the DNA sequence encodes an antibody or antibody fragment thereof.

[0395] More recently, the Sleeping Beauty (SB) transposon has been used as a transposable element. This transposon was reconstructed from extinct transposase sequences obtained from genome DNA of salmon. It is a member of the Tcl / mariner class of transposons found in the genomes of some fish. The transposase genes found in fish have been inactive for more than 10 million years. Using the sequences of many inactive fish transposases, an approximation of an ancestral (and functional) transposase was designed and constructed.

[0396] The SB transposase is a polypeptide with an amino-terminal DNA-recognition binding domain that binds the direct repeats, a nuclear localization sequence, and a domain that catalyzes the cut-and-paste reactions that are transposition. The DNA-recognition domain has two paired box sequences that can bind to DNA and are related to various motifs found on some transcription factors. The catalytic domain has the hallmark amino acids that are found in many transposase and recombinase enzymes.

[0397] SB transposons have been developed as non-viral vectors to introduce recombinant genes into host cells and organisms, which avoids triggering the cells' defense mechanisms against viruses. The genetic cargo can be an expression cassette — a gene and associated elements that grant the ability to regulate the expression of the gene at a desired level. The SB transposons are integrated into host cells with greater ease and efficiency than plasmids.

[0398] A specific embodiment of the SB transposon has been described, for example, in U. S. Patent Publication No. 2015 / 0072064, which describes the SB transposon as a suitable vector for integrating transgenes into a genome.

[0399] In some embodiments, the DNA cassette contains a constitutive promoter.

[0400] In some embodiments, the DNA cassette contains a promoter that achieves inducible gene expression.

[0401] In some embodiments, the DNA cassette undergoes transcription and translation to express the protein construct.

[0402] In preferred embodiments, the protein construct expresses an antibody or antibody fragment thereof.

[0403] In some embodiments, the DNA cassette encodes a fusion protein comprising enzyme replacement therapy (ERT) enzymes and methods of use thereof for treating various diseases or conditions.

[0404] In some aspects, provided herein is a DNA construct encoding an Fc polypeptide that is linked to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof.

[0405] In some embodiments, the protein expression titer is 1000ng / mL-500 ng / ml per cell.

[0406] In some embodiments, the protein expression titer is 500ng / mL-100 ng / mL per cell.

[0407] In some embodiments, the protein expression titer is 100ng / mL-10ng / mL per cell.

[0408] In some embodiments, the protein expression titer is 10ng / mL-1ng / mL per cell.

[0409] In some embodiments, the protein expression titer is 1pg / mL per cell.

[0410] In some embodiments, the protein expression titer is 2 pg / mL per cell.

[0411] In some embodiments, the protein expression titer is 3 pg / mL per cell.

[0412] In some embodiments, the protein expression titer is 4 pg / mL per cell.

[0413] In some embodiments, the protein expression titer is 5 pg / mL per cell.

[0414] In some embodiments, the protein expression titer is 6 pg / mL per cell.

[0415] In some embodiments, the protein expression titer is 7 pg / mL per cell.

[0416] In some embodiments, the protein expression titer is 8 pg / mL per cell.

[0417] In some embodiments, the protein expression titer is 9 pg / mL per cell.

[0418] In some embodiments, the protein expression titer is 10 pg / mL per cell.

[0419] In some embodiments, the protein expression titer is greater than 20 pg / mL per cell.

[0420] In some embodiments, the amount of antibodies expressed are at a rate of 1000ng / mL-500 ng / ml, 500ng / mL-100 ng / mL, 100ng / mL-10ng / mL, 10ng / mL-1ng / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 30 pg / mL to 85 pg / mL or greater at 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 3 months, 6 months, 9 months, 1 year, or greater.GENE EDITING

[0421] Illustrative payloads for the polymer nanoparticle or nanostructure DNA delivery compositions described herein can include any one or a combination of compositions selected from the group comprising: nucleic acids (e.g., DNA or RNA), pDNA, oligodeoxyribonucleic acids (ODNs), dsDNA, ssDNA, antisense oligonucleotides, antisense RNA, siRNA, messenger RNA, guide RNA (e.g., small guide RNA), ribonucleoproteins, donor DNA strands used in the CRISPR / Cas9 system, and enzymes, such as CRISPR-associated enzymes, e.g., Cas9, enzymes used in other gene editing systems, such as ZFNs, custom designed homing endonucleases, TALENS systems, other gene editing endonucleases, and reverse transcriptase.

[0422] In another embodiment, the payload can be a nucleic acid construct (e.g., DNA or RNA) with a size selected from the group consisting of 3 kB or more, 3.1 kB or more, 3.2 kB or more, 3.3 kB or more, 3.4 KB or more, 3.5 kB or more, 3.6 kB or more, 3.7 kB or more, 3.8 KB or more, 3.9 kB or more, 4 kB or more, 4.1 kB or more, 4.2 kB or more, 4.3 kB or more, 4.4 KB or more, 4.5 KB or more, 4.6 kB or more, 4.7 kB or more, 4.8 kB or more, 4.9 KB or more, 5 kB or more, 5.1 kB or more, 5.2 kB or more, 5.3 kB or more, 5.4 kB or more, 5.5 kB or more, 5.6 kB or more, 5.7 KB or more, 5.8 kB or more, 5.9 kB or more, 6 kB or more, 6.1 kB or more, 6.2 kB or more, 6.3 kB or more, 6.4 kB or more, 6.5 kB or more, 6.6 kB or more, 6.7 kB or more, 6.8 KB or more, 6.9 kB or more, 7 KB or more, 7.1 kB or more, 7.2 kB or more, 7.3 kB or more, 7.4 KB or more, 7.5 KB or more, 7.6 kB or more, 7.7 KB or more, 7.8 KB or more, 7.9 KB or more, 8 kB or more, 8.1 kB or more, 8.2 kB or more, 8.3 kB or more, 8.4 KB or more, and 8.5 kB or more.

[0423] In various embodiments, the payload can be any one or more of the components of the CRISPR RNP system including a CRISPR-associated enzyme (e.g., Cas9), a short guide RNA (sgRNA), and a donor DNA strand. In an embodiment where the payload comprises Cas9, Cas9 can be fused to a deaminase. In yet another embodiment, the payload can comprise an sgRNA used for targeting an enzyme to a specific genomic sequence. In another aspect, the targeted enzyme can be a CRISPR-associated enzyme. In another illustrative aspect, the payload can comprise one molecule each of CRISPR / Cas9, an sgRNA, and a donor DNA strand in the DNA nanostructure, polymer nanoparticle or polymer nucleotide complex delivery compositions described herein. In another embodiment, the payloads can be nucleic acids used for homology directed repair or as transposable elements. In yet another embodiment, the payloads can be anyof the payloads described herein in the form of a plasmid construct, as described in US20240189338.

[0424] As discussed in Bartesaghi et al., many barriers have prevented the clinical utilization of mRNA for patients with chronic diseases. The major challenges include the instability of mRNA in vivo; it is susceptible to degradation by endo- and exo-nucleases. In addition, cellular membrane lipid bilayers block entry of highly charged, high molecular weight mRNAs. Further, innate immune pattern recognition receptors, like Toll-like receptors have evolved to mount an immune response to RNA invasion (Bartesaghi S, et al., (2022). Mol Ther Nucleic Acids, 28:500-513). Substantial investment has been made in the last decades to modify the structural elements of the mRNA (including modifications of the nucleotides and cap structure) to enable increased protein expression and reduced immunogenicity. However, there are still some fundamental challenges with the use of mRNA as therapeutics, including stability, duration of action, in vivo pharmacokinetic / pharmacodynamic (PK / PD), and effective delivery to the target cell type or tissue (Davies N, et al., (2021). Mol Ther Nucleic Acids, 24:369-384).

[0425] In one aspect, the polymer nucleotide complex delivery composition described herein can encapsulate a payload that is used for gene editing. In one aspect, the CRISPR / Cas9 system can be the payload and can be used for gene editing. In another embodiment, another gene editing system can be the payload, such as ZFNs, custom designed homing endonucleases, and TALENS systems. In the embodiment where the CRISPR / Cas9 system is the payload, the Cas9 endonuclease is capable of introducing a double strand break into a DNA target sequence. In this aspect, the Cas9 endonuclease is guided by the guide polynucleotide (e.g., sgRNA) to recognize and optionally introduce a double strand break at a specific target site into the genome of a cell. In this illustrative embodiment, the Cas9 endonuclease can unwind the DNA duplex in close proximity to the genomic target site and can cleave both target DNA strands upon recognition of a target sequence by a guide polynucleotide (e.g., sgRNA), but only if the correct protospacer-adjacent motif (PAM) is approximately oriented at the 3' end of the target. In this embodiment, the donor DNA strand can then be incorporated into the genomic target site. The CRISPR / Cas9 system for gene editing is well-known in the art.

[0426] In various embodiments, the payload can be any one or more of the components of the CRISPR RNP system including a CRISPR-associated enzyme (e.g., Cas9), a short guide RNA (sgRNA), and a donor DNA strand. In an embodiment where the payload comprises Cas9, Cas9can be fused to a deaminase. In yet another embodiment, the payload can comprise an sgRNA used for targeting an enzyme to a specific genomic sequence. In another aspect, the targeted enzyme can be a CRISPR-associated enzyme. In another illustrative aspect, the payload can comprise one molecule each of CRISPR / Cas9, an sgRNA, and a donor DNA strand in the polymer nanoparticle or polymer nucleotide complex delivery compositions described herein. In another embodiment, the payloads can be nucleic acids used for homology directed repair or as transposable elements. In yet another embodiment, the payloads can be any of the payloads described herein in the form of a plasmid construct.

[0427] In one aspect, the polymer polynucleotide complex delivery composition described herein can encapsulate a payload that is used for gene editing. In one aspect, the CRISPR / Cas9 system can be the payload and can be used for gene editing. In another embodiment, another gene editing system can be the payload, such as ZFNs, custom designed homing endonucleases, and TALENS systems. In the embodiment where the CRISPR / Cas9 system is the payload, the Cas9 endonuclease is capable of introducing a double strand break into a DNA target sequence. In this aspect, the Cas9 endonuclease is guided by the guide polynucleotide (e.g., sgRNA) to recognize and optionally introduce a double strand break at a specific target site into the genome of a cell. In this illustrative embodiment, the Cas9 endonuclease can unwind the DNA duplex in close proximity to the genomic target site and can cleave both target DNA strands upon recognition of a target sequence by a guide polynucleotide (e.g., sgRNA), but only if the correct protospacer-adjacent motif (PAM) is approximately oriented at the 3' end of the target. In this embodiment, the donor DNA strand can then be incorporated into the genomic target site. The CRISPR / Cas9 system for gene editing is well-known in the art.

[0428] In some embodiments, the polymer nanoparticle comprises nucleic acids. In some embodiments, the nucleic acids comprise DNA or RNA. In some embodiments, the nucleic acids comprise a ribonucleoprotein. In some embodiments, the payload nucleic acids are used for homology-directed repair or as transposable elements. In some embodiments, the payload nucleic acids comprise a short guide RNA (sgRNA) and a donor DNA strand. In some embodiments, the sgRNA is used for targeting an enzyme to a specific genomic sequence. In some embodiments, the payloads comprise a CRISPR-associated enzyme. In some embodiments, the targeted enzyme is a CRISPR-associated enzyme. In some embodiments, the payloads comprise a CRISPR-associated enzyme, a sgRNA, and a donor DNA strand. In someembodiments, the payloads comprise CRISPR / Cas9. In some embodiments, the payloads comprise CRISPR / Cas9, a sgRNA, and a donor DNA strand. In some embodiments, the payload comprises CRISPR / Cas9 and Cas9 is fused with a deaminase. In some embodiments, the payloads comprise a coding sequence for Cas9, an sgRNA, and a donor DNA strand in the form of a plasmid.

[0429] In another illustrative embodiment, the payload may include DNA segments that serve as nuclear localization signals, enhancing nuclear delivery of the polymer nanoparticles or polymer nucleotide complex delivery compositions upon endosomal escape. In another aspect, the payload may include a nucleotide sequence designed to bind as an aptamer to endosomal receptors, enhancing intracellular trafficking of the polymer nanoparticles or polymer nucleotide complex delivery compositions.

[0430] In another illustrative embodiment, the payload may include DNA segments that serve as nuclear localization signals, enhancing nuclear delivery of the polymer nanoparticle or polymer nucleotide complex delivery compositions upon endosomal escape. In another aspect, the payload may include a nucleotide sequence designed to bind as an aptamer to endosomal receptors, enhancing intracellular trafficking of the polymer nanoparticles or polymer nucleotide complex delivery compositions.

[0431] In some embodiments, optionally the nuclear localization signal is included in the pDNA operon.

[0432] In other embodiments, the nuclear localization signal is not present in the pDNA operon.

[0433] In another illustrative embodiment, the payload may include DNA segments that serve as nuclear localization signals, enhancing nuclear delivery of the polymer nanoparticles or polymer nucleotide complex delivery compositions upon endosomal escape. In another aspect, the payload may include a nucleotide sequence designed to bind as an aptamer to endosomal receptors, enhancing intracellular trafficking of the polymer nanoparticles or polymer nucleotide complex delivery compositions.

[0434] In some embodiments, the polymer nanoparticle delivers DNA cargo to the nucleus.

[0435] In some embodiments, the DNA cargo is transfected or transiently transfected into the host cell nucleus.

[0436] In preferred embodiments, the DNA cargo is not transfected or transiently transfected into the host cell nucleus.COMPLEXES

[0437] The polymers described herein may bind with a biological agent. The biological agent may be bound to the polymer by a variety of methods. In some embodiments, the biological agent is bound noncovalently to the polymer, e.g., electrostatically to the polymer. In some embodiments, the polymer may be complexed with the biological agent. In some embodiments, the polymer may be condensed with the biological agent. Without wishing to be bound to theory, the polymers described herein, including those bound, complexed, or condensed to negatively charged biological agents, may be able to evade the immune system by mimicking bacteria-like, less-foreign morphologies, thereby showing great promise as a multiplexable system.

[0438] The present disclosure provides a complex including a polymer described herein, e.g., of formula (I), (II), (III), or (IV) and a biological agent. In some embodiments, the biological agent is a negatively charged biological agent. The biological agent may include a therapeutic agent. The biological agent may include a nucleic acid, a peptide, a protein, or a small molecule. The biological agent may include a small molecule. Small molecules are compounds with low molecular weight that are capable of modulating biochemical processes to diagnose, treat, or prevent diseases. The present polymeric delivery systems may be used to transport smallmolecule therapeutics to cells.

[0439] The biological agent may include a small molecule. Small molecules are compounds with low molecular weight that are capable of modulating biochemical processes to diagnose, treat, or prevent diseases. The present polymeric delivery systems may be used to transport smallmolecule therapeutics to cells.

[0440] The biological agent may include a nucleic acid. Without wishing to be bound to theory, when negatively charged biological agents (e.g., nucleic acids such as RNA or other small molecule therapeutics), the circulation time of the cargo may be increased in vivo. The nucleic acid may be DNA, RNA, or chimeric. In some embodiments, the nucleic acid includes gRNA, mRNA (e.g., that encodes for proteins (fluorescent or therapeutic), tmRNA, tRNA, rRNA, siRNA, shRNA, PNA, ssRNA, dsRNA, pDNA (e.g., that encodes for proteins (fluorescent or therapeutic), ssDNA, dsDNA, a DNA: RNA hybrid molecule, DNA editing templates, miRNA, an artificial chromosome, oligonucleotide, a nucleic acid encoding a nuclease, cDNA, a PCR product, a restriction fragment, a ribozyme, an antisense construct, or a combination thereof.Nucleic acids may include modifications to the sugar, backbone, and / or base and may include synthetic or non-canonical bases.

[0441] A gRNA includes an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gRNA that hybridizes with a target nucleic acid sequence of interest. In various embodiments, DNA editing templates include an exogenous strand of DNA that bears homology arms to a section of genomic DNA that has been cut by a nuclease (for example, Cas9, TALEN or zinc finger) along with an intervening sequence between these homology arms that differs with the natural segment of genomic DNA that has been cut. This intervening segment serves as the template for repair of the cut genomic DNA and, in so doing, the cell corrects its own DNA to match that of the DNA template. The DNA template may be included in a single DNA expression vector that also encodes the nuclease. In various embodiments, DNA editing templates include an exogenous strand of DNA that bears homology arms to a section of genomic DNA that has been cut by a nuclease (for example, Cas9, TALEN or zinc finger) along with an intervening sequence between these homology arms that differs with the natural segment of genomic DNA that has been cut. This intervening segment serves as the template for repair of the cut genomic DNA and, in so doing, the cell corrects its own DNA to match that of the DNA template. The DNA template may be included in a single DNA expression vector that also encodes the nuclease.

[0442] siRNAs refer to a double-stranded interfering RNA. In addition to siRNA molecules, other interfering RNA molecules and RNA-like molecules may be used. Examples of other interfering RNA molecules that may to inhibit target biomolecules include, but are not limited to, short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), piwiRNA, Dicer- substrate 27-mer duplexes, and variants thereof containing one or more chemically modified nucleotides, one or more non- nucleotides, one or more deoxyribonucleotides, and / or one or more non-phosphodiester linkages. Typically, all RNA or RNA-like molecules that may interact with transcripts RISC complexes and participate in RISC-related changes in gene expression may be referred to as interfering RNAs or "interfering” RNA molecules.

[0443] Suitable interfering RNAs may readily be produced based on the well-known nucleotide sequences of target biomolecules. In various embodiments interfering RNAs that inhibit target biomolecules may include partially purified RNA, substantially pure RNA, synthetic RNA, recombinant produced RNA, as well as altered RNA that differs from naturally occurring RNAby the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include, for example, addition of non-nucleotide material, such as to the end(s) of the interfering RNAs or to one or more internal nucleotides of the interfering RNAs, including modifications that make the interfering RNAs resistant to nuclease digestion. Such alterations result in sequences that are generally at least about 80%, or more, or even 100% identical to the sequence of the target biomolecule. When the gene to be downregulated is in a family of highly conserved genes, the sequence of the duplex region may be chosen with the aid of sequence comparison to target only the desired gene. On the other hand, if there is sufficient identity among a family of homologous genes within an organism, a duplex region may be designed that would downregulate a plurality of genes simultaneously.

[0444] The N / P ratio of a complex is the ratio of positively charged polymer amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. The N / P character of a polymer / nucleic acid complex may influence complex properties, such as its net surface charge, size, and stability of the complex. The N / P ratio of the present complexes may be from 0 to 10, e.g., from 0 to 1, 0 to 2, 0 to 3, 0 to 4, 0 to 5, 0 to 6, 0 to 7, 0 to 8, 0 to 9, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, 9 to 10, or about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0445] Alternatively, or in addition, the biological agent may include a peptide. In various embodiments, peptide fragments include two or more amino acids covalently linked by at least one amide bond. For example, in some embodiments the peptide fragments may include pDNA-encoded fluorescent or therapeutic peptides.

[0446] Alternatively, or in addition, the biological agent may include a protein. In some embodiments, the protein may include an antibody. The antibody may be a monoclonal antibody.

[0447] In some embodiments, the protein includes a ribonucleoprotein. The ribonucleoprotein may include a ribosome, telomerase, ribonuclease P (Rnase P), a heterogeneous ribonucleoprotein particle (hnRNP), or a small nuclear ribonucleoprotein particle (snRNP). sgRNA may be chemically modified to improve stability and prevent intracellular degradation.

[0448] In some embodiments, the protein may include a nuclease. The nuclease may include a zinc finger nuclease (ZFNs), a transcription-activator like effector nucleases (TALEN), or a Cas protein. The Cas protein may be a Cas2, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Casio, CaslOd, CasF, CasG, CasH, CjCas9, SpCas9, Casl2, Casl3, Casl4, Cfpl, Casi, CaslB, Cpfl, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966, modified versions thereof, or combinations thereof. In some embodiments, the Cas protein is Cas9.

[0449] In some embodiments, the biological agent includes a nucleic acid and a nuclease. The negatively charged biological agent may be gRNA and a Cas protein, as in the CRISPR-Cas system. The CRISPR-Cas system is useful for precise editing of genomic nucleic acids (e g., for creating null mutations). For example, a composition containing only the guide RNA can be administered to an animal or cells transgenic for the Cas9 enzyme. Similar strategies may be used (e.g., zinc finger, transcription activator-like effectors (TALEs) or homing meganucleases).

[0450] The CRISPR-Cas system is known in the art for deleting, modifying genome sequences or incorporating transgenes. Transgene refers to any nucleotide sequence, particularly a DNA sequence, that is integrated into one or more chromosomes of a host cell by human intervention, such as by the methods of the present invention. For example, a transgene can be an RNA coding region or a gene of interest, or a nucleotide sequence, preferably a DNA sequence, that is used to mark the chromosome where it has integrated or may indicate a position where nucleic acid editing, such as by the CRISPR-CAS system, may occur. In this situation, the transgene does not have to include a gene that encodes a protein that may be expressed. CRISPR-Cas genome editing has rapidly emerged as a multi-faceted technology to enable gene insertion, deletion, activation, suppression, and even single-base editing of target genes within the nucleus of any cell. This highly efficient and facile technique has broad utility from white biotechnology and agriculture to biomedical research, pharmaceutics, and regenerative medicine.

[0451] Currently, the CRISPR / Cas9 system can be delivered in vitro, ex vivo, and in vivo in three different payload forms: i) pDNA that encodes Cas9 protein and / or sgRNA ii) mRNA that encodes for Cas9 nuclease and a separate sgRNA, or iii) a ribonucleoprotein (RNP) that consists of recombinant Cas9 protein precomplexed directly with a sgRNA.

[0452] CRISPR-Cas9 pDNA needs to enter the cellular nucleus to express, and consistent expression produces an overabundance of Cas9 protein, which can lead to increased off-target editing and mutagenesis. Researchers have utilized the CRISPR / Cas9 system in mRNA form to circumvent the barrier of nuclear entry, which has been reported with polymer-based nanoparticles. However, sgRNA often needs to be delivered separately, presenting challenges in trafficking kinetics of different payloads.

[0453] Direct delivery of CRISPR / Cas9 ribonucleoprotein (RNP) has several benefits, including precision in endonuclease dosing and the potential to avoid uncontrolled integration of the transgene into the cellular genome.DELIVERY

[0454] The non-viral delivery vehicles needed for translation of gene editing treatments can be synthesized with repeatable, versatile and scalable methods to meet GMP requirements. For these reasons, lipid nanoparticles have been used for most non-viral nucleic acid delivery vehicles in clinical trials. Although polymer nanoparticles have been less commonly used for nucleic acid delivery in the clinic, polymer synthesis methods provide these types of scalable manufacturing benefits in ways that cannot be matched by other biomaterials such as lipids. Given the robustness, versatility and sophistication of the use of polymer nanoparticles, it is an attractive route to synthesizing non-viral delivery vehicle alternatives to AAVs.

[0455] As is the case with lipid nanoparticles, polymer nanoparticle delivery vehicles are designed using cationic polymers that form complexes with negatively charged nucleic acids and enhance interaction with the negative resting potential of the cellular membrane, as discussed in US20240189338. However, the same positive surface charge may also lead to cytotoxicity in high enough doses. The polymer nanoparticles prepared according to the methods described herein are well tolerated by cells as provided in the examples described herein.

[0456] Cationic polymers are capable of forming polyplexes with negatively charged genes such as siRNA and plasmid DNA (pDNA) via electrostatic interaction (Rose VL, et al., (2017). Polym Chen, 8(2):353-360). These polyplexes with a size around a few hundred nanometers are further taken up by cells through endocytosis mechanisms, for example, phagocytosis, clathrin-mediated endocytosis and micropinocytosis or a fusion mechanism (Rinkenauer AC, et al., (2015). J Mater Chem B., 3(38):7477-7493). After cell uptake of the complex, the PNP complex has to evade acidic intracellular compartments such as lysosome and endosome via the proton-spongemechanism or the swelling and local mechanical disruption mechanism (Rinkenauer AC, et al., (2011). J Controlled Release, 15 l(3):220-228; Rehman ZU, et al., (2013). ACS Nano, 7(5):3767-3777). After the evasion of the cell degradation techniques, the PNP unpacks its DNA content through degradation, and the DNA cassette has to translocate to the cell nucleus for transcription and protein expression (Rinkenauer AC, et al., (2015). J Mater Chem B., 3(38):7477-7493).

[0457] Favorable characteristics of polymeric nanoparticle delivery such as nanoformulation, including size, charge, loading efficiency, stability, cytotoxicity, and gene-silencing ability, have been fully analyzed, suggesting PNP delivery as a strong candidate for delivering DNA into cells (Youngren SR, et al., (2013). Biomed Res Int, 858946).

[0458] In some embodiments, the therapeutic agent includes a drug. In some embodiments, the therapeutic agent includes genetic material. In some embodiments, the genetic material includes, without limitation, one or more of a non-viral gene vector, DNA, RNA, RNAi, a viral particle, combinations thereof, or the like.

[0459] In preferred embodiments, the polymer nanoparticle delivers genetic material such as DNA.

[0460] In some embodiments, a particle may deliver a cargo such as a biologically active cargo to treat a disease. In some embodiments, a particle is delivered to a desired location in a patient. In some embodiments, the particle is delivered to a patient where the particle crosses a cellular membrane into intracellular space and releases the cargo to treat a disease. The particle may release the cargo actively or passively.

[0461] In preferred embodiments, the polymer nanoparticle comprising the polymer polynucleotide complex delivers pDNA that translocates to the cell nucleus for transcription and protein expression

[0462] In particular embodiments, for parenteral administration, the antibodies or antigen binding fragments thereof are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. In certain embodiments, the antibodies or antigen-binding fragments thereof are formulated in suchvehicles at concentrations of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 50 mg / ml, about 1 mg / ml to about 25 mg / ml, about 1 mg / ml to about 10 mg / ml, e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml about 25 mg / ml about 50 mg / ml about 100 mg / ml, about 150 mg / ml, or about 200 mg / ml.

[0463] The dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection, the characteristics of the subject, and the subject's history. In particular embodiments, the amount of antibody or antigen-binding fragment thereof administered or provided to the subject is in the range of about 0.1 mg / kg to about 50 mg / kg of the subject's body weight. Depending on the type and severity of the infection, in certain embodiments, about 0.1 mg / kg to about 50 mg / kg body weight (e.g., about 0.1-15 mg / kg / dose) of antibody or antigen-binding fragment thereof may be provided as an initial candidate dosage to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of the therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

[0464] In certain embodiments, the pharmaceutical composition is formulated for subcutaneous injection and has a concentration of polymer and DNA of 50-500 mg / ml, 50-250 mg / ml, or 100 to 150 mg / ml, and a viscosity less than cP, less than 30 cP, less than 20 cP, or about 10 cP. In particular embodiments, the pharmaceutical compositions are liquids or solids. In particular embodiments, the pharmaceutical compositions are formulated for parenteral, e.g., intravenous, subcutaneous, or oral administration.

[0465] In one aspect, the polymer nanoparticle composition or the polymer nucleotide complex delivery composition may be administered, for example, directly into the blood stream of a patient, into muscle, into an internal organ, or can be administered in a topical formulation. In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular and subcutaneous delivery. In one embodiment, means for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0466] The formulation of and delivery methods of pharmaceutical compositions will generally be adapted according to the site and the disease to be treated. Exemplary formulations include,but are not limited to, those suitable for parenteral administration, e.g., intravenous, intra-arterial, intramuscular, 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 creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays.

[0467] In preferred embodiments, the composition described herein is delivered intravenously, with tropism to target cells such as kidney and liver cells.

[0468] In some embodiments, the composition described herein can be re-dosed. In certain embodiments, redosing occurs every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.FORMULATIONS

[0469] In various aspects, the present disclosure is directed to compositions comprising the polymer polynucleotide complexes described herein which have been dispersed in a solution. In some embodiments, the composition includes a plurality of polymers described herein which have been dispersed in a solution, e.g., a mixture of 2, 3, 4, 5, or more. When multiple polymers are employed, they may differ in the fraction of monomeric units, i.e., in o and n, or they may differ in the chemical structure of the monomeric units. In some embodiments, the complexes may be added to a liquid carrier and stored in liquid form until needed or may be dried and introduced into and dispersed in the liquid carrier before use, e.g., administration to a subject.

[0470] In some embodiments, the liquid carrier is a pharmaceutically acceptable carrier. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions. The formulation may include solvents, dispersing medium (containing, e.g., water, cell culture medium, buffers (e.g., phosphate buffered saline), polyol (for example, glycerol, propylene glycol, or liquid polyethylene glycol), wetting agents, emulsifying agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, colors, preservatives, or antioxidants. Other examples include DMSO and ethanol. Complexes may be stored frozen when suspended in a liquid.

[0471] The polymers, e.g., of formula (I), (II), (III), or (IV) may be ionized in dry or liquid formulation. For example, amine or basic nitrogen groups may be protonated. The polymers,e.g., of formula (I), (II), (III), or (IV) may be in salt form with one or more anions, e.g., acetate, ascorbate, benzoate, bicarbonate, bisulfate, carbonate, cholate, citrate, dihydrogen citrate, glycocholate, halide (such as F, Cl, Br, or I), hydrogen citrate, hydroxide, mandelate, methanesulfonate, nitrate, oxalate, p-toluenesulfonate, persulfate, phosphate, lactate, succinate, sulfate, sulfite, tartrate, taurocholate, or trifluoroacetate.METHODS OF USE

[0472] The present disclosure provides methods of contacting a cell with the herein-described polymer polynucleotide complex, wherein the complex is delivered into the cell.

[0473] For example, after a composition including the polymer and bound polynucleotide are contacted with a cell, the complex is taken up and internalized by the cell, and the polynucleotide disassociate partially or completely from the polymeric carrier.

[0474] The formulations described herein can be delivered to a cell or an organism via any administration mode known to a skilled practitioner. For example, the formulations described herein can be delivered via administration routes such as, but not limited to, in vitro, oral, intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous. In some embodiments, the compositions described herein are in a form that is suitable for injection. In other embodiments, the formulations described herein are formulated for oral administration. In various embodiments, for in vivo administration a delivery device can be used to facilitate the administration of any composition described herein to a subject, e.g., a syringe, a dry powder injector, a nasal spray, a nebulizer, or an implant such as a microchip, e.g., for sustained release or controlled release of any formulation described herein.

[0475] In various embodiments, the compositions may be administered to a cell in vitro by removing a cell from a subject, culturing the cells, applying to the cells a composition including polymer vehicles and bonded biological agent to deliver a therapeutic amount of the biological agent into at least a portion of the cells, and optionally re-introducing the cell to the subject.

[0476] In another embodiment, a tissue cell therapy technique may be used in which a tissue sample is removed from a subject, a composition including a polymer and a bonded biological agent is applied to the tissue to deliver a therapeutic amount of the biological agent to modify a selected cell or region of the tissue, and the modified tissue is transplanted into the subject.

[0477] In some embodiments, disclosed herein is a method of treating a disease, such as an infectious disease, wherein the disease is selected from the group consisting of Acute FlaccidMyelitis, Alpha-gal Syndrome, Anaplasmosis, Anthrax, Avian Influenza, Babesiosis, Botulism, Brucellosis, Campylobacteriosis, Carbapenem-resistant Infection, Chancroid, Chikungunya Virus Infection (Chikungunya), Chlamydia, Ciguatera (Harmful Algae Blooms (HABs)), Clostridium Difficile Infection, Clostridium Perfringens (Epsilon Toxin), Coccidioidomycosis fungal infection (Valley fever), COVID-19 (Coronavirus Disease 2019), Creutzfeldt-Iacob Disease, transmissible spongiform encephalopathy, Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue 1-4 (Dengue Fever), Diphtheria, E. coli infection, Shiga toxin-producing (STEC), Eastern Equine Encephalitis (EEE), Ebola Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Enterovirus Infection, Non-Polio (Non-Polio Enterovirus), Enterovirus Infection, D68 (EV-D68), Giardiasis (Giardia), Glanders, Gonococcal Infection (Gonorrhea), Granuloma inguinale, Haemophilus Influenza disease, Type B (Hib or H-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C / HCV), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes Zoster, zoster VZV (Shingles), Histoplasmosis infection (Histoplasmosis), Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Lead Poisoning, Legionellosis (Legionnaires Disease), Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma venereum infection (LGV), Malaria, Measles, Melioidosis, Meningitis, Viral (Meningitis, viral), Meningococcal Disease, Bacterial (Meningitis, bacterial), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Monkeypox Virus (Mpox), Multisystem Inflammatory Syndrome in Children (MIS-C), Mumps, Norovirus, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning, Ciguatera), Pediculosis (Lice, Head and Body Lice), Pelvic Inflammatory Disease (PID), Pertussis (Whooping Cough), Plague; Bubonic, Septicemic, Pneumonic (Plague), Pneumococcal Disease (Pneumonia), Poliomyelitis (Polio), Powassan, Psittacosis (Parrot Fever), Pthiriasis (Crabs; Pubic Lice Infestation), Pustular Rash diseases (Smallpox, monkeypox, cowpox), Q-Fever, Rabies, Respiratory Syncytial Virus (RSV), Ricin Poisoning, Rickettsiosis (Rocky Mountain Spotted Fever), Rubella, Including congenital (German Measles), Salmonellosis gastroenteritis (Salmonella), Scabies Infestation (Scabies), Scombroid, Septic Shock (Sepsis), Severe Acute Respiratory Syndrome (SARS), Shigellosis gastroenteritis (Shigella), Smallpox, Staphylococcal Infection, Methicillin-resistant (MRSA), Staphylococcal Food Poisoning, Enterotoxin-B Poisoning (Staph Food Poisoning), Staphylococcal Infection, VancomycinIntermediate (VISA), Staphylococcal Infection, Vancomycin Resistant (VRSA), Streptococcal Disease, Group A (Group A Strep), Streptococcal Disease, Group B (Strep-B), Streptococcal Toxic-Shock Syndrome, STSS, Toxic Shock (STSS, TSS), Syphilis (primary, secondary, early latent, late latent, congenital), Tetanus Infection, tetani (Lock Jaw), Trichomoniasis (Trichomonas infection), Trichinosis Infection (Trichinosis), Tuberculosis (Latent), Tuberculosis, Mycobacteriosis, Tularemia (Rabbit fever), Typhoid Fever, Group D, Typhus, Vaginosis bacterial (Yeast Infection), Vaping-Associated Lung Injury (e-Cigarette Associated Lung Injury), Varicella (Chickenpox), Vibrio cholerae (Cholera), Vibriosis (Vibrio), Viral Hemorrhagic Fever (Ebola, Lassa, Marburg), West Nile Virus, Yellow Fever, Yersinia (Yersinia), and Zika Virus Infection (Zika).

[0478] In some embodiments, disclosed herein is a method of treating cancer, including but not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma,chondrosarcoma, cancer of hematological tissues, and the like. Other nonlimiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer,bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.

[0479] In other embodiments, disclosed herein is a method of treating disease, such as an autoimmune disease. Examples of autoimmune disorders that may be treated by use of the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathyjuvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, Neuromyelitis optica (NMO), type 1 or immune -mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis / giant cell arteritis,transverse myelitis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

[0480] In other embodiments, disclosed herein is a method of treating disease, such as a respiratory disease, wherein the disease is selected from the group consisting Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic cough, Chronic obstructive pulmonary disease (COPD), Common cold, COVID-19, Croup, Cystic fibrosis, Hantavirus pulmonary syndrome, Influenza (flu), Interstitial lung disease (ILD), Long COVID, Lung cancer, Pertussis (whooping cough), Pleurisy, Pneumonia, Pulmonary embolism, Pulmonary fibrosis, Pulmonary hypertension, Pulmonary sarcoidosis, Respiratory syncytial virus (RSV), Sleep apnea, Sudden infant death syndrome (SIDS), Tuberculosis, Adult Cystic Fibrosis, Chest Wall Cancer, Emphysema, Cryptogenic Organizing Pneumonia (COP), Dyspnea, Empyema and Lung Infection, End-Stage Lung Disease, Eosinophilic Granulomatosis with Polyangiitis (EGPA), Germ Cell Tumors, Lymphangioleiomyomatosis (LAM), Lung Nodules, Lymphoma, Mesothelioma, Neurogenic Tumors, Occupational and Environmental Lung Diseases, Pectus Malformation, Pleural Effusion, Pneumonia, Pneumothorax, Pulmonary Vascular Disease, Respiratory Failure, Rib Fractures, Sarcoidosis, Severe Asthma, Thoracic Outlet Syndrome (TOS), Thymoma and Thymic Cancer, Tracheal Disorders, Tracheobronchomalacia, Tuberculosis, and Nontuberculosis Mycobacteria.

[0481] In some embodiments, the invention described herein is applicable towards CNS-targeting moieties, polycistronic expression cassettes, multi-modality approaches, cancers, viral infections, immune disorders, neurological disorders, cardiometabolic and autoimmune diseases (e.g., obesity, inflammation, diabetes, using therapeutic agents like secreted anti-TNF, GLP-1, anti-VEGF, and anti-C5 (aHUS, CHAPLE syndrome, NASH)), inborn errors of metabolism (e g., PKU, Al AT deficiency, ADA deficiency), lysosomal storage disorders (e g., Mucopolysaccharidoses (e.g., Hunter syndrome, Hurler syndrome, Sanfilippo syndrome, Scheie syndrome), Sphingolipidoses (e.g., Gaucher's disease, Tay-Sachs, Niemann-Pick disease, Fabry disease), and Glycoproteinoses (e.g., Pompe disease, Fucosidosis)), and coagulation disorders (e.g., using vectorized factors like Factor IX, VII, and VIII for managing coagulation disorders and / or Hemophilia).MACHINE LEARNING APPLICATIONS

[0482] In certain aspects, Generative Adversarial Network (GAN) can be employed to generate desired polynucleotide sequences encoding one or more antibodies having certain desirable characteristics. For instance, studies by Amimeur et al. bioRxiv 2020.04.12.024844 investigated Wasserstein-GANs (Gulrajani, I., Ahmed, F., Arjovsky, M., Dumoulin, V. & Courville, A. Improved training of wasserstein GANs. Adv. Neural Inf. Process. Syst. 2017-Decem, 5768-5778 (2017)) by training the network on 400,000 heavy- or light-chain sequences from human antibodies to generate regions of 148 amino acids of the respective chain. After initial training, by biasing further input data on desired properties (length, size of a negatively charged region, isoelectronic point, and estimated immunogenicity), the estimated properties of the generated examples shift in the desired direction. While it is not known what fraction of the 100,000 generated constructs is functional from the experimental validation, extensive biophysical characterization of two of the successful designs show promising signs of retaining the designed properties in vitro. An alternative study developing a Feedback GAN (FBGAN) framework extends this by iteratively generating sequences from a GAN, scoring them with an oracle, and replacing the lowest scoring members of the training set with the highest scoring generated sequences (Zachary Wu, Kadina E. Johnston, Frances H. Arnold, Kevin K. Yang, Protein sequence design with deep generative models, Current Opinion in Chemical Biology, Volume 65, 2021, Pages 18-27).

[0483] Another approach for model-based optimization is reinforcement learning (RL). The RL framework is typically applied when a decision maker is asked to choose an action that is available, given the current state. From this action, the state changes through the transition function with some reward. When a given state and action are independent of all previous states and actions (the Markov property), the system can be modeled with Markov decision processes. This requirement is satisfied by interpreting the protein sequence generation as a process wherein the sequence is generated from left to right. At each time step, the sequence as generated to that point (the current state) is used as a starting point, then select the next amino acid (the action), and add that amino acid to the sequence (the transition function). The reward remains 0 until generation is complete, and the final reward is the fitness measurement for the generated sequence. The action (picking the next amino acid) is decided by a policy network, which is trained to output a probability over all available actions based on the sequence thus far and the expected future reward. Notably, the transition function is simple (adding an amino acid) (Wu Z,et al., (2021). Curr Opin Chem Biol, 65:18-27). Nanite’s PGT121 polymer optimization using the SAYER platform can be seen in FIG. 6.KITS

[0484] In still further embodiments, the present disclosure concerns kits for use with the methods described above. The kits will thus comprise, in suitable container means for providing the pharmaceutical composition of the invention. The components of the kits may be packaged either in aqueous media or in lyophilized form. Such kits may also include instructions for using the pharmaceutical composition of the invention and notification of approved therapeutic use by regulatory agencies.

[0485] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the product may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.EXAMPLES

[0486] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice.

[0487] However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0488] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.EXAMPLE 1: SYNTHESIS OF POLYMER SCAFFOLDS

[0489] Synthesis of polymer reactive backbones:

[0490] A Schlenk flask was filled with Mg(BHT)2(THF)2 (0.25 equiv.), propargyl alcohol (1 equiv.), allyl glycidyl ether (n equiv.) and succinic anhydride (n equiv.), thereby forming reactive backbone Polymer 1.

[0491] A Schlenk flask was filled with Mg(BHT)2(THF)2 (0.25 equiv.), propargyl alcohol (1 equiv.), 2-((allyloxy)methyl)oxirane (n equiv.) and diglycolic anhydride (n equiv.), thereby forming reactive backbone Polymer 2.

[0492] A Schlenk flask was filled with Mg(BHT)2(THF)2 (0.25 equiv.), propargyl alcohol (1 equiv.), maleic anhydride (n equiv.) and propylene oxide (n equiv.), thereby forming reactive backbone Polymer 3.

[0493] Variable n is the targeted degree of polymerization. In various, non-limiting examples, reactive backbones investigated have had degrees of polymerization between 8 and 77. The solution was dissolved into toluene to a total monomer concentration of 5 M. The ampoule was sealed and heated at 100 °C for 48-72 hours. The resultant polymer was recovered by precipitation in excess heptane and dried under vacuum prior to use.

[0494] Synthesis of reactive backbone Polymer 1.

[0495] Synthesis of Reactive Backbone Polymer 2.Mg(BHT)2(THF)2, propargyl alcohol100 C toluene '-i

[0496] Synthesis of Reactive Backbone Polymer 3.O A^O Mg(BHT)2(THF)2, propargyl alcohol o100 C toluene

[0497] Synthesis of Reactive Backbone Polymer 4.O Mg(BHT)2(THF)2, propargyl alcohol...100 C toluene

[0498] Synthesis of polymer 1: First, 11-mercaptoundecanoic acid (MU A) was attached to the reactive backbone polymer using a UV light mediated thiol-ene reaction. Reactions were performed under UV light in a combination of DMSO and methanol in the presence of 0.05 molar equivalents (relative to the alkene) of 2,2-dimethoxy-2-phenylacetophenone (DMPA) to obtain compositions ranging from 0-50% functionality. Next, the reaction was flooded with 3 equivalents (relative to the alkene) of cysteamine hydrochloride and an additional 0.05 equivalents of DMPA prior to reacting again under UV light. The excess thiol was removed by dialysis in water and the sample was dried by lyophilization.

[0499] Polymer 1 was a polyampholyte with a degree of polymerization of 8, wherein 30% of the side chains are formed from 11-mercaptoundecanoic acid (MU A) and 70% of the side chains are formed from cysteamine (CYS).Polymer 1.

[0500] Synthesis ofM11163

[0501] Polymer M11163 was prepared from high-throughput synthesis runs using an Opentron OT-2 equipped with P300 and P1000 pipettes. All monomers were passed through an aluminiumoxide column prior to use and prepared in 3.5 M stock solutions. Liquid handling calibrations and accurate aspiration of viscous monomer stock solutions were accounted for using standard Opentron protocols. In general, to 4 mL glass vials in 24-well plates, pre-calculated volumes of monomer, initiator, chain transfer agent, and solvent (1.5 M total 3 monomer) were transferred. Stir bars were added before sealing the vials for sparging with inert anhydrous nitrogen gas for 20 min. Vials were then individually moved to a StarFish PolyBlocks Workstation (Heidolph, USA) for well-mixed heating at 60 °C. RAFT reactions were quenched by cooling to room temperature and exposure to air with the addition of 1 mL of 6 N HCl. Low volume aliquots of the crude sample were purified by gel filtration column and lyophilized for 2 days to afford light yellow free-flowing solid polymer.

[0502] Polymer size, uniformity and stability studies:Polymer size and uniformity was confirmed using dynamic light scattering (DLS). For DLS measurements, PNPs were formulated with PGT121 GenCircle. A DynaPro plate reader (Wyatt Technology) was used to quantify the size (hydrodynamic radius) and uniformity(poly dispersity) of PGT121 PNPs at various N / P ratios (FIG. 23). Polymers formed small (< 100 nm) and uniform (polydispersity index (PDI) < 0.30) particles that were stable across time. Figure 23(A) represents intensity-weighted distributions of PNPs approximately 45 minutes after PNP formulation. Figure 23B plots the hydrodynamic radius of PNPs across a range of N / P ratios from experiments in FIG. 23 A. Figure 23C plots polydispersity of PNPs across a range of N / P ratios from experiments in FIG. 23 A. n=3, error bars=SEM for data in FIG. 23B, and FIG.23C.

[0503] Transfection studies:

[0504] Polymer 1 was able to transfect Jurkat cells as shown in FIG. 19. The ability of Polymer 1 to transfect Jurkat cells can also be enhanced by complexing it with an antibody conjugate. Antibody conjugates were prepared at 100:1, 10:1, and 1:1 ratios, respectively. The addition of the antibody conjugate enabled transfection efficiencies that far surpassed those achieved by a benchmark LNP while maintaining or surpassing cell viability rates (FIG. 20). Polymer 8 was able to transfect 16HBE-G542X cells with as shown in FIG. 35, and primary human basal cells with dual reporter cargo (pDNA and mRNA) as shown in FIG. 36.

[0505] Polymers of different sizes with different compositions of MUA and CYS have also been synthesized and tested. The results of these studies are summarized in Table 5 and Table 6. A check mark ( ) is used to designate binding detected and an “X” being used to denote no binding detected. In Table 5, short refers to 8 monomer units, medium refers to 18 monomer units, long refers to 45 monomer units.Table 7: Polymers Synthesized Containing MUA and CYSPolymer Polymer ID pDNA mRNA HEK TF binding binding mRNA 1 SAcoAGE Short 30% MUA 70% CYS z z2 SAcoAGE Short 40% MUA 60% CYS does not bind 3 SAcoAGE Short 20% MUA 80% CYS z z - 4 SAcoAGE Short 10% MUA 90% CYS z Z - 5 SAcoAGE Medium 30% MUA 70% CYS z Z6 SAcoAGE Medium 20% MUA 80% CYS Z7 SAcoAGE Medium 10% MUA 90% CYS z Z Z8 SAcoAGE Long 30% MUA 70% CYS Z9 SAcoAGE Long 20% MUA 80% CYS z10 SAcoAGE Long 10% MUA 90% CYS z Z ZTable 6: pDNA binding and HEK transfection pDNApDNAPolymer binding HEK transfection pDNAD2592D42 ZD126 ZD51D164D2397 Z ZD2386 - D1167D4172D1169D5588pDNAPolymer binding HEK transfection pDNAD3859D3576D4764D4894DI 462D4175D156D4800D5580D1163D188D178D3560D3503D2598D5386D4895D5584D5585D5586D5587D5592D5366D11883D11344D6014D11910D11491DI 1273D6352DI 1052DI 1042D11867

[0506] Polymer 1 was able to transfect Jurkat cells. The ability of Polymer 1 to transfect Jurkat cells can also be enhanced by complexing it with an antibody conjugate. Antibody conjugates were prepared at 100:1, 10:1, and 1:1 ratios. The addition of the antibody conjugate enabled transfection efficiencies that far surpassed those achieved by a benchmark LNP whilemaintaining or surpassing cell viability rates. Jurkat cell transfection by LNP can be seen in FIG.20, and Jurkat cells transfection with mRNA can be seen in FIG. 19. The affinity of Polymer 1 with pDNA and mRNA can be seen in FIG. 18.To a dried 4 mL glass vial, the reactants (e.g., APMAm / MAETMA / PhHPMA, chain transfer agent, thermal initiator; 800 to 10 to 1 eq., respectively at desired mmol for targeted monomer compositions) were dissolved in acetic acid / methanol (1:1 v / v). The solution was sealed, degassed with nitrogen for at least 30 min, and set at 60 °C under constant stirring for 18 h. The reaction was quenched by cooling to room temperature and exposure to air. Crude solutions were purified with dialysis against filtered Milli-Q water for 3 cycles of 8 h each, followed by lyophilization to afford free flowing yellow solids.

[0507] Polymer stock solutions were prepared in 4 mL glass vials by direct dissolution of polymer in filtered Milli-Q water at 150 mM total concentration. Samples were vortexed for at least 30 sec. Polyplex assemblies were prepared by mixing polymer stock solutions with nucleic acid stock solutions at specific N / P ratios (number of nitrogen to phosphate between polymer and nucleic acid, respectively). Solutions are allowed to equilibrate for at least 30 min before further bioanalysis. Results of various A-2 polyplexes are shown below in Table 8, and all four A-2 polyplexes were successful at delivering a biological agent. The biodistribution of polymers can be seen in FIG. 5, FIG. 9, and FIG. 10. FIG 1-4 show the polymers and delivery efficiency for both pDNA and mRNA.Table 8: Components of A-2 (Y = yes, included; N = no, not included)Sample 1 Sample 2 Sample 3 Sample 4 (3 different (1 (1 (1 compositions) compositio composition composition n) ) )N-(3- Y YAmi nopropyl )methacry 1 ami dehydrochloride (APMAm)2-(Methacryloyloxy)ethyl) Y Y Y Y trimethylammonium chloride(MAETMA)3 -Phenoxy-2-hy droxypropyl Y Y Y Y methacrylate (PhHPMA)Poly(ethylene glycol) 2000 Da N Y N Y (PEG2k)Alpha lipoic acid (ALA) N N Y Y Successful at delivering Successful Successful Successful Successfulbiological agent?EXAMPLE 2: IN VITRO GENCIRCLE TRANSFECTION

[0508] HEK293T cells were grown in DMEM media supplemented with 10% fetal bovine serum and Penicillin-Streptomycin. Cells were maintained in a humidified incubator (Thermo Forma Direct Heat CO2 Incubator) set to 5% CO2 and 37 °C. Approximately 24 h before transfection, 7,500 HEK293T cells (in DMEM media supplemented with 10% fetal bovine serum) were seeded in each well of a poly-L-Lysine coated 96 well plate. PNPs were made by complexing polymer with 250 ng of pCMV-PGT121(P2A) or pCMV-Elipovimab(P2A) in 0.9% NaCl for 45 min and then 50 pL of solution was added dropwise to each well. 6-7 days after transfection, media was removed from each well, spun down at 2,000 RCF, and the resulting supernatant was flash frozen in liquid nitrogen for ELISA analysis.

[0509] HEK293T cell supernatant or mouse serum was assayed for the human IgG using either the Novus Human IgG ELISA Kit Colorimetric (NBP3-14790) or the Cayman Chemical Human Therapeutic IgGl ELISA Kit (Item No. 500910) according to the manufacturer’s protocols. Standard curves were plotted in Prism 10 software and interpolated to calculate Human IgG sample concentrations. FIG. 11 depicts the PGT121 GenCircle delivered in vitro, and FIG. 12 confirms the delivery of lead polymers in vitro. FIG. 7 shows the human IgG concentration inHEK293 supernatant. FIGs. 1-4 depict polymers complexed with biological agents of pDNA or mRNA evaluated for efficient delivery.EXAMPLE 3: IN VIVO GENCIRCLE TRANSFECTION

[0510] The in vivo animal work was performed by Biomere Biomedical Research Models, Inc. (Worcester, MA, USA) following standard protocols of lateral tail vein or intramuscular injections for gene delivery applications. All animal work was carried out in accordance with NIH Office of Laboratory Animal Welfare (OLAW) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) assurance. Briefly, 5-7 week-old female BALB / c mice were subject to intravenous injection or intramuscular injection of 50-200 pL prepared PNP working solution containing 0.75-2.0 mg / kg pCMV-PGT121(P2A) construct, monitored over time, and sacrificed after 24 h. Animals were intracardially perfused with PBS to remove blood before tissue collection and tissues were flash frozen and stored at -80 °C until processing.

[0511] Tissue DNA or RNA extraction was performed with DNeasy Blood & Tissue Kit (Qiagen, MD, USA) or RNeasy Mini Kit (Qiagen, MD, USA). RNA samples were DNase treated with TURBO DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove residual pDNA from RNA samples. TURBO-DNAse treated samples then underwent cDNA synthesis using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Custom TaqMan qPCR forward primers, reverse primers, and probes with specificity to PGT121, EGFP, or mouse B-actin were designed to quantify the relative delivery of pDNA construct to each mouse organ. qPCR was run with TaqMan™ Fast Advanced Master Mix for qPCR (Thermo Fisher Scientific, Waltham, MA, USA) on a QuantStudio 7 Pro Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the -AACT method. Three technical replicates were done for each qPCR. To calculate RNA -AACT values (-AACT*), -AACT signal from a no reverse transcriptase qPCR reaction from the same sample was subtracted from the raw RNA -AACT values. The average ACT values for a 5% glucose-only injection were used as a control injection to calculate -AACT values. In vivo-jetPEI serum levels and PGT121 serum and transfection levels can be viewed in FIG. 13, FIG. 14, and FIG. 15.EXAMPLE 4: RAG2 KO IV EXPERIMENT

[0512] The in vivo animal work was performed by Biomere Biomedical Research Models, Inc. (Worcester, MA, USA) following standard protocols of lateral tail vein or intramuscular injections for gene delivery applications. All animal work was carried out in accordance with NIH Office of Laboratory Animal Welfare (OLAW) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) assurance. Briefly, 5-7 week-old female rag2- / - KO mice supplied by Taconic were subject to intravenous injection or intramuscular injection of 50-200 pL prepared PNP working solution containing 0.75-2.0 mg / kg pCMV-PGT121(P2A) construct, monitored over time, and sacrificed after 24 h. Animals were intracardially perfused with PBS to remove blood before tissue collection and tissues were flash frozen and stored at -80 °C until processing.

[0513] Tissue DNA or RNA extraction was performed with DNeasy Blood & Tissue Kit (Qiagen, MD, USA) or RNeasy Mini Kit (Qiagen, MD, USA). RNA samples were DNase treated with TURBO DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove residual pDNA from RNA samples. TURBO-DNAse treated samples then underwent cDNA synthesis using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Custom TaqMan qPCR forward primers, reverse primers, and probes with specificity to PGT121, EGFP, or mouse B-actin were designed to quantify the relative delivery of pDNA construct to each mouse organ. qPCR was run with TaqMan™ Fast Advanced Master Mix for qPCR (Thermo Fisher Scientific, Waltham, MA, USA) on a QuantStudio 7 Pro Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the -AACT method. Three technical replicates were done for each qPCR. To calculate RNA -AACT values (-AACT*), - ACT signal from a no reverse transcriptase qPCR reaction from the same sample was subtracted from the raw RNA - CT values. The average ACT values for a 5% glucose-only injection were used as a control injection to calculate -AACT values. In vivo PGT121 tissue transfection and serum levels can be seen in FIG. 16. FIG.8 shows the IgG concentration in serum levels, and FIG. 17 shows the IM mouse injections. EXAMPLE 5: POLYMERS DELIVER DNA-ENCODED PGT121 VIA INTRAMUSCULAR INJECTION

[0514] ~7-week-old RAG2- / - mice (n=2) were injected in the right gastrocnemius and the left tibialis anterior at Day 0 with PNPs containing a GenCircle construct encoding PGT121 and serially bled. PGT121 serum protein levels were quantified at each time point using an anti-human IgG ELISA kit. The polymer, N / P ratio, and dose of PGT121 GenCircle is detailed for each group in FIG. 21. The final two groups were redosed at Day 8 with another 2.0 mg / kg of the PGT121 GenCircle.EXAMPLE 6: IN VITRO VALIDATION OF PNPS USING DNA CARGOS

[0515] The first goal was to validate the DNA-delivery efficiency and cytotoxicity of SAYER-optimized PNPs using a ZsGreen reporter plasmid. Polymer stocks were added to a fixed mass of ZsGreen plasmid at varying N / P ratios, the ratio of the positively charged nitrogren (N) groups from the cationic polymer to the negatively charged phosphate (P) groups from the backbone of the plasmid DNA. Simple electrostatic interactions between these molecules allow the PNPs to immediately self-assemble at room temperature. Approximately 45 minutes after PNP formulation, the particles were added to HEK293T cells, a common mammalian cell line used to investigate recombinant protein expression. For ZsGreen protein expression to occur, the PNPs must first enter the cell through electrostatic interactions with the negatively charged cell membrane. Following cellular entry via endocytosis, the PNPs must escape the endosome to avoid degradation within lysosomes. Once the PNPs are released into the cytosol, the plasmid must transport to the nuclear pore complex where it can finally enter the nucleus and begin transcription of the endcoded protein. We observed that all four lead polymers efficiently delivered the ZsGreen reporter plasmid in vitro across a range of different N / P ratios (FIG. 22A). For PNPs, there can be a tradeoff between transfection efficiency and toxicity when increasing the N / P ratio of the PNP, and thus the charge of the PNP. This tradeoff was quantified by multiplying the transfection efficiency and the cell viability of the PNP to obtain the effective transfection efficiency (FIG. 22B). All PNPs showed peak effective transfection efficiencies between N / P=3 and N / P=12. These differences in PNP function can be attributed to the structural differences of the polymer chemistries, and thus the structural differences in the PNPs. The observed effects are achieved by varying the properties of the polymer including but not limited to: the composition of cationic monomers, the composition of non-cationic monomers, the relative abundance of each monomer, the physical structure of the polymer (linear vs. branched), and the molecular weight of the polymer. All of the above factors contribute to the physical properties of the PNPs by changing the size, polydispersity, zeta potential (surface charge), and functional delivery capabilities. With even a small but repeatable dataset of in vitro transfection values, polymers (and PNPs) can be tuned to increase delivery efficiency using machine learningtechniques. Although these PNPs follow a general trend of increasing transfection efficiency with N / P ratio and eventual toxicity at higher N / P ratios, this is not always the case. The Sayer platform can be seen in FIG. 6.

[0516] Following validation that these PNPs could efficiently deliver a cytosolic reporter plasmid, the aim was to determine whether these same polymers could deliver a GenCircle encoding the anti-HIV antibody PGT121, a secreted protein. PGT121 is a human IgG (hlgG) antibody which consists of four polypeptides, two identical heavy chains and two identical light chains which self-assemble into a functional antibody molecule via disulfide bonds. To address the need to encode two distinct polypeptides to deliver a functional antibody, a previously developed single plasmid strategy was used (Patel A, et al., (2018). Cell Rep, 25: 1982-1993. e4. Briefly, a GenCircle was engineered to encode both the heavy chain and the light chain of PGT121, separated by a self-cleaving P2A linker. Once expressed, the PGT121 fusion protein self-cleaves, forming one heavy chain and one light chain. To validate this PGT121 GenCircle, PNPs were formulated and transfected into HEK293T cells. Following a period of 6-7 days posttransfection, supernatant was collected and run on an anti-hlgG ELISA to quantify the amount of secreted PGT121 antibody (FIG. 22C). All PNPs produced high levels of secreted PGT121 protein with similar trends to the delivery of the ZsGreen reporter. Due to the similar size of the ZsGreen plasmid (3.6 kb) and PGT121 GenCircle (3.6 kb) cargos, this result is not completely unexpected.

[0517] After confirming the high delivery efficiency and low toxicity of PNPs carrying the PGT121 GenCircle, the aim was to confirm the size and uniformity of the particles using dynamic light scattering (DLS). For DLS measurements, PNPs were formulated with PGT121 GenCircle. A DynaPro plate reader (Wyatt Technology) was used to quantify the size (hydrodynamic radius) and uniformity (poly dispersity) of PGT121 PNPs at various N / P ratios (FIG. 23). It was observed that these polymers formed small (< 100 nm) and uniform(poly dispersity index (PDI) < 0.30) particles that were stable across time. Confirmation that small, uniform, and stable PNPs were created to efficiently deliver PGT121 GenCircle without toxicity enabled the testing of whether these PNPs could deliver PGT121 in vivo. This stage was also the culmination of the machine learning-guided process which identified the selected polymers shown in FIG. 22-23.EXAMPLE 7: IN VIVO PNP DELIVERY OF A DNA-ENCODED ANTI-HIV ANTIBODY

[0518] Unlike an in vitro environment, there are many physiological barriers PNPs must overcome in vivo to successfully deliver genetic cargo to the nucleus. Different routes of PNP administration in vivo can significantly affect delivery efficiency, just as polymer structure can. To address this issue, two separate routes of administration commonly used to evaluate gene delivery vehicles in mice were investigated: systemic i.v. administration of PNPs into the bloodstream via lateral tail vein injection, and local administration of PNPs to skeletal muscle cells via i.m. injection. When PNPs are injected intravenously, they immediately encounter proteins in the bloodstream which can coat PNPs (the protein corona effect) and influence which organs and cell types they target (Dilliard s, et al., (2023). J Control. Rel., 361:361-372). When PNPs are injected intramuscularly, they reach their muscle cell targets immediately, but the intracellular barriers of post-mitotic multi-nuclear skeletal muscle cells pose new obstacles for gene expression.

[0519] The first goal of the investigation is whether the PNPs could deliver PGT121 GenCircle intravenously. When administered to a wild-type mouse, human antibodies like PGT121 elicit a predictable immune response. The mouse will produce anti-hlgG antibodies against the foreign human protein, complicating quantitative analysis of gene delivery efficiency. To circumvent this challenge, the RAG2 knock-out (RAG2- / -) immunodeficient mouse model was utilized to carry out in vivo gene delivery experiments. The RAG2 gene is necessary for V(D)J recombination during B and T cell development. RAG2- / - mice produce no mature B or T cells and therefore cannot mount an immune response against exogenous human antibodies. RAG2- / -mice were intravenously injected with PNPs containing 1.8 mg / kg of PGT121 GenCircle. Serial blood draws were taken from each animal out to day 28, processed to serum, and run on an anti-hlgG ELISA (FIG 24A, FIG. 24B). For all PNPs, a peak circulating PGT121 expression levels were observed in the 20-100 ng / mL range at 1-day post-injection. An approximate 1-2 week half-life of PGT121 in RAG2- / - mice was extrapolated when the GenCircle was delivered with the PNPs. These results suggest the PNPs quickly transfected cells in vivo, and that the GenCircle was subsequently silenced (CpG silencing) or cleared from the transfected cells. No toxicity was observed. It is anticipated that optimization of the GenCircle promoter / enhancers, 3’ UTRs, codon-optimization, signal peptide, or other genetic engineering could further boostserum PGT121 levels via i.v. PNP administration. Additionally, polymer / PNP optimization to specifically target high-transfection cell types for PGT121 DNA delivery would likely achieve clinically relevant levels of protein antibody expression.

[0520] The delivery performance of the PNPs were evaluated via an i.m. route of administration. Although targeting and biodistribution is localized with an i.m. injection, skeletal muscle cells have unique nanoparticle delivery challenges. The RAG2- / - mouse model was used to carry out the i.m. injection experiments. PNPs were formulated and injected into the right tibialis anterior and the left gastrocnemius muscles for all animals. Serial blood draws were taken out to day 56 post-injection. Interestingly, higher circulating PGT121 titers and different pharmacokinetic profiles were observed when compared to intravenous injection (FIG. 25 A). When PNPs were delivered to mouse skeletal muscle, we achieved peak serum PGT121 levels between 0.400-1.100 pg / mL with an average peak expression time of 28-42 days post-injection (FIG. 25B). The antibody expression profiles were unique to the polymer, N / P ratio, and dose of GenCircle administered (FIG. 25C-F). The PGT121 expression levels were high (> 1.0 pg / mL), durable (sustained across many weeks), but also transient (decreased from peak levels). No local toxicity at the site of injection was observed, nor any evidence of systemic toxicity. The ability of PNPs to tune antibody expression levels and profiles in vivo from DNA-encoded cargo based on polymer structure provides a foundation for further advances.

[0521] Finally, the redosability of the PNPs were investigated, which is a challenge for many gene delivery vehicles, such as AAVs. Following an initial i.m. 2.0 mg / kg GenCircle PNP injection on day 0, D3503 PNPs were intramuscularly redosed with 2.0 mg / kg GenCircle at day 8 (FIG. 25F). Higher and more persistent PGT121 serum levels in the redosing group were observed when compared to a single dose on day 0 (FIG. 25F). The ability to safely redose PNPs and improve circulating antibody titers in vivo is critical for genetically encoded antibody therapies where constant and predictable antibody levels are required. These data establish PNPs as safe, degradable, effective, and redosable gene delivery vehicles for DNA-encoded antibodies. Further, this DNA-encoded antibody PNP strategy could be applied to other clinically relevant secreted proteins.EXAMPLE 8: IN VITRO TRANSFECTION OF DNA-ENCODED OSTEOCALCIN PEPTIDE

[0522] For measurement of in vitro secreted protein levels from cell supernatant, HEK293T transfections were performed as previously described, with the exception that the DNA cargo used in the transfection was a plasmid encoding either pCMV-Osteocalcin-FLAG-Fc or pCMV-Osteocalcin-FLAG-ABD (albumin binding domain). Media was removed at 3 days posttransfection and spun down at 2000 RCF. The resulting supernatant was either frozen at -80C or immediately analyzed by an MSD immunoassay.

[0523] For the MSD immunoassay, the R-PLEX Human Osteocalcin Assay (MSD Catalog No. K151ZIR-2) was used. The assay was performed according to the protocol in the R-PLEX Singleplex Insert. Plates were read using a MESO SECTOR S 600MM instrument (MSD) and data was analyzed using Discovery Workbench (MSD) and Prism (GraphPad) software. Each data point was generated using n=3 replicates (FIG. 26), plasmid maps can be seen in FIG. 37 and FIG. 38.EXAMPLE 9: IN VIVO TRANSFECTION OF DNA-ENCODED PEPTIDES AND SMALL PROTEINS

[0524] All in vivo work was performed by Biomere Biomedical Research Models, Inc.(Worcester, MA, USA) and all study protocols were approved by an Institutional Animal Care and Use Committee (IACUC). PNPs were formulated as previously described using a polymer and plasmid DNA cargo encoding one of the following fusion proteins: GLPl-ABD-6xHis, FGF21-ABD, or Follistatin-Fc-6xHis, where ABD is an albumin binding domain and Fc is the Fc region of human IgG. PNPs were delivered intramuscularly into 5-7-week-old female C57B1 / 6J mice at a dose level of 1.0 or 1.5 mg / kg. Blood draws were taken 1, 3, 5, 7, 14, and 21 days post-injection, converted to either plasma or serum, then flash-frozen and shipped to Nanite for analysis. Plasma or serum was analyzed by MSD immunoassay, using either a custom MSD assay for GLP- 1, the U-PLEX Human FGF-21 Assay (MSD Catalog No. K 1515WK- 1 ), or the R-PLEX Human Follistatin Assay (MSD Catalog No. K151E5R-2). In Figure 27, each data point is generated using 1 technical replicate from n=l mouse. A plasmid map can be seen in FIG. 39. In Figure 28, each data point is generated using 4 technical replicates from n=l mouse. A plasmid map can be seen in FIG. 40. In Figure 29, each data point is generated using 2 technical replicates from each of n=3 mice. A plasmid map can be seen in FIG. 41.EXAMPLE 10: IN VIVO DELIVERY OF DB-DNA-FLUC

[0525] All in vivo work was performed by Biomere Biomedical Research Models, Inc.(Worcester, MA, USA) and all study protocols were approved by an Institutional Animal Care and Use Committee (IACUC). PNPs were formulated in 5% glucose as previously described using a polymer and a double-stranded linear DoggyBone-DNA (db-DNA) cargo (Touchlight, Hampton, UK) encoding firefly luciferase. db-DNA-CMV-fLuc (dbO 100-022) and db-DNA-CAG-fLuc constructs were first buffer exchanged from Tris buffer to nuclease-free water prior to complexation with polymers. PNPs were delivered via bilateral subcutaneous flank injections targeting the inguinal white adipose tissue (iWAT) into 7-8-week-old female C57B1 / 6J or BALB / c mice at a dose level of 0.1 to 2.5 mg / kg at a volume of 7.5 mL / kg. db-DNA dosages in all figures are reported per injection and not the total db-DNA dose. Mouse body weights were taken on Day 0 (prior to injection) and on following days, as reported in Figures 3 IB and 32B. Plasmid maps can be seen in FIG. 42 and FIG. 43.EXAMPLE 11: IN VIVO AND EX VIVO I VIS IMAGING

[0526] Prior to in vivo IVIS imaging, animals were shaved on the dorsal regions to remove hair. Dorsal in vivo IVIS imaging was conducted on all animals ~15 minutes following a 150 mg / kg single intraperitoneal injection at 10 mL / kg. For ex vivo IVIS imaging, animals were euthanized following in vivo IVIS imaging and the liver, left iWAT, and right iWAT were removed and imaged again to confirm the specificity of in vivo IVIS signal. All IVIS images were taken using auto-image settings. For IVIS image analysis, ROIs (shown in overlay images as white boxes or ovals) were placed around the dorsal part of the animal or the entire tissues for in vivo and ex vivo IVIS imaging, respectively. Total flux (photons / second) within the ROIs was quantified and reported in all IVIS figures. In vivo IVIS data is shown in Figures 30A, 30B, 31 A, 32A, 32B, 33A, 33B, 34A, and 34B. Ex vivo IVIS data is shown in Figure 34C. Plasmid maps can be seen in FIG. 42 and FIG. 43.EXAMPLE 12: EX VIVO QPCR

[0527] For quantitative real-time PCR (qPCR) analysis of db-DNA tissue biodistribution, animals were euthanized and liver, left iWAT, and right iWAT were removed and flash frozen. Tissue DNA extraction was performed with a DNAeasy Blood & Tissue Kit (Qiagen, MD, USA). Custom primers with specificity to mouse beta-actin and firefly luciferase were designed to quantify the relative delivery of db-DNA-fLuc to each organ. qPCR was run with PowerUP SYBR Green Master Mix for qPCR (Thermo Fisher Scientific, Waltham, MA, USA) on aQuantStudio 7 Pro Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Raw CT values were analyzed using the - AACT method and reported as RQ values (2-ΔΔCT). Ex vivo qPCR data is shown in Figures 31C and 34D.EXAMPLE 13: CGAS-STING CYTOKINE ANALYSIS

[0528] For cGAS-STING cytokine analysis, blood draws were taken ~24 hours pre-PNP-inj ection and 4 hours post-PNP-inj ection, converted to plasma, then flash-frozen and shipped to Nanite for analysis. Plasma was analyzed by a custom Biomarker Group 1 U-PLEX MSD Immunoassay (K15069M, Meso Scale Diagnostics, Rockville, MD, USA) for all analytes reported in Figure 33C.REFERENCES

[0529] The references provided herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.SEQUENCE DISCLOSURESEQ ID SEQUENCE DESCRIPTION NO:1 ATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCT PGT121 coding ATTCCATGCCACCCAGGCCCAGATGCAGTTACAGGAG sequence TCGGGCCCCGGACTGGTGAAGCCTTCGGAAACCCTGT CCCTCACGTGCAGTGTGTCTGGTGCCTCCATAAGTGA CAGTTACTGGAGCTGGATCCGGCGGTCCCCAGGGAA GGGACTTGAGTGGATTGGGTATGTCCACAAAAGCGG CGACACAAATTACAGCCCCTCCCTCAAGAGTCGAGTC AACTTGTCGTTAGACACGTCCAAAAATCAGGTGTCCC TGAGCCTTGTGGCCGCGACCGCTGCGGACTCGGGCAA ATATTATTGCGCGAGAACACTGCACGGGAGGAGAAT TTATGGAATCGTTGCCTTCAATGAGTGGTTCACCTAC TTCTACATGGACGTCTGGGGCAATGGGACTCAGGTCA CCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCAGCAACACCAAGGTGGA CAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTA CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAAAGAGCCAGACGGGGCTCTGGCGCCA CCAACTTCTCCCTTCTGAAGCAAGCAGGGGACGTTGAAGAAAACCCTGGCCCTATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGCCACCCAGGCCTCCGA TATATCTGTGGCCCCAGGAGAGACGGCCAGGATTTCC TGTGGGGAAAAGAGCCTTGGAAGTAGAGCTGTACAA TGGTATCAACACAGGGCCGGCCAGGCCCCCTCTTTAA TCATATATAATAATCAGGACCGGCCCTCAGGGATCCC TGAGCGATTCTCTGGCTCCCCTGACTCCCCTTTTGGGA CCACGGCCACCCTGACCATCACCAGTGTCGAAGCCGG GGATGAGGCCGACTATTACTGTCATATATGGGATAGT AGAGTTCCCACCAAATGGGTCTTCGGCGGAGGGACC ACGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCT CGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCA AGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGAC TTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCA GATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACC ACACCCTCCAAACAAAGCAACAACAAGTACGCGGCC AGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAG TCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAA GGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAA TGTTCATGA ATGGGGTGGTCGTGCATCATCCTGTTCCTCGTGGCTA elipovimab CTGCCACGGGAGTACACAGTCAGATGCAGTTGCAGG coding sequence AGTCTGGCCCTGGTCTGGTTAAGCCTTCTGAAACTCT GAGCTTAACCTGCAGCGTGTCAGGCGCCAGCATTTCT GATTCGTACTGGAGCTGGATCAGGAGAAGCCCTGGG AAGGGCCTGGAGTGGATTGGCTACGTGCACAAATCT GGTGACACAAATTACAACCCCAGCCTGAAGTCCAGA GTTCACTTATCCCTGGACACAAGCAAGAACCAGGTGT CTCTATCTCTCACCGGCGTGACCGCTGCAGACTCCGG AAAGTACTATTGTGCTCGGACCTTACATGGACGGCGT ATCTATGGAATAGTAGCCTTCAATGAATGGTTTACTT ACTTCTACATGGATGTGTGGGGAACGGGGACACAGG TGACCGTCAGTTCAGCCAGCACCAAAGGACCCTCCGT GTTTCCTCTTGCCCCTTCTTCCAAAAGCACCTCCGGTG GCACAGCAGCCCTTGGCTGCCTGGTCAAGGATTATTT CCCTGAGCCCGTAACTGTGTCCTGGAACTCTGGCGCA CTGACCAGTGGGGTGCATACCTTTCCAGCCGTACTCC AAAGCAGCGGCTTGTACTCCCTCTCCAGTGTGGTGAC AGTGCCTAGTTCGTCGCTGGGCACCCAGACTTATATC TGCAACGTGAACCACAAGCCGAGCAACACAAAGGTA GACAAGAAGGTGGAGCCCAAGTCCTGTGACAAAACC CACACATGCCCCCCCTGTCCCGCCCCGGAGCTGCTGG CCGGCCCAGATGTCTTCTTATTCCCTCCCAAACCAAA GGACACCCTGATGATCAGTCGGACACCAGAGGTAAC GTGTGTGGTTGTGGACGTGAGCCACGAAGACCCCGA GGTGAAGTTCAACTGGTACGTGGATGGCGTTGAGGTCCACAATGCGAAGACCAAACCCAGAGAAGAACAGTATAACTCCACATACAGAGTCGTGTCAGTGCTGACCGTCC TCCACCAGGACTGGCTCAACGGCAAGGAGTACAAAT GCAAAGTATCGAACAAGGCCCTTCCCCTGCCTGAGGA AAAGACTATCTCCAAGGCAAAAGGTCAGCCCCGAGA ACCACAAGTCTACACATTGCCCCCTTCGAGAGAAGAG ATGACCAAAAATCAAGTAAGTCTGACTTGCCTGGTGA AGGGCTTCTATCCATCGGACATAGCAGTCGAATGGGA GTCCAATGGCCAGCCAGAAAACAACTACAAGACCAC CCCCCCCGTGCTGGACAGTGACGGGAGCTTTTTCCTC TATAGTAAGCTGACCGTGGATAAGAGCAGGTGGCAG CAGGGAAATGTGTTCTCGTGCTCTGTGCTGCATGAAG CCCTCCACTCTCATTACACTCAGAAGTCTCTCTCCCTG AGTCCTGGGAAGAGAGCCCGTAGAGGAAGCGGGGCC ACCAACTTTAGCCTGCTGAAACAGGCCGGCGATGTCG AAGAAAACCCTGGTCCCATGGGCTGGAGCTGCATCAT TCTCTTCCTAGTGGCCACAGCCACCGGTGTTCACTCCT CGGATATTAGTGTGGCCCCTGGAGAGACAGCTCGGAT CTCTTGTGGGGAGAAGAGCCTGGGCAGTAGAGCTGT CCAGTGGTACCAGCACCGGGCCGGGCAGGCTCCTAG TTTGATCATCTACAATAACCAAGACCGCCCTTCCGGC ATTCCAGAGCGCTTCAGCGGCTCCCCTGATAGTCGGC CAGGCACTACGGCCACACTGACCATCACCAGTGTCGA GGCTGGCGATGAAGCAGACTATTACTGCCACATCTGG GATTCCCGGGTGCCTACCAAGTGGGTGTTTGGAGGGG GCACCACGCTCACCGTGCTGGGTCAGCCTAAAGCTGC CCCCAGCGTGACTCTGTTCCCTCCAAGCAGCGAGGAG TTGCAAGCCAACAAAGCCACGCTGGTTTGCCTCATCA GCGACTTTTACCCGGGTGCCGTCACGGTGGCCTGGAA GGCTGACAGCAGCCCTGTGAAAGCTGGCGTGGAAAC CACCACACCATCTAAGCAGTCCAACAACAAGTATGCT GCTTCGAGCTACCTGTCTCTGACCCCAGAACAATGGA AATCCCACAGATCCTACAGCTGCCAGGTAACCCATGA GGGCAGTACGGTAGAGAAGACCGTGGCCCCCACAGA GTGCTCATGA EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWV Adalimumab RQAPGKGLEWVSAITWNSGHIDADSVEGRFTISRDNAK Heavy Chain NSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI< FNWYVDGVEVHNAI< TI< PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPP SRDELTKNQ VSLTCL VKGF YP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SC SVMHE ALHNHYTQKSLSLSPGK DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQ Adalimumab KPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSL Light Chain QPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECgaagtgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgcg Adalimumab cctgagctgcgcggcgagcggctttacctttgatgattatgcgatgcattgggtgcgcca Heavy Chain ggcgccgggcaaaggcctggaatgggtgagcgcgattacctggaacagcggccatat tgattatgcggatagcgtggaaggccgctttaccattagccgcgataacgcgaaaaaca gcctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcg aaagtgagctatctgagcaccgcgagcagcctggattattggggccagggcaccctgg tgaccgtgagcagcgcgagcaccaaaggcccgagcgtgtttccgctggcgccgagca gcaaaagcaccagcggcggcaccgcggcgctgggctgcctggtgaaagattattttcc ggaaccggtgaccgtgagctggaacagcggcgcgctgaccagcggcgtgcatacctt tccggcggtgctgcagagcagcggcctgtatagcctgagcagcgtggtgaccgtgccg agcagcagcctgggcacccagacctatatttgcaacgtgaaccataaaccgagcaaca ccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgcc gtgcccggcgccggaactgctgggcggcccgagcgtgtttctgtttccgccgaaaccg aaagataccctgatgattagccgcaccccggaagtgacctgcgtggtggtggatgtgag ccatgaagatccggaagtgaaatttaactggtatgtggatggcgtggaagtgcataacgc gaaaaccaaaccgcgcgaagaacagtataacagcacctatcgcgtggtgagcgtgctg accgtgctgcatcaggattggctgaacggcaaagaatataaatgcaaagtgagcaacaa agcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggccagccgcgcg aaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaaaaccaggtga gcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgggaaagc aacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgatggc agcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaacgtg tttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctgagc ctgagcccgggcaaa gatattcagatgacccagagcccgagcagcctgagcgcgagcgtgggcgatcgcgtg Adalimumab accattacctgccgcgcgagccagggcattcgcaactatctggcgtggtatcagcagaa Light Chain accgggcaaagcgccgaaactgctgatttatgcggcgagcaccctgcagagcggcgt gccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagc ctgcagccggaagatgtggcgacctattattgccagcgctataaccgcgcgccgtatac ctttggccagggcaccaaagtggaaattaaacgcaccgtggcggcgccgagcgtgttt atttttccgccgagcgatgaacagctgaaaagcggcaccgcgagcgtggtgtgcctgct gaacaacttttatccgcgcgaagcgaaagtgcagtggaaagtggataacgcgctgcag agcggcaacagccaggaaagcgtgaccgaacaggatagcaaagatagcacctatagc ctgagcagcaccctgaccctgagcaaagcggattatgaaaaacataaagtgtatgcgtg cgaagtgacccatcagggcctgagcagcccggtgaccaaaagctttaaccgcggcgaatgcMALPVTALLLPLALLLHAARPDIQLTQSPASLAVSLGQR blinatumomabAT1SCKASQSVDYDGDSYLNWYQQ1PGQPPKLL1YDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQST EDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQS GAELVRPGSS VKISCKASGYAF S S YWMNWVKQRPGQG LEWIGQIWPGDGDTNYNGKFKGKATLT ADES S ST AYM QLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGT TVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGY TFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKF KDK ATLTTDK S S S T A YMQL S SLT SED S A V Y YC ARY YDD HYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDI QLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKS GTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSME AEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHHatggcgctgccggtgaccgcgctgctgctgccgctggcgctgctgctgcatgcggcgc blinatumomab gcccggatattcagctgacccagagcccggcgagcctggcggtgagcctgggccagc gcgcgaccattagctgcaaagcgagccagagcgtggattatgatggcgatagctatctg aactggtatcagcagattccgggccagccgccgaaactgctgatttatgatgcgagcaa cctggtgagcggcattccgccgcgctttagcggcagcggcagcggcaccgattttacc ctgaacattcatccggtggaaaaagtggatgcggcgacctatcattgccagcagagcac cgaagatccgtggacctttggcggcggcaccaaactggaaattaaaggcggcggcgg cagcggcggcggcggcagcggcggcggcggcagccaggtgcagctgcagcagag cggcgcggaactggtgcgcccgggcagcagcgtgaaaattagctgcaaagcgagcg gctatgcgtttagcagctattggatgaactgggtgaaacagcgcccgggccagggcctg gaatggattggccagatttggccgggcgatggcgataccaactataacggcaaatttaa aggcaaagcgaccctgaccgcggatgaaagcagcagcaccgcgtatatgcagctgag cagcctggcgagcgaagatagcgcggtgtattttgcgcgcgccgcgaaaccaccacc gtgggccgctattattatgcgatggattattggggccagggcaccaccgtgaccgtgag cagcggcggcggcggcagcgatattaaactgcagcagagcggcgcggaactggcgc gcccgggcgcgagcgtgaaaatgagctgcaaaaccagcggctatacctttacccgcta taccatgcattgggtgaaacagcgcccgggccagggcctggaatggattggctatatta acccgagccgcggctataccaactataaccagaaatttaaagataaagcgaccctgacc accgataaaagcagcagcaccgcgtatatgcagctgagcagcctgaccagcgaagat agcgcggtgtattattgcgcgcgctattatgatgatcatattgcctggattattggggcca gggcaccaccctgaccgtgagcagcgtggaaggcggcagcggcggcagcggcggc agcggcggcagcggcggcgtggatgatattcagctgacccagagcccggcgattatg agcgcgagcccgggcgaaaaagtgaccatgacctgccgcgcgagcagcagcgtgag ctatatgaactggtatcagcagaaaagcggcaccagcccgaaacgctggatttatgata ccagcaaagtggcgagcggcgtgccgtatcgctttagcggcagcggcagcggcacca gctatagcctgaccattagcagcatggaagcggaagatgcggcgacctattattgccagcagtggagcagcaacccgctgacctttggcgcgggcaccaaactggaactgaaacatcatcatcatcatcat

Claims

CLAIMSWe claim:

1. A pharmaceutical composition comprisinga. a first polymer comprising:i. a polymer comprising a backbone and one or more charged pendant groups,ii. a polymer comprising a backbone and one or more charged pendant groups, and one or more uncharged pendant groups, iii. a polymer comprising a backbone derived from one or more than one first monomer, which is a ionizable N-alkyl or N, N-dialkyl substituted (alkyl) methacrylamide, methacrylate, styrenic, acrylamide, or acrylate, iv. a polymer comprising a backbone derived from one or more than one first monomer, which is a ionizable N-alkyl or N, N-dialkyl substituted (alkyl) methacrylamide, methacrylate, styrenic, acrylamide, or acrylate, and one or more than one second monomer comprising non-ionizable structural units,v. a polymer having a backbone comprising a plurality of structural units derived from one or more than one first monomer, which is an N-alkyl or N, N-dialkyl substituted (alkyl)aciylamide monomer, and a plurality of hydrophilic structural units derived from one or more than one second monomer, which is an ethylenically unsaturated monomer,vi. a polymer comprising a backbone and one or more pendant groups selected from aminoalkyl group or amino aryl group, a dialkyl group, a neutral substituted alkyl group, or an aryl group or a neutral heteroalkyl group,vii. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant groupcomprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, orviii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. a heterologous first nucleic acid encoding one or more proteins.

2. The pharmaceutical composition of claim 1 wherein the heterologous first nucleic acid is complexed with the first polymer.

3. The pharmaceutical composition of claim 1 wherein the ratio of the heterologous first nucleic acid to the first polymer to the is selected from 1 to 150.

4. The pharmaceutical composition of claim 1 wherein the ratio of the heterologous first nucleic acid to the first polymer to the is selected from 3 to 15.

5. The pharmaceutical composition of claim 1 wherein the first polymer comprises ai. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5-95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains.

6. The pharmaceutical composition of claim 1 wherein the heterologous first nucleic acid is operably linked to a regulatory sequence.

7. The pharmaceutical composition of claim 6 wherein the regulatory sequence comprises an inducible promoter or a constitutive promoter.The pharmaceutical composition of claim 7 wherein the promoter is selected from human cytomegalovirus (CMV) promoter, chicken beta-actin (CAG) promoter cysteine synthase (cysK), ORF upstream of cysK (cysZ), ATP sulfurylase (cysN), sulfate adenylyltransferase (cysD), adenylylsulfate kinase (cysC), Periplasmic sulfate-binding protein (sbp), phosphoenolpyruvate carboxylase (ppc), phosphoenolpyruvate synthase (pps), pyruvate carboxylase (pyc), acetyl-CoA synthetase (acs), homoserine O-transsuccinylase (metA), cystathionine gamma-synthase (metB), cystathionine beta-lyase (metC), 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (metE), 5,10-methylenetetrahydrofolate reductase (metF), B12-dependent homocysteine-N5 -methyltetrahydrofolate transmethylase (metH), methionine adenosyltransferase (metK), aspartokinase II / homoserine dehydrogenase II (metL), aspartate-semialdehyde dehydrogenase (asd), aspartate aminotransferase (aspC), aspartokinase III (lysC), pyruvate kinase I (pykA), pyruvate kinase II (pykF) formyltetrahydrofolate deformylase (purU), the operons cysPUWAM (periplasmic sulphate binding protein, a component of sulphate ABC transporter, a membrane bound sulphate transport protein, a sulphate permease and an 0-acetyl serine sulfhydralase), cysJIH (alpha and beta subunits of a sulfite reductase and an adenylylsulfate reductase) and gcvTHP (Tetrahydrofolate dependent aminomethyl transferase, a glycine cleavage, carrier of aminomethyl group and a glycine dehydrogenase), phosphoglycerate dehydrogenase (serA), phosphoserine phosphatase (serB), phosphoserine aminotransferase (serC), serine hydroxymethyl transferase (glyA), acetate kinase (ackA), phosphotransacetylase (pta), pyruvate dehydrogenase El (ace), pyruvate dehydrogenase E2 (aceF), lipoamide dehydrogenase (Ipd), succinyl-CoA synthetase beta subunit (sucC), succinyl-CoA synthetase alpha subunit (sucD), phosphoenolpyruvate carboxykinase (pck), malate dehydrogenase (maeB), pyruvate oxidase (poxB), acetohydroxy acid synthase I large subunit (ilvB), acetohydroxy acid synthase I small subunit (ilvN), acetohydroxy acid synthase II large subunit (ilvG), acetohydroxy acid synthase II small subunit (ilvM), acetohydroxy acid synthase III large subunit (ilvl), acetohydroxy acid synthase III small subunit (ilvH), DAHP synthetase (aroF), DAHP synthetase (aroG), DAHP synthetase (aroH), homoserine kinase (thrB), threonine synthase (thrC), serine deaminase (sdaA), serine deaminase (sdaB), S-Adenosylmethionine decarboxylase (speD), ornithine decarboxylase(speC), arginine succinyltransf erase (astA), dihydrodipicolinate synthase (dapA), malate dehydrogenase (mdh), malate dehydrogenase FAD / NAD(P)-binding domain (mqo), citrate synthase (gltA), araBAD, prpBCDE, rhaSR, xlyA, araA, araB, prpB, prpD, rhaA, rhaB, rhaD, xylA, xylB, scpA / sbm, argK / ygfD, scpB / ygfG, scpC / ygfH, rmlA, rmlB, rmlC, T7, anhydrotetracycline-inducible promoter, IPTG-inducible promoter, rhamnose- inducible promoter, arabinose-inducible promoter, a tetracycline-inducible promoter, a light-inducible promoter, a heat-inducible promoter, a phage shock promoter (PSP), ADH1, GALI, CaMKIIa,rtTA, tTA, TRE, LacO, rm ID, a constitutive RNA polymerase III promoter, SP6, SV40, EF-1 alpha, PGK1, UBI, human beta actin, Ac5, Polyhedrin, TEF1, GDS, CaMV35S, or a UBC promoter.

9. The pharmaceutical composition of claim 1 wherein the heterologous first nucleic acid is controlled by a synthetic gene circuit.

10. The pharmaceutical composition of claim 9 wherein the synthetic gene circuit comprises a modular genetic switch.

11. The pharmaceutical composition of claim 10 wherein the modular genetic switch regulates expression of the heterologous first nucleic acid.

12. The pharmaceutical composition of claim 1 wherein the heterologous first nucleic acid comprises a heavy chain (HC) and a light chain (LC) of an immunoglobulin molecule.

13. The pharmaceutical composition of claim 1 wherein the pharmaceutical composition comprises a heterologous second nucleic acid.

14. The pharmaceutical composition of claim 13 wherein the heterologous first nucleic acid comprises a heavy chain (HC) and the second nucleic acid comprises light chain (LC).

15. The pharmaceutical composition of claim 13 wherein the heterologous first nucleic acid and the heterologous second nucleic acid are on a single plasmid.

16. The pharmaceutical composition of claim 13 wherein the heterologous first nucleic acid and the heterologous second nucleic acid are on separate plasmids.

17. The pharmaceutical composition of claim 13 wherein the heterologous first nucleic acid and / or the heterologous second nucleic acid optionally includes a nuclear localization sequence (NLS) and / or a nuclear export sequence (NES).

18. The pharmaceutical composition of claim 13 wherein the heterologous first nucleic acid and / or the heterologous second nucleic acid are codon optimized.

19. The pharmaceutical composition of claim 13 wherein the heterologous first nucleic acid and / or the heterologous second nucleic acid are optimized for in vivo expression.

20. The pharmaceutical composition of claim 1 further comprising a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 1 wherein the composition is formulated with a recombinant human hyaluronidase.

22. The pharmaceutical composition of claim 1, wherein the first polymer comprises formula (I) or formula (II)(II),or a salt or ion thereof,whereineach X is independentlywherein R1is -NRARBor ORA, wherein RAand RBare each independently H, aminoalkyl, or aminoheteroalkyl, provided that at least one of RAand RBis aminoalkyl or aminoheteroalkyl, R1is amino, aminoalkyl, or aminoheteroalkyl, and u is 1 to 5, and wherein R2is H or CH3;R3is -NRCRDor -ORC, wherein Rc and RD are each independently H or quaternary aminoalkyl or quaternary aminoheteroalkyl, provided that at least one Rcand RDis quaternary aminoalkyl or quaternary aminoheteroalkyl, R3is quaternary amino,quaternary aminoalkyl, or quaternary aminoheteroalkyl, and v is 1 to 5, and wherein R4is H or CH3;each Z is independentlyNREREor -ORE, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand R1is neutral substituted alkyl or neutral heteroalkyl, R3is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3;each W is independently, wherein n is from 1 to 8 and each R7is independently H, alkyl, alkenyl, alkynyl, or heteroalkyl;each Q is independently, -ORE, -CH2SO3, -CH2CH2SO3, or -CH2CH2CH2SO3, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFis neutral substituted alkyl or neutral heteroalkyl, R3is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3m is from 10 to 100;a is 0 - 0.8, b is 0 - 0.5, c is 0 - 0.9, d is 0 - 0.15, and e is 0 - 0.3, and each represents a fraction of m, and the sum of a, b, c, d and e is 1, provided that at least two of a, b, c, d and e are not 0.

23. The pharmaceutical composition of claim 1, wherein the first polymer comprises a polyester copolymer comprising formula (A):(A),or an ion or salt thereof;wherein:n is from 2 to 100,m is 0 or 1,X is O, CH or C,R1, R2R3, R4and R5are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, or an ionizable side chain,R1and R2, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain,R3and R4, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain, and wherein at least one of R1, R2R3, R4and R5comprises an ionizable side chain.

24. A modified cell comprising:a. a first polymer comprising;i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. an expression cassette comprising a heterologous polynucleotide sequence encoding one or more therapeutic proteins and a regulatory sequence operably linked to the one or more therapeutic proteins.

25. The modified cell of claim 24 wherein the heterologous polynucleotide transiently expresses the one or more therapeutic proteins.

26. The modified cell of claim 24 wherein the one or more therapeutic proteins is selected from:i) Factor IX, Factor VII, Factor VIII, glycoproteinoses, angiotensin II, anti- GM- CSF, anti- XP01, anti-TNF, GLP-1, anti-VEGF, anti-C5, vectorized PROTACs, vectorized siRNA and / or miRNA + / - genes; andii) GLP-1 receptor agonists, GIP receptor agonists, glucagon analogs, dual incretin agonists, triple / tri-agonists (GLP-l / GIP / glucagon), FGF21 analogs, FGF19 analogs, amylin analogs, adiponectin mimetics, leptin and leptin analogs, insulin, insulin analogs, insulin fusion proteins, Follistatin, myostatin inhibitors, activin inhibitors, GDF-8 inhibitors, osteocalcin, osteoprotegerin (OPG), sclerostin inhibitors, parathyroid hormone (PTH), PTHrP analogs, BMP2, BMP4, BMP6, BMP7, Wnt ligands, sFRP inhibitors, R-spondin proteins, irisin, myonectin, myokines, IL- 10, IL-22, IL-2 muteins, IL-1 receptor antagonist (IL-IRa), IL-6 receptor blockers (e.g., tocilizumab), TNF inhibitors (e.g., etanercept, adalimumab), TGF-0 inhibitors, GM-CSF, G-CSF, M-CSF, erythropoietin (EPO), thrombopoietin (TPO), Factor VIII, Factor IX, von Willebrand factor (vWF), fibrinogen, protein C, protein S, antithrombin III, hepcidin, complement C l -INH, complement C3 inhibitors, complement C5 inhibitors, HGF, VEGF, PDGF, FGF2, IGF-1, OSK / OSKM reprogramming factors, GDF-11, GDF-15, klotho, neuregulin-1, relaxin, natriuretic peptides (ANP, BNP, CNP), apelin, defensins, cathelicidins, interferons (IFN-a, IFN-P, IFN-y), antiviral antibodies (RSV mAbs, influenza mAbs, COVID- 19 mAbs), broadly neutralizing antibodies (bnAbs), antibacterial antibodies, anti -toxin antibodies, alpha- 1 antitrypsin (AAT),lysosomal enzymes (IDUA, IDS, GALC, GAA, ASB, NAGLU, GLA, SGSH, ARSB, HEXA, HEXB), GALC (galactocerebrosidase), GAA (acid alphaglucosidase), Cl esterase inhibitor (Cl -INH), GH (growth hormone), prolactin, ACTH, TSH, thyroid hormone-binding proteins, AMH, inhibin, activin, GnRH, FSH, LH, hCG, oxytocin, vasopressin, somatostatin analogs, osteopontin inhibitors, anti-fibrotic proteins (L0XL2 inhibitors, CTGF inhibitors), PCSK9 inhibitors, ANGPTL3 inhibitors, apoA-I mimetics, anti-IL-13 antibodies, anti-IL- 17 antibodies, anti-IL-23 antibodies, neuregulin, KIM-1 antagonists, antimicrobial peptides, immune checkpoint ligand traps (e.g., PD-L1 traps), immunoglobulins, bispecific antibodies, fusion cytokines, enzyme replacement therapies, complement regulatory proteins, renal protective peptides, cardioprotective peptides, digestive enzymes (pancrelipase), transferrin, ceruloplasmin, hemojuvelin, fibrinolytic proteins (tPA, uPA), anti-plasmin inhibitors, osteoanabolic proteins, anti -re sorptive proteins, metabolic regulators (e.g., glucagon receptor antagonists as proteins), chemokines, chemokine receptor ligands, antiviral interferons, anti-infective fusion proteins, and / or antiinflammatory cytokines.

27. The modified cell of claim 24 wherein the one or more proteins is selected from one or more antibodies selected from 1443_C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN- 119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480108 (PG16), 1456P20 (PG20), 1460G14 (PGG14), 1495_C14 (PGC14), 1496 C09 (PG9) (TCN-109),4838 L06 (PGT-121),4873 E03 (PGT- 121),4877_D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125), 5141_B17 (PGT-126), 5145_B14 (PGT-127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136 HO1 (PGT-131), 5343 B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT- 136), 5366P21 (PGT-136), 4964_G22 (PGT-141), 5345101 (PGT-137), 4993 K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131 A17 (PGT-132), 5138G07 (PGT-138), 5120_N10 (PGT-139), 6831A21 (PGT-151), 6889 117 (PGT-152), 6891_F06 (PGT-153), 6843_G20 (PGT-154), 6892D19 (PGT- 155), 6808 B09 (PGT-156), 6892C23 (PGT-157), 6881NO5 (PGT-158), elipovimab,monoclonal antibody 12-4.1, monoclonal antibody 33-7.1, monoclonal antibody 43-1.3, monoclonal antibody 22-1.5, ribavirin, palivizumab, motavizumab, RSV-IGIV ( RespiGam® ), MEDI-557, A-60444, MDT -637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola convalescent plasma (ECP), TKM- 100201, BCX4430 ((2S,3 S, 4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), method Pilavir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (1-N,7-N-bisphosphate) [3-(dimethylamino)propyl]-3,9-dimethylquinoline[8,7-h]quinolinone-l,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2, VRC-EBOADC076-00-VP, OS-2966, MVA-BN fdo, brincidofovir, Ebola vaccine based on Vaxart adenovirus vector 5, Ad26-ZEBOV, FiloVax vaccine, GOVX-E301, GOVX-E302, Ebolavirus entry inhibitor (NPC1 inhibitor), rVSV-EBOV, GS-5734, TAK-888, BN162, Ad5-nCoV, INO-4800, mRNA-1273, torcilla tocilizumab, sarilumab, TZLS-501, siltuximab, Favipravir, PRO 140, SAB-185, COVID-HIG, COVID-EIG, COVIDTRAP, APN1, STI-4920, CMAB020, ruxolitinib, baricitinib, TMPRSS2, lopinavir / ritonavir, dalunavir, cobicistat, eculizumab, danoprevir, gimsilumab, IZN-101, selinexor, celecoxib, BN162, INO-4800, NVX-CoV2373, IN04800, BNT-162, apiimod, ritalide, DAS 181, CM-4620-IE, CAP- 1002, IFX-1, D25, MPE8, palivizumab, motavizumab, nirsevimab, 12C6, 3D3, 2D10, 5C4, RBI, MPE8, RV01, RV10, RV11, RV15, AM-14, AM-16, AM-22, AM-23, abciximab, adalimumab, alemtuzumab, alirocumab, avibactam, basiliximab, benralizumab, bezlotoxumab, blinatumomab, brodalumab, burosumab, canakinumab, caplacizumab, certolizumab pegol, daclizumab, denosumab, dupilumab, eculizumab, emicizumab, erenumab, evolocumab, fremanezumab, galcanezumab, golimumab, guselkumab, ibalizumab, idarucizumab, infliximab, itolizumab, ixekizumab, lanadelumab, lokivetmab, mepolizumab, natalizumab, obiltoxaximab, ocrelizumab, omalizumab, palivizumab, ranibizumab, raxibacumab, reslizumab, rmab, rovelizumab, ruplizumab, sarilumab, secukinumab, tildrakizumab, thiomab, tocilizumab, ustekinumab, vedolizumab, abrilumab, actoxumab, aducanumab, afasevikumab, afelimomab, anifrolumab, anrukinzumab (IMA-638), aselizumab, atorolimumab, bapineuzumab, BCD- 100, bertilimumab, besilesomab, biciromab, bimagrumab, bimekizumab, birtamimab, bleselumab, blosozumab, bococizumab, brazikumab, briakinumab, brolucizumab,carlumab, carotuximab, cedelizumab, clazakizumab, clenoliximab, concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab, crotedumab, depatuxizumab, mafodotin, derlotuximab biotin, dezamizumab, diridavumab, domagrozumab, dusigitumab, ecromeximab, edobacomab, efalizumab, efungumab, eldelumab, elezanumab, enokizumab, eptinezumab, erlizumab, etrolizumab, evinacumab, exbivirumab, fanolesomab, faralimomab, faricimab, fasinumab, felvizumab, fezakinumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foravirumab, frovocimab, fulranumab, gantenerumab, gavilimomab, gevokizumab, gimsilumab, gomiliximab, gosuranemab, ianalumab, inclacumab, inolimomab, iomab-B, keliximab, lampalizumab, landogrozumab, larcaviximab, lebrikizumab, lenvervimab, lerdelimumab, letolizumab, libivirumab, ligelizumab, lodelcizumab, lulizumab pegol, marstacimab, mavrilimumab, metelimumab, mirikizumab, motavizumab, muromonab CD3, nebacumab, nemolizumab, NEODOO 1, nirsevimab, odulimomab, olendalizumab, olokizumab, OMS721, opicinumab, orticumab, otelixizumab, otilimab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, panobacumab, pascolizumab, pateclizumab, PDR001, perakizumab, pexelizumab, placulumab, plozalizumab, ponezumab, porgaviximab, prasinezumab, priliximab, PRO 140, quilizumab, rafivirumab, ralpancizumab, ranevetmab, ravagalimab, ravulizumab, refanezumab, regavirumab, relatlimab, rinucumab, risankizumab, roledumab, romosozumab, rontalizumab, SA237, satralizumab, sevirumab, SHP647, sifalimumab, simtuzumab, siplizumab, sirukumab, solanezumab, sonepcizumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tadocizumab, talizumab, tamtuvetmab, tanezumab, tefibazumab, telimomab aritox, teneliximab, teplizumab, teprotumumab, tezepelumab, tibulizumab, toralizumab, tralokinumab, trevogrumab, tuvirumab, ulocuplumab, urtoxazumab, varisacumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, zolimomab aritox, trastuzumab, gemtuzumab, brentuximab, vorsetuzumab, lorvotuzumab, cantuzumab, bivatuzumabor inotuzumab, or vadastuximab.

28. The modified cell of claim 24 wherein the expression cassette optionally comprises a nuclear localization sequence (NLS) and / or a nuclear export sequence (NES).

29. The modified cell of claim 24 wherein the expression cassette is on a plasmid DNA.

30. The modified cell of claim 24 wherein the plasmid DNA is taken up in the nucleus of the cell.

31. The modified cell of claim 24 wherein the antibody expression titer is characterized to exhibit antibody concentrations of 500ng / mL-100 ng / mL, 100ng / mL-10ng / mL, 10ng / mL-1ng / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 30 pg / mL to 85 pg / mL or greater.

32. The modified cell of claim 24, wherein the first polymer comprises formula (I) or formula (II)(II),or a salt or ion thereof,whereineach X is independentlywherein R1is -NRARBor ORA, wherein RAand RBare each independently H, aminoalkyl, or aminoheteroalkyl, provided that at least one of RAand RBis aminoalkyl or aminoheteroalkyl, R1is amino, aminoalkyl, or aminoheteroalkyl, and u is 1 to 5, and wherein R2is H or CH3;R3is -NRCRDor -ORC, wherein Rc and RD are each independently H or quaternary aminoalkyl or quaternary aminoheteroalkyl, provided that at least one Rcand RDis quaternary aminoalkyl or quaternary aminoheteroalkyl, R3is quaternary amino,quaternary aminoalkyl, or quaternary aminoheteroalkyl, and v is 1 to 5, and wherein R4is H or CH3;each Z is independentlyNREREor -ORE, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand R1is neutral substituted alkyl or neutral heteroalkyl, R3is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3;each W is independently, wherein n is from 1 to 8 and each R7is independently H, alkyl, alkenyl, alkynyl, or heteroalkyl;each Q is independently, -ORE, -CH2SO3, -CH2CH2SO3, or -CH2CH2CH2SO3, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFis neutral substituted alkyl or neutral heteroalkyl, R3is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3m is from 10 to 100;a is 0 - 0.8, b is 0 - 0.5, c is 0 - 0.9, d is 0 - 0.15, and e is 0 - 0.3, and each represents a fraction of m, and the sum of a, b, c, d and e is 1, provided that at least two of a, b, c, d and e are not 0.

33. The modified cell of claim 24, wherein the first polymer comprises a polyester copolymer comprising formula (I):R1R2R3R4wherein n is from 2-50;R1and R2are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, carbocyclyl, or heterocyclyl, or R1and R2, together with the atoms to which they are attached, combine to form a 5-7 membered ring;R3and R4are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, carbocyclyl, heterocyclyl, or heterocyclyl, or R3and R4, together with the atoms to which they are attached, combine to form a 5-7 membered ring; andthe dashed bond is absent or a bond of either E or Z orientation,wherein at least one of R1, R2, R3and R4comprises a conjugated moiety.

34. A method for administering a polymer polynucleotide complex into a cell in a subject in need thereof comprising:a. introducing into the cell a polymer polynucleotide complex comprising:i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side chains; andb. recombinantly expressing a polynucleotide sequence encoding a therapeutic protein and a regulatory sequence operably linked to the therapeutic protein.

35. The method of claim 34 wherein the method is characterized to exhibit nuclear uptake of the polynucleotide sequence encoding a therapeutic protein in the cell in the subject.

36. The method of claim 34 wherein the method is characterized to exhibit enhanced in vivo transfection efficiency in the cell in the subject approximately 10- to 100 fold, 100- to 500 fold, 500- to 1000-fold or greater.

37. The method of claim 34 wherein the method is characterized to increase therapeutic protein expression in the cell in the subject.

38. The method of claim 34 wherein the polynucleotide sequence encodes an antibody.

39. The method of claim 38 wherein the antibody is selected from a broadly neutralizing HIV antibody, PGT121, 1443 C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480108 (PG16), 1456P20 (PG20), 1460G14 (PGG14), 1495_C14 (PGC14),1496_C09 (PG9) (TCN-109),4838_L06 (PGT-121),4873_E03 (PGT-121),4877_D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125), 5141_B17 (PGT-126), 5145_B14 (PGT- 127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136_HO1 (PGT-131), 5343_B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT-136), 5366P21 (PGT-136), 4964 G22 (PGT-141), 5345101 (PGT-137), 4993_K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138G07 (PGT-138), 5120 N1O (PGT-139), 6831A21 (PGT-151), 6889 117 (PGT-152), 6891 F06 (PGT- 153), 6843_G20 (PGT-154), 6892D19 (PGT-155), elipovimab, 6808_B09 (PGT-156), 6892C23 (PGT-157), and 6881 NO5 (PGT-158).

40. The method of claim 38 wherein the antibody is characterized to exhibit neutralization in an in vitro HIV neutralization assay.

41. The method of claim 38 wherein the cell produces one or more antibodies in circulation in the subject.

42. The method of claim 38 wherein the method is characterized to exhibit serum concentrations of the antibody in the subject of about 500ng / mL-100 ng / mL, 100ng / mL-10ng / mL, 10ng / mL-1ng / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 30 pg / mL to 85 pg / mL or greater.

43. The method of claim 38 wherein the antibody expressed in the subject is detected for 10, 20, 30, 40, 50, 60...365 days or greater.

44. The method of claim 38 wherein the antibody is optimized to increase antibody expression in the subject.

45. The method of claim 38 wherein the administration of the antibody is optimized for dose in the subject.

46. The method of claim 34 wherein the polymer polynucleotide complex is formulated with a pharmaceutically acceptable carrier.

47. The method of claim 34 wherein the polymer polynucleotide complex is administered in the subject via parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, intratumoral administration or subcutaneous administration.

48. The method of claim 34 wherein the polymer polynucleotide complex is administered in an effective amount.

49. The method of claim 34, wherein the first polymer comprises formula (I) or formula (II)(II),or a salt or ion thereof,whereineach X is independentlyis -NRARBor ORA, wherein RAand RBare each independently H, aminoalkyl, or aminoheteroalkyl, provided that at least one of RAand RBis aminoalkyl or aminoheteroalkyl, R1is amino, aminoalkyl, or aminoheteroalkyl, and u is 1 to 5, and wherein R2is H or CH3;each Y is independently, wherein R3is -NRCRDor -ORC, wherein Rc and RD are each independently H or quaternary aminoalkyl or quaternary aminoheteroalkyl, provided that at least one Rcand RDis quaternary aminoalkyl or quaternary aminoheteroalkyl, R3is quaternary amino, quaternary aminoalkyl, or quaternary aminoheteroalkyl, and v is 1 to 5, and wherein R4is H or CH3;each Z is independentlyNRERFor -ORE, wherein REand RFare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFis neutral substituted alkyl or neutral heteroalkyl, R5is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3;each W is independentlywherein n is from 1 to 8 and each R7is independently H, alkyl, alkenyl, alkynyl, or heteroalkyl;each Q is independentlyAR5, wherein R is H, -NRERF, -ORE, -CH2SO3, -CH2CH2SO3, or -CH2CH2CH2SO3, wherein REand RFare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFisneutral substituted alkyl or neutral heteroalkyl, R5is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3 m is from 10 to 100;a is 0 - 0.8, b is 0 - 0.5, c is 0 - 0.9, d is 0 - 0.15, and e is 0 - 0.3, and each represents a fraction of m, and the sum of a, b, c, d and e is 1, provided that at least two of a, b, c, d and e are not 0.

50. The method of claim 34, wherein the first polymer comprises a polyester copolymer comprising formula (A):or an ion or salt thereof;wherein:n is from 2 to 100,m is 0 or 1,X is O, CH or C,R1, R2R3, R4and R5are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, or an ionizable side chain,R1and R2, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain,R3and R4, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain, and wherein at least one of R1, R2R3, R4and R5comprises an ionizable side chain.

51. A method of treating a disease or disorder in a subject in need thereof comprising:administering a polymer nucleic acid complex comprising:a. a means for transfecting a cell with the polymer nucleic acid complex comprising:i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable side;b. a means for encoding a nucleic acid to one or more proteins; andc. a means for binding a target of interest with the one or more proteins.

52. The method of claim 51 wherein the protein is selected from Factor IX, Factor VII, Factor VIII, glycoproteinoses, angiotensin II, anti- GM-CSF, anti- XP01, anti-TNF, GLP-1, anti-VEGF, anti-C5, vectorized PROTACs, vectorized siRNA and / or miRNA + / - genes.

53. The method of claim 51 wherein the protein is selected from one or more antibodies selected from 1443_C16 (PG16) (TCN-116), 1503 H05 (PG16) (TCN-119), 1456 A12 (PG16) (TCN-117), 1469 M23 (PG16) (TCN-118), 1489 113 (PG16) (TCN-120), 1480108 (PG16), 1456P20 (PG20), 1460G14 (PGG14), 1495_C14 (PGC14),1496_C09 (PG9) (TCN-109),4838 L06 (PGT- 121), 4873 E03 (PGT-121),4877 D15 (PGT-122), 4858P08 (PGT-123), 6123_A06 (PGT-125), 5141_B17 (PGT-126), 5145_B14 (PGT- 127), 5114A19 (PGT-128), 5147_N06 (PGT-130), 5136_HO1 (PGT-131), 5343_B08 (PGT-135), 5344E16 (PGT-135), 5329 C19 (PGT-136), 5366P21 (PGT-136), 4964 G22 (PGT-141), 5345101 (PGT-137), 4993_K13 (PGT-141), 4995E20 (PGT-142), 4980_N08 (PGT-143), 4970K22 (PGT-144), 4995_P16 (PGT-145), 4835F12 (PGT-124), 4869-KI5 (PGT-133), 4876M06 (PGT-134), 5131_A17 (PGT-132), 5138G07 (PGT-138), 5120 N1O (PGT-139), 6831A21 (PGT-151), 6889 117 (PGT-152), 6891 F06 (PGT- 153), 6843_G20 (PGT-154), 6892D19 (PGT-155), 6808_B09 (PGT-156), 6892C23 (PGT-157), 6881NO5 (PGT-158), elipovimab, monoclonal antibody 12-4.1,monoclonal antibody 33-7.1, monoclonal antibody 43-1.3, monoclonal antibody 22-1.5, ribavirin, palivizumab, motavizumab, RSV-IGIV ( RespiGam® ), MEDI-557, A-60444, MDT -637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola convalescent plasma (ECP), TKM-100201, BCX4430 ((2S,3S, 4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), method Pilavir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (l-N,7-N-bisphosphate) [3-(dimethylamino)propyl]-3,9-dimethylquinoline[8,7-h]quinolinone-l,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2, VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, brincidofovir, Ebola vaccine based on Vaxart adenovirus vector 5, Ad26-ZEBOV, FiloVax vaccine, GOVX-E301, GOVX-E302, Ebola virus entry inhibitor (NPC1 inhibitor), rVSV-EBOV, GS-5734, TAK-888, BN162, Ad5-nCoV, INO-4800, mRNA-1273, torcilla tocilizumab, sarilumab, TZLS-501, siltuximab, Favipravir, PRO 140, SAB-185, COVID-HIG, COVID-EIG, COVIDTRAP, APN1, STI-4920, CMAB020, ruxolitinib, baricitinib, TMPRSS2, lopinavir / ritonavir, dalunavir, cobicistat, eculizumab, danoprevir, gimsilumab, IZN-101, selinexor, celecoxib, BN162, INO-4800, NVX-CoV2373, IN04800, BNT-162, apiimod, ritalide, DAS 181, CM-4620-IE, CAP- 1002, IFX-1, D25, MPE8, palivizumab, motavizumab, nirsevimab, 12C6, 3D3, 2D10, 5C4, RBI, MPE8, RV01, RV10, RV11, RV15, AM-14, AM- 16, AM-22, AM-23, abciximab, adalimumab, alemtuzumab, alirocumab, avibactam, basiliximab, benralizumab, bezlotoxumab, blinatumomab, brodalumab, burosumab, canakinumab, caplacizumab, certolizumab pegol, daclizumab, denosumab, dupilumab, eculizumab, emicizumab, erenumab, evolocumab, fremanezumab, galcanezumab, golimumab, guselkumab, ibalizumab, idarucizumab, infliximab, itolizumab, ixekizumab, lanadelumab, lokivetmab, mepolizumab, natalizumab, obiltoxaximab, ocrelizumab, omalizumab, palivizumab, ranibizumab, raxibacumab, reslizumab, rmab, rovelizumab, ruplizumab, sarilumab, secukinumab, tildrakizumab, thiomab, tocilizumab, ustekinumab, vedolizumab, abrilumab, actoxumab, aducanumab, afasevikumab, afelimomab, anifrolumab, anrukinzumab (IMA-638), aselizumab, atorolimumab, bapineuzumab, BCD- 100, bertilimumab, besilesomab, biciromab, bimagrumab, bimekizumab, birtamimab, bleselumab, blosozumab, bococizumab, brazikumab, briakinumab, brolucizumab, carlumab, carotuximab, cedelizumab, clazakizumab, clenoliximab,concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab, crotedumab, depatuxizumab, mafodotin, derlotuximab biotin, dezamizumab, diridavumab, domagrozumab, dusigitumab, ecromeximab, edobacomab, efalizumab, efungumab, eldelumab, elezanumab, enokizumab, eptinezumab, erlizumab, etrolizumab, evinacumab, exbivirumab, fanolesomab, faralimomab, faricimab, fasinumab, felvizumab, fezakinumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foravirumab, frovocimab, fulranumab, gantenerumab, gavilimomab, gevokizumab, gimsilumab, gomiliximab, gosuranemab, ianalumab, inclacumab, inolimomab, iomab-B, keliximab, lampalizumab, landogrozumab, larcaviximab, lebrikizumab, lenvervimab, lerdelimumab, letolizumab, libivirumab, ligelizumab, lodelcizumab, lulizumab pegol, marstacimab, mavrilimumab, metelimumab, mirikizumab, motavizumab, muromonab CD3, nebacumab, nemolizumab, NEODOO 1, nirsevimab, odulimomab, olendalizumab, olokizumab, OMS721, opicinumab, orticumab, otelixizumab, otilimab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, panobacumab, pascolizumab, pateclizumab, PDR001, perakizumab, pexelizumab, placulumab, plozalizumab, ponezumab, porgaviximab, prasinezumab, priliximab, PRO 140, quilizumab, rafivirumab, ralpancizumab, ranevetmab, ravagalimab, ravulizumab, refanezumab, regavirumab, relatlimab, rinucumab, risankizumab, roledumab, romosozumab, rontalizumab, SA237, satralizumab, sevirumab, SHP647, sifalimumab, simtuzumab, siplizumab, sirukumab, solanezumab, sonepcizumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tadocizumab, talizumab, tamtuvetmab, tanezumab, tefibazumab, telimomab aritox, teneliximab, teplizumab, teprotumumab, tezepelumab, tibulizumab, toralizumab, tralokinumab, trevogrumab, tuvirumab, ulocuplumab, urtoxazumab, varisacumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, zolimomab aritox, trastuzumab, gemtuzumab, brentuximab, vorsetuzumab, lorvotuzumab, cantuzumab, bivatuzumabor inotuzumab, or vadastuximab.

54. The method of claim 51 wherein the antibodies are produced in the patient at a level of [serum concentrations of the antibody expression is characterized to exhibit 500ng / mL- lOO ng / mL, lOOng / mL-lOng / mL, lOng / mL-lng / mL, 10 pg / mL, 15 pg / mL, 30 pg / mL to 85 pg / mL or greater.

55. The method of claim 51 wherein the target of interest is selected from ACE2, CD4 receptor, CCR5, CXCR5, gpl20, fusion F protein, CX3CR1, RBD of spike (S) protein, envelope glycoprotein, and EBOV GP head epitope.

56. A method of treating, preventing, or ameliorating an immune response to an antigen comprising administering to a subject in need thereof a pharmaceutical composition comprisinga. a polymer nucleic acid complex comprising:i. a polymer comprising a backbone and first and second pendant groups or an ion or salt thereof, wherein the first pendant group comprises an aminoalkyl group or amino aryl group, the second pendant group comprises an aminoalkyl group, a dialkyl group, a neutral substituted alkyl group, an aryl group or a neutral heteroalkyl group, wherein the first pendant group comprises 5-95%, the second pendant group comprises 5- 95% of the total number of first and second pendant groups and the first and second pendant groups are different, or a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the side chains contain at least one conjugated moiety, orii. a polyester copolymer comprising of a backbone formed from a cyclic anhydride monomer and an epoxide monomer, wherein the polyester copolymer comprises one or more ionizable; andb. a polynucleotide encoding a protein or an antibody.

57. The method of claim 56, wherein the first polymer comprises formula (I) or formula (II)or a salt or ion thereof,whereineach X is independently, wherein R1is -NRARBor ORA, wherein RAand RBare each independently H, aminoalkyl, or aminoheteroalkyl, provided that at least one of RAand RBis aminoalkyl or aminoheteroalkyl, R1is amino, aminoalkyl, or aminoheteroalkyl, and u is 1 to 5, and wherein R2is H or CH3;each Y is independentlyR3is -NRCRDor -ORC, wherein Rc and RD are each independently H or quaternary aminoalkyl or quaternary aminoheteroalkyl, provided that at least one Rcand RDis quaternary aminoalkyl or quaternary aminoheteroalkyl, R3is quaternary amino, quaternary aminoalkyl, or quaternary aminoheteroalkyl, and v is 1 to 5, and wherein R4is H or CH3;R5each Z is independentlyor', wherein R5is - NRERFor -ORE, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one RFand RFis neutral substituted alkyl or neutral heteroalkyl, R5is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3;each W is independently, wherein n is from 1 to 8 and each R7is independently H, alkyl, alkenyl, alkynyl, or heteroalkyl;each Q is independently, -ORE, -CH2SO3, -CH2CH2SO3, or -CH2CH2CH2SO3, wherein REand REare each independently H or neutral substituted alkyl or neutral heteroalkyl, wherein alkyl is substituted with a neutral hydrophilic group, provided that at least one REand RFis neutral substituted alkyl or neutral heteroalkyl, R5is a neutral hydrophilic group, neutral substituted alkyl or neutral heteroalkyl, and w is 0 to 5, and wherein R6is H or CH3 m is from 10 to 100;a is 0 - 0.8, b is 0 - 0.5, c is 0 - 0.9, d is 0 - 0.15, and e is 0 - 0.3, and each represents a fraction of m, and the sum of a, b, c, d and e is 1, provided that at least two of a, b, c, d and e are not 0.

58. The method of claim 56, wherein the first polymer comprises a polyester copolymer comprising formula (A):R2or an ion or salt thereof;wherein:n is from 2 to 100,m is 0 or 1,X is O, CH or C,R1, R2R3, R4and R5, are each independently H, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, or an ionizable side chain,R1and R2, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain,R3and R4, together with the atoms to which they are attached, optionally combine to form a 5-7 membered ring optionally comprising an ionizable side chain, and wherein at least one of R1, R2R3, R4and R5comprises an ionizable side chain.

59. The method of claim 56, wherein the protein is selected from GLP-1 receptor agonists, GIP receptor agonists, glucagon analogs, dual incretin agonists, triple / tri-agonists (GLP- 1 / GIP / glucagon), FGF21 analogs, FGF19 analogs, amylin analogs, adiponectin mimetics, leptin and leptin analogs, insulin, insulin analogs, insulin fusion proteins, Follistatin, myostatin inhibitors, activin inhibitors, GDF-8 inhibitors, osteocalcin, osteoprotegerin (OPG), sclerostin inhibitors, parathyroid hormone (PTH), PTHrP analogs, BMP2, BMP4, BMP6, BMP7, Wnt ligands, sFRP inhibitors, R-spondin proteins, irisin, myonectin, myokines, IL- 10, IL-22, IL-2 muteins, IL-1 receptor antagonist (IL-IRa), IL-6 receptor blockers (e.g., tocilizumab), TNF inhibitors (e.g., etanercept, adalimumab), TGF-0 inhibitors, GM-CSF, G-CSF, M-CSF, erythropoietin (EPO), thrombopoietin (TPO), Factor VIII, Factor IX, von Willebrand factor (vWF), fibrinogen, protein C, protein S, antithrombin III, hepcidin, complement Cl -INH, complement C3 inhibitors, complement C5 inhibitors, HGF, VEGF, PDGF, FGF2, IGF-1, OSK / OSKM reprogramming factors, GDF-11, GDF-15, klotho, neuregulin-1, relaxin, natriuretic peptides (ANP, BNP, CNP), apelin, defensins, cathelicidins, interferons (IFN-a, IFN-0, IFN-y), antiviral antibodies (RSV mAbs, influenza mAbs, COVID- 19 mAbs), broadly neutralizing antibodies (bnAbs), antibacterial antibodies, anti-toxin antibodies, alpha-1 antitrypsin (AAT), lysosomal enzymes (IDUA, IDS, GALC, GAA, ASB, NAGLU, GLA, SGSH, ARSB, HEXA, HEXB), GALC (galactocerebrosidase), GAA (acid alpha-glucosidase), C 1 esterase inhibitor (Cl -INH), GH (growth hormone), prolactin, ACTH, TSH, thyroid hormone-binding proteins, AMH, inhibin, activin, GnRH, FSH, LH, hCG, oxytocin, vasopressin, somatostatin analogs, osteopontin inhibitors, anti-fibrotic proteins (LOXL2 inhibitors, CTGF inhibitors), PCSK9 inhibitors, ANGPTL3 inhibitors, apoA-I mimetics, anti-IL-13 antibodies, anti-IL-17 antibodies, anti-IL-23 antibodies, neuregulin, KIM-1 antagonists, antimicrobial peptides, immune checkpoint ligand traps (e.g., PD-L1 traps), immunoglobulins, bispecific antibodies, fusion cytokines, enzyme replacement therapies, complement regulatory proteins, renal protective peptides, cardioprotective peptides, digestive enzymes (pancrelipase), transferrin, ceruloplasmin, hemojuvelin, fibrinolyticproteins (tPA, uPA), anti-plasmin inhibitors, osteoanabolic proteins, anti -resorptive proteins, metabolic regulators (e.g., glucagon receptor antagonists as proteins), chemokines, chemokine receptor ligands, antiviral interferons, anti-infective fusion proteins, and / or anti-inflammatory cytokines.

60. The method of claim 34 or claim 56, wherein the protein is GLP-ls, tri-agonists, FGF21 / 19, amylin, adiponectin mimetics, leptin, insulin / insulin fusions, Follistatin, myostatin inhibitors, osteocalcin, OPG, sclerostin inhibitors, IL- 10, IL-22, IL-2 muteins, IL-IRa, HGF, relaxin, EPO, Factor VIII / IX, vWF, hepcidin, OSK / OSKM, GDF-11 / 15, klotho, PCSK9, IL-6R, TNF, RSV / flu / COVID, bnAbs, AAT, GALC, GAA, lysosomal enzymes, ANP / BNP / CNP, apelin, defensins, interferons, Cl-INH, GH, IGF-1, PTH, AMH, GnRH, or FSH / LH / hCG.

61. The pharmaceutical composition of claim 1 wherein the protein is selected from GLP-1 receptor agonists, GIP receptor agonists, glucagon analogs, dual incretin agonists, triple / tri-agonists (GLP-l / GIP / glucagon), FGF21 analogs, FGF19 analogs, amylin analogs, adiponectin mimetics, leptin and leptin analogs, insulin, insulin analogs, insulin fusion proteins, Follistatin, myostatin inhibitors, activin inhibitors, GDF-8 inhibitors, osteocalcin, osteoprotegerin (OPG), sclerostin inhibitors, parathyroid hormone (PTH), PTHrP analogs, BMP2, BMP4, BMP6, BMP7, Wnt ligands, sFRP inhibitors, R-spondin proteins, irisin, myonectin, myokines, IL-10, IL -22, IL-2 muteins, IL-1 receptor antagonist (IL-IRa), IL-6 receptor blockers (e.g., tocilizumab), TNF inhibitors (e g., etanercept, adalimumab), TGF-P inhibitors, GM-CSF, G-CSF, M-CSF, erythropoietin (EPO), thrombopoietin (TPO), Factor VIII, Factor IX, von Willebrand factor (vWF), fibrinogen, protein C, protein S, antithrombin III, hepcidin, complement Cl-INH, complement C3 inhibitors, complement C5 inhibitors, HGF, VEGF, PDGF, FGF2, IGF- 1, OSK / OSKM reprogramming factors, GDF-11, GDF-15, klotho, neuregulin-1, relaxin, natriuretic peptides (ANP, BNP, CNP), apelin, defensins, cathelicidins, interferons (IFN- a, IFN-P, IFN-y), antiviral antibodies (RSV mAbs, influenza mAbs, COVID-19 mAbs), broadly neutralizing antibodies (bnAbs), antibacterial antibodies, anti-toxin antibodies, alpha- 1 antitrypsin (AAT), lysosomal enzymes (IDUA, IDS, GALC, GAA, ASB, NAGLU, GLA, SGSH, ARSB, HEXA, HEXB), GALC (galactocerebrosidase), GAA (acid alpha-glucosidase), Cl esterase inhibitor (Cl-INH), GH (growth hormone),prolactin, ACTH, TSH, thyroid hormone-binding proteins, AMH, inhibin, activin, GnRH, FSH, LH, hCG, oxytocin, vasopressin, somatostatin analogs, osteopontin inhibitors, anti-fibrotic proteins (L0XL2 inhibitors, CTGF inhibitors), PCSK9 inhibitors, ANGPTL3 inhibitors, apoA-I mimetics, anti-IL-13 antibodies, anti-IL-17 antibodies, anti-IL-23 antibodies, neuregulin, KIM-1 antagonists, antimicrobial peptides, immune checkpoint ligand traps (e.g., PD-L1 traps), immunoglobulins, bispecific antibodies, fusion cytokines, enzyme replacement therapies, complement regulatory proteins, renal protective peptides, cardioprotective peptides, digestive enzymes (pancrelipase), transferrin, ceruloplasmin, hemojuvelin, fibrinolytic proteins (tPA, uPA), anti-plasmin inhibitors, osteoanabolic proteins, anti-resorptive proteins, metabolic regulators (e.g., glucagon receptor antagonists as proteins), chemokines, chemokine receptor ligands, antiviral interferons, anti-infective fusion proteins, and / or anti-inflammatory cytokines.

62. The pharmaceutical composition of claim 1 wherein the protein is GLP-ls, tri-agonists, FGF21 / 19, amylin, adiponectin mimetics, leptin, insulin / insulin fusions, Follistatin, myostatin inhibitors, osteocalcin, OPG, sclerostin inhibitors, IL- 10, IL-22, IL-2 muteins, IL-IRa, HGF, relaxin, EPO, Factor VIII / IX, vWF, hepcidin, OSK / OSKM, GDF-11 / 15, klotho, PCSK9, IL-6R, TNF, RSV / flu / COVID, bnAbs, AAT, GALC, GAA, lysosomal enzymes, ANP / BNP / CNP, apelin, defensins, interferons, Cl-INH, GH, IGF-1, PTH, AMH, GnRH, or FSH / LH / hCG.

63. The modified cell of claim 24, wherein the protein is GLP-1 receptor agonists, GIP receptor agonists, glucagon analogs, dual incretin agonists, triple / tri-agonists (GLP- 1 / GIP / glucagon), FGF21 analogs, FGF19 analogs, amylin analogs, adiponectin mimetics, leptin and leptin analogs, insulin, insulin analogs, insulin fusion proteins, Follistatin, myostatin inhibitors, activin inhibitors, GDF-8 inhibitors, osteocalcin, osteoprotegerin (OPG), sclerostin inhibitors, parathyroid hormone (PTH), PTHrP analogs, BMP2, BMP4, BMP6, BMP7, Wnt ligands, sFRP inhibitors, R-spondin proteins, irisin, myonectin, myokines, IL- 10, IL-22, IL-2 muteins, IL-1 receptor antagonist (IL-IRa), IL-6 receptor blockers (e.g., tocilizumab), TNF inhibitors (e.g., etanercept, adalimumab), TGF- inhibitors, GM-CSF, G-CSF, M-CSF, erythropoietin (EPO), thrombopoietin (TPO), Factor VIII, Factor IX, von Willebrand factor (vWF), fibrinogen, protein C, protein S,antithrombin TIT, hepcidin, complement Cl -INH, complement C3 inhibitors, complement C5 inhibitors, HGF, VEGF, PDGF, FGF2, IGF-1, OSK / OSKM reprogramming factors, GDF-11, GDF-15, klotho, neuregulin-1, relaxin, natriuretic peptides (ANP, BNP, CNP), apelin, defensins, cathelicidins, interferons (IFN-a, IFN-P, IFN-y), antiviral antibodies (RSV mAbs, influenza mAbs, COVID- 19 mAbs), broadly neutralizing antibodies (bnAbs), antibacterial antibodies, anti-toxin antibodies, alpha-1 antitrypsin (AAT), lysosomal enzymes (IDUA, IDS, GALC, GAA, ASB, NAGLU, GLA, SGSH, ARSB, HEXA, HEXB), GALC (galactocerebrosidase), GAA (acid alpha-glucosidase), Cl esterase inhibitor (Cl -INH), GH (growth hormone), prolactin, ACTH, TSH, thyroid hormone-binding proteins, AMH, inhibin, activin, GnRH, FSH, LH, hCG, oxytocin, vasopressin, somatostatin analogs, osteopontin inhibitors, anti-fibrotic proteins (L0XL2 inhibitors, CTGF inhibitors), PCSK9 inhibitors, ANGPTL3 inhibitors, apoA-I mimetics, anti-IL-13 antibodies, anti -IL- 17 antibodies, anti-IL-23 antibodies, neuregulin, KIM-1 antagonists, antimicrobial peptides, immune checkpoint ligand traps (e.g., PD-L1 traps), immunoglobulins, bispecific antibodies, fusion cytokines, enzyme replacement therapies, complement regulatory proteins, renal protective peptides, cardioprotective peptides, digestive enzymes (pancrelipase), transferrin, ceruloplasmin, hemojuvelin, fibrinolytic proteins (tPA, uPA), anti-plasmin inhibitors, osteoanabolic proteins, anti -re sorptive proteins, metabolic regulators (e.g., glucagon receptor antagonists as proteins), chemokines, chemokine receptor ligands, antiviral interferons, anti-infective fusion proteins, and / or anti-inflammatory cytokines.

64. The modified cell of claim 24 wherein the protein is GLP-ls, tri-agonists, FGF21 / 19, amylin, adiponectin mimetics, leptin, insulin / insulin fusions, Follistatin, myostatin inhibitors, osteocalcin, OPG, sclerostin inhibitors, IL-10, IL-22, IL-2 muteins, IL-IRa, HGF, relaxin, EPO, Factor VI1I / 1X, vWF, hepcidin, OSK / OSKM, GDF-11 / 15, klotho, PCSK9, IL-6R, TNF, RSV / flu / COVID, bnAbs, AAT, GALC, GAA, lysosomal enzymes, ANP / BNP / CNP, apelin, defensins, interferons, Cl -INH, GH, IGF-1, PTH, AMH, GnRH, or FSH / LH / hCG.

Images