Compositions and methods for transfecting cells

Abstract

To address the issue where development of a reliable non-viral gene delivery system to transfect fibroblasts with high efficiency and safety is imperative and of great significance.SOLUTION: The present disclosure relates to branched polymers and polyplexes which find use in gene therapy applications as safe and non-toxic nucleic acid transfection agents.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 744,994, filed October 12, 2018, and U.S. Provisional Application No. 62 / 826,461, filed March 29, 2019, the contents of which are incorporated herein by reference in their entireties.

[0002] Field of the Disclosure Various embodiments of the present disclosure relate to branched polymers that are used as safe and non-toxic nucleic acid transfection agents, for example in gene therapy applications. [Background technology]

[0003] Delivery of functional genetic material to cells (e.g., skin fibroblasts) to manipulate transgene expression is of great importance in nanomedicine. Despite the development of numerous polymeric gene delivery systems, highly safe and efficient gene transfection (e.g., fibroblast gene transfection) has yet to be achieved.

[0004] After nearly three decades of development, gene therapy has become a key part of the burgeoning nanomedicine armamentarium for improving health and correcting genetic disorders. [1] Although several clinical trials are underway with viral gene delivery vectors, the risks of triggering immunogenic responses and mutagenesis due to the transgene insertion, limitations associated with large-scale production of genetic material and low "cargo capacity", as well as unpredictable vector mobility remain to be addressed. [2、3]From this perspective, non-viral gene delivery vectors seem more promising due to their minimal immunogenicity, non-tumorigenicity, cost-effective manufacturing, high payload of nucleic acid and the possibility of localized gene expression. Since 2010, the number of clinical trials of gene therapy using non-viral gene vectors has increased significantly, with plasmid DNA and small interfering RNA (siRNA) being formulated in at least 40 nanoparticle-based gene therapies for gene correction, therapeutic protein expression, and antigen vaccination, 12 major liposomal systems being investigated in 27 clinical trials, and 7 polymer-based systems being investigated in 13 clinical trials. [3] Among polymer-based gene therapy clinical trials, the commercially available cationic polymer polyethyleneimine (PEI) has shown some promise, but PEI is non-degradable and concerns about its safety have seriously hindered its use. [4] Therefore, in order to bring polymer-based gene therapy closer to clinical application, considerable efforts are being made to improve the gene transfection efficiency and safety of polymer gene vectors.

[0005] Among polymeric gene delivery vectors, poly(β-amino esters) (PAEs) are one type of most promising candidate. PAEs were first designed and synthesized by Langer and coworkers by copolymerization of amines with diacrylates through a one-step Michael addition process. [5] The tertiary amines on the backbone and the terminal primary amines act as cationic units to condense DNA into nanoparticles through electrostatic interactions and facilitate polyplex escape from the endo / lysosomes via the "proton sponge effect". The ester bonds on the backbone can be hydrolytically degraded under aqueous conditions to dissociate the polyplexes, release the DNA, and further reduce cytotoxicity after gene transfection.[6,7] After extensive structure / property optimization, [8-10] Several PAEs have been identified for DNA transfection with favorable safety profiles and high transfection efficiencies both in vitro and in vivo. [6、11、12]However, until 2015, almost all studies with PAEs focused on polymers with linear structures. Branched polymers are believed to have greater potential for gene transfection because their three-dimensional (3D) structure and multiple terminal functional groups provide additional advantages to polymer gene vectors. We have successfully developed highly branched poly(β-amino esters) (HPAEs) via a facile one-pot "A2+B3+C2" Michael addition strategy. [13-16] Across a wide range of cell types, HPAEs exhibited much higher gene transfection capacity than their linear counterparts, demonstrating their greater potential in gene delivery. The high gene transfection capacity of HPAEs was further demonstrated in vivo using a recessive dystrophic epidermolysis bullosa (RDEB) skin disease model. RDEB is a rare and devastating inherited mechanical bullous disease caused by mutations in the COL7A1 gene, which encodes type VII collagen (C7), a critical component of anchoring fibers (AFs) that function to anchor epidermal-dermal adhesions.

[17] Deficiency of C7 leads to fragile skin, extensive blisters, and sores that heal with excessive scarring and milia formation.

[18] In both RDEB knockout and transplanted mouse models, HPAE mediated high levels of C7 expression and restoration of C7 expression for up to 10 weeks. [13、15、19] , highlighting its great potential for clinical cutaneous gene delivery. HPAEs are further described in U.S. Patent Publication No. 2017 / 0216455, which is hereby incorporated by reference in its entirety for all purposes.

[0006] Fibroblasts play a central role in maintaining skin tissue integrity and biological functions, controlling the cellular microenvironment, and are associated with multiple skin diseases such as hypertrophic scarring, aging / photoaging, diabetic wound healing, cancer, and hypertrophic dermatomyositis. The ability to manipulate gene expression in fibroblasts is fundamental for functional genomics, pathway analysis, and biomedical applications. For example, primary human dermal fibroblasts (HPDFs) are an accessible source of phenotypically and karyotypically normal human skin cells and are biologically more relevant for in vivo applications compared to immortalized cell lines.

[20] In the past, HPDF was injected directly into the skin for C7 repair in RDEB. Nevertheless, direct intradermal injection of HPDF did not result in the abnormal morphology of AF in RDEB patients.

[21] and transient protein replacement

[22] It has been shown that fibroblasts can be diversified and more suitable for clinical gene therapy after genetic manipulation by transfection. C7 enrichment of HPDFs is expected to have a significant effect on improving the strength and stability of reconstituted AF, optimizing the administration schedule, and reducing the frequency of RDEB administration. However, non-viral gene transfection of fibroblasts has always been difficult. The most common methods include electroporation, magnetofection, and chemical preparations, which are relatively inefficient and toxic, all of which are expensive. [20、23、24] For example, enhanced green fluorescent protein (EGFP) delivery efficiency was significantly improved in human dermal fibroblasts by different electroporation systems.

[25] 27% in human primary fibroblasts

[26] The maximum transfection efficiency in mouse embryonic fibroblasts was 15.7%, 11.8%, and 48.1% for the potent cationic lipid reagents TransFectin, Lipofectamine LTX, and electroporation, respectively.

[24] .

[0007] Therefore, the development of a reliable non-viral gene delivery system for transfecting fibroblasts with high efficiency and safety is essential and of great importance. Summary of the Invention

[0008] In some embodiments, the present disclosure provides branched polymers suitable for forming polyplexes useful for gene transfection therapy, the polyplexes being prepared by the following process: (a) A compound of formula (A) [ka] with a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1; (b) reacting the product of step (a) with a second amine having the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2; (c) reacting the product of step (b) with a compound of formula (B), [ka] During the ceremony, each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; Each Q is C1-C 10 is a linear or branched alkyl group, Each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl), and R1 is unsubstituted or is halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10) aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; A branched polymer is provided, wherein each n is at least 1.

[0009] In some embodiments, the present disclosure provides a method of making a polymer, comprising: (a) A compound of formula (A) [ka] with a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1; (b) reacting the product of (a) with a second amine having the formula R2-NH2 or R1-N(H)-Z''-N(H)-R2; (c) reacting the product of (b) with a compound of formula (B), [ka] During the ceremony, each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; Each Q is H or C1-C 10 is a linear or branched alkyl group, Each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl), and R1 is unsubstituted or is halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 ) aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; wherein each n is at least 1.

[0010] In some embodiments, the present disclosure provides a method for the preparation of a nucleic acid component and a polymer prepared by the methods described herein, or a polymer having formula (I): [ka] and a polymer comprising the formula: each A is independently a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; each B is independently a first binding moiety; Each X is independently [ka] and Each Y is independently [ka] and each L is independently a second linking moiety; Each R, R, and R, independently in each occurrence, is H, C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C6 alkyl, C2-C8 alkenyl, C4-C8 cycloalkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2; or R2 and R3 together with the atom to which they are attached may form a heterocyclyl or heteroaryl containing 1 to 3 heteroatoms selected from the group consisting of N, S, P, and O; a is 1 to 1000; b is 1 to 4; c is 1 to 3; z is 1 to 100; provided that at least one of R2 and R3 is not H.

[0011] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a polyplex according to certain embodiments of the present disclosure in combination with a pharma- ceutically acceptable carrier.

[0012] In some embodiments, the present disclosure provides a method of cell transfection comprising contacting one or more target cells with a pharmaceutical composition comprising at least one polyplex according to certain embodiments of the present disclosure under conditions suitable for transfecting the target cells with the one or more polyplexes.

[0013] In some embodiments, the present disclosure provides a method of treating a disease in a patient in need of such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one polyplex according to certain embodiments of the present disclosure, such that one or more of the patient's cells are transfected with the polyplex nucleic acid components.

[0014] In some embodiments, the present disclosure provides a method of treating a disease in a patient in need of such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising at least one polyplex according to certain embodiments of the present disclosure, wherein administration of the composition corrects defective translation of a target gene in the subject. [Brief description of the drawings]

[0015] [Figure 1] Evaluation of transfection efficiency and cell viability is shown. a) Gluc activity and cell viability of HPDF 48 hours after transfection with LBPAE / DNA, PEI / DNA, and SuperFect / DNA polyplexes at a range of w / w ratios. b) Gluc activity and cell viability of 3T3. *Significant difference from PEI and #SuperFect groups in Gluc activity (p<0.05, two-tailed Student's t-test). [Diagram 2] LC50 evaluation of LBPAE / DNA polyplexes and SuperFect / DNA polyplexes in HPDF and 3T3. a, Representative live / dead images of untreated cells or cells transfected with LBPAE / DNA polyplexes at a concentration of 555 μg / mL-1 or SuperFect / DNA polyplexes at a concentration of 35 μg / mL-1. Scale bar is 50 μm. b, LBPAE / DNA polyplex concentration-dependent cell viability measured by Alamar Blue assay. c, SuperFect / DNA polyplex concentration-dependent cell viability measured by Alamar Blue assay. [Diagram 3]Figure 1 shows a comparison of GFP expression and MFI mediated by different gene delivery systems. a shows GFP images of HPDF cells after treatment with LBPAE / DNA, PEI / DNA, and SuperFect / DNA polyplexes. Untreated (UT) cells were used as a negative control. Scale bar 200 μm. b shows histogram distribution of HPDF population after transfection with different polyplexes. c shows the percentage and MFI of GFP-positive HPDF cells after transfection. d shows GFP images of 3T3. Scale bar 200 μm. e shows histogram distribution of 3T3 population after transfection with different polyplexes. f shows the percentage and MFI of GFP-positive 3T3 cells after transfection. *Significant difference from commercial reagent group in percentage and #MFI of GFP-positive cells (p<0.05, two-tailed Student's t-test). [Figure 4] Physicochemical characterization of LBPAE / polyplexes. a) DNA condensation ability quantification by agarose gel electrophoresis. b) DNA binding affinity measurement by PicoGreen assay. c) Polyplex size and zeta potential measurements. d) Polyplex morphology observation by TEM. Scale bar 200 nM. [Diagram 5] Figure 1 shows the cellular uptake of various polyplexes. a) Fluorescence images of cells 4 hours after transfection with different polyplexes. Nuclei were stained with DAPI (blue) and DNA was labeled with Cy3 (red). Scale bar 20 μm. b) Polyplex uptake efficiency in HPDFs quantified by flow cytometry. c) Polyplex uptake efficiency in 3T3s quantified by flow cytometry. d) Percentage of Cy3-positive HPDFs and normalized MFI of cells. e) Percentage of Cy3-positive 3T3s and normalized MFI of cells. *Significant difference from SuperFect in MFI quantification (p<0.05, two-tailed Student's t-test). [Figure 6]Figure 1 shows the proton buffering capacity, degradation, and DNA release evaluation of LBPAE. a) Proton buffering capacity quantified by acid-base titration. b) Degradation profile quantified using GPC. c) DNA release from polyplexes evaluated by PicoGreen assay. [Figure 7] Immunofluorescence staining of C7 expression in HPDFs. a) Fluorescence images of HPDFs 4 days after transfection with LBPAE / MCC7 polyplexes. Nuclei were stained with DAPI (blue), and C7 was incubated with monoclonal anti-collagen VII primary antibody and stained with Alexa-568 goat anti-mouse secondary antibody (red). Scale bar 20 μm. b) Flow cytometry quantification of C7 expression in HPDFs. c) Degree of upregulation of C7 expression and MFI of HPDFs after transfection with LBPAE / MCC7 and SuperFect / DNA polyplexes. *Percentage of C7 upregulation and #Significant difference from SuperFect in MFI (p<0.05, two-tailed Student's t-test). [Figure 8] Figure 1 is a schematic diagram of the synthesis of LBPAEs through a combinatorial strategy of linear oligomers. In step 1, A2-type amines react with C2-type diacrylates to form linear A2-C2 base oligomers, which are further end-capped with a second amine to generate linear A2-C2 oligomers. In step 2, the linear A2-C2 oligomers are combined with B3-type triacrylates via branching to give LBPAEs. Boxes indicate the monomers and end-capping agents used in the synthesis of LBPAEs in this work. [Figure 9] 4 shows the GPC results for linear oligomers and LBPAE. [Figure 10] The MH alpha curve and LBPAE values ​​are shown. [Figure 11] 1 shows the chemical composition analysis of LBPAE by 1H NMR. [Figure 12] Agarose gel results of MCC7 and pcDNA3.1COL7A1 are shown. [Figure 13] a shows the synthesis of an HPAE via the "A2+B3+C2" Michael addition strategy; b shows the GPC curve and calculated Mw of an HPAE of the present disclosure; c shows the MH alpha curve and calculated Mw of an HPAE of the present disclosure. (Figures 13 and 16-19 use "HC32-122" to define the HPAE polymer of the present disclosure. HC32-122 corresponds to HC32-DATOU used in the examples.) [Figure 14] Transfection of RDEB keratinocytes with polyplexes containing HPAEs with MW of 11 kDa, 21 kDa, 34 kDa, and 41 kDa is shown using HPAE / DNA ratios of 10:1, 30:1, and 50:1 (wt% / wt%). [Figure 15] 1 shows cellular DNA following gene transfection of RDEB keratinocytes with polyplexes containing HPAEs with MW of 11 kDa, 21 kDa, 34 kDa, and 41 kDa using polymer / DNA ratios of 10:1, 30:1, and 50:1 (wt% / wt%). [Figure 16]Figure 1 shows reporter gene transfection studies in RDEBK cells using HPAE / DNA polyplexes. a shows the relative Gluc activity of RDEBK cells 48 hours after transfection with HPAE / DNA and PEI / DNA polyplexes. Data are presented as percentages normalized to the Gluc activity of RDEBK cells transfected with HPAE / DNA polyplexes (30:1 wt% / wt%). *Significant difference from the HPAE group (w / w=30:1) (p<0.05, Student's t-test). b shows the viability of RDEBK cells after transfection with HPAE / DNA and PEI / DNA polyplexes, and c shows GFP images of untreated (UT) cells, cells treated with HPAE / DNA (w / w=30:1) or PEI / DNA (w / w=1:1) polyplexes. Scale bar 200 μm. d shows representative histogram distribution of UT and transfected cell populations, and e shows the percentage of GFP-positive RDEBK cells and MFI quantified by flow cytometry. *Percentage of GFP-positive cells and #Significant difference from PEI in cell MFI (p<0.05, Student's t-test). [Figure 17] Figure 1 shows MCC7 biosynthesis and cellular uptake of HPAE / MCC7 polyplexes. a) MCC7 biosynthesis using phiC31 and 1-scel digestion systems. b) Agarose gel electrophoresis of three COL7A1-encoding plasmid DNAs after EcoR1 digestion. The backbone lengths of pcDNA3.1COL7A1 regular plasmid (RP), MN511A-1-COL7A1 parental plasmid (PP), and MCC7 are 5 kb, 8 kb, and 3 kb, respectively. c) Fluorescence images of RDEBK cells after transfection with different polyplexes. Nuclei were stained with DAPI (blue) and DNA was labeled with Cy3 (red). Scale bar is 20 μm. d) Polyplex cellular uptake efficiency quantified by flow cytometry, and e) percentage of Cy3 positive cells and MFI. *Significant difference from the PEI / MCC7 group in cell MFI (p<0.05, Student's t-test). [Figure 18]Figure 1 shows COL7A1m RNA and recombinant C7 expression after transfection with HPAE / MCC7 polyplex. a shows the amplification plot of endogenous control GAPDH obtained by RT-qPCR. b shows the amplification plot of COL7A1 mRNA in RDEBK cells after transfection obtained by RT-qPCR. c shows the quantification of COL7A1 mRNA. *Significant difference from PEI group (p<0.05, Student's t-test). d shows the cell immunofluorescence image of C7 staining (red fluorescence) (scale bar is 20 μm). e shows the western blotting result of C7 expression. 42 kDa β-actin was used as a loading control. [Figure 19] Figure 1 shows the physicochemical characterization of HPAE / MCC7 polyplexes with a wt% / wt% ratio of HPAE / DNA of 30:1. a) HPAE / MCC7 polyplex formation. b) Agarose gel results of DNA condensation and heparin competition assay 2 hours after polyplex preparation. c) DNA binding ability test in the presence or absence of heparin by PicoGreen assay 2 hours after polyplex preparation. d) Size of HPAE / MCC7 polyplexes measured by NTA. e) Zeta potential distribution of HPAE / MCC7 polyplexes. f) TEM image of HPAE / MCC7 polyplexes. Scale bar 500 nM. [Figure 20]Figure 1 shows gene transfection performance of formulations containing HPAE polyplexes of the present disclosure. (a) Polyplex freeze-drying and further transfection test in RDEBK cells. (b) GFP images of cells after transfection with polyplexes from different storage methods and freeze-drying conditions. FZ: freeze-drying; Suc: sucrose. Scale bar 200 μm. (c) Representative histogram distribution of UT and transfected cell populations. (d) GFP expression efficiency of cells after transfection quantified by flow cytometry. *Significant difference from freshly prepared polyplex group (p<0.05, Student's t-test). (e) Normalized MFI quantified by flow cytometry. *Significant difference from freshly prepared polyplex group (p<0.05, Student's t-test). [Figure 21] 1 shows transfection of HPAE polyplexes of the present disclosure into RDEBK cells after long-term storage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] As used above, and throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. In the absence of a term, conventional terms known to those skilled in the art shall prevail.

[0017] As used herein, the terms "including," "containing," and "comprising" are used in their open, non-limiting sense.

[0018] The articles "a" and "an" are used in this disclosure to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0019] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0020] For more concise explanation, some of the quantitative expressions shown herein are not modified with the term "about". It should be understood that all amounts shown herein are intended to mean the actual given value, whether or not the term "about" is explicitly used, and also to mean the approximation to such given value that is reasonably inferred based on the ordinary skill in the art, including the equivalent value and approximation by experimental and / or measurement conditions to such given value. Whenever a yield is shown as a percentage, such a yield means the mass of the entity for which the yield is shown with respect to the maximum amount of the same entity that can be obtained under specific stoichiometric conditions. Unless otherwise indicated, a concentration shown as a percentage means a mass ratio.

[0021] A "patient" is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or a non-human primate, such as a monkey, chimpanzee, baboon, or rhesus monkey. A "patient" includes both humans and animals.

[0022] When used in relation to a compound, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound sufficient to provide a desired biological result. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition containing a compound as disclosed herein that is required to cause a clinically significant reduction in a disease. The appropriate "effective amount" in each individual case can be determined by one of ordinary skill in the art using routine experimentation. Thus, the term "effective amount" generally refers to an amount of an active agent that has a therapeutic effect.

[0023] As used herein, the term "treat" or "treatment" is synonymous with the term "prevent" and is intended to refer to postponing the onset of a disease, preventing the onset of a disease, and / or reducing the severity of such symptoms that have developed or are expected to develop. Thus, these terms include improving existing disease symptoms, preventing additional symptoms, improving or preventing the underlying cause of a symptom, inhibiting a disorder or disease, for example, arresting the onset of a disorder or disease, relieving a disorder or disease, causing the regression of a disorder or disease, relieving a condition caused by a disease or disease, or arresting or alleviating a disease or disease symptom. In certain embodiments, "treat" or "treatment" refers to promoting a healthy skin phenotype.

[0024] The term "disorder" is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

[0025] By use of the terms "pharmacologically acceptable" or "pharmacologically acceptable" is intended to mean a substance that is biologically or otherwise undesirable, which can be administered to an individual without causing substantial undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0026] As used in this disclosure, the term "carrier" encompasses carriers, excipients, and diluents, and refers to a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a pharmaceutical agent from one organ or part of the body of a subject to another. Excipients should be selected based on the compatibility and release profile of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, spray-dried dispersions, and the like.

[0027] The term "pharmaceutical compatible carrier material" may include, for example, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. See, e.g., Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975.

[0028] As used herein, the term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other ape and monkey species; livestock animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the present disclosure, the mammal is a human.

[0029] As used in this disclosure, the terms "administered," "administration," or "administering" refer to either administering directly to a subject a disclosed compound, or a pharma- ceutical acceptable salt of a disclosed compound, or a composition, or administering to a subject a prodrug derivative or analog of a disclosed compound, or a pharma- ceutical acceptable salt of a disclosed compound, or a composition, that is capable of forming an equivalent amount of an active compound in a subject (including an animal) in need of treatment by contacting or otherwise exposing such individual to such compound.

[0030] As used herein, "alkyl" refers to a straight or branched saturated chain having from 1 to 40 carbon atoms. Representative saturated alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like, as well as longer alkyl groups (e.g., heptyl, octyl, and the like). Alkyl groups can be unsubstituted or substituted. Alkyl groups containing 3 or more carbon atoms can be straight or branched. As used herein, "lower alkyl" means an alkyl having 1 to 10 carbon atoms.

[0031] As used herein, "alkenyl" includes unbranched or branched hydrocarbon chains containing 2 to 40 carbon atoms. An "alkenyl" group contains at least one double bond. The double bond of an alkenyl group may be unbonded or bonded to another unsaturated group. Examples of alkenyl groups include, but are not limited to, ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl, and the like. Alkenyl groups may be unsubstituted or substituted. Additionally, as defined herein, alkenyl may be branched or straight chain.

[0032] As used herein, "alkynyl" includes unbranched or branched unsaturated hydrocarbon chains containing 2 to 40 carbon atoms. An "alkynyl" group contains at least one triple bond. The triple bond of an alkynyl group may be unbonded or bonded to another unsaturated group. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, 4-butyl-2-hexynyl, and the like. An alkynyl group may be unsubstituted or substituted.

[0033] It should also be noted that any carbon and heteroatoms with unsatisfied valences in the text, schemes, examples, and tables herein are assumed to have sufficient hydrogen atom(s) to satisfy the valences.

[0034] As used herein, references to hydrogen may also refer to deuterium substitution, if desired. As used herein, the term "deuterium" refers to a stable isotope of hydrogen having an odd number of protons and neutrons.

[0035] The term "halo" or "halogen" means fluorine, chlorine, bromine, or iodine.

[0036] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, and the like.

[0037] Unless otherwise specified, the term "aryl" refers to a cyclic aromatic hydrocarbon group having one to three aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. When containing two aromatic rings (such as bicyclic), the aromatic rings of the aryl group may be linked at a single point (e.g., biphenyl) or fused (e.g., naphthyl). An aryl group may be optionally substituted at any point of attachment with one or more substituents (e.g., one to five substituents). The substituents may themselves be optionally substituted. Additionally, when containing two fused rings, an aryl group as defined herein may have an unsaturated or partially saturated ring fused with a fully saturated ring. Exemplary ring systems of such aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthalenyl, tetrahydrobenzoannulenyl, and the like.

[0038] Unless otherwise specified, "heteroaryl" refers to a monovalent monocyclic or polycyclic aromatic radical of 5-18 ring atoms containing one or more ring heteroatoms selected from N, O, or S, with the remaining ring atoms being C. Heteroaryl, as defined herein, also refers to bicyclic heteroaromatic groups in which the heteroatoms are selected from N, O, or S. The aromatic radicals are optionally substituted independently with one or more substituents described herein. The substituents may themselves be optionally substituted. Examples include, but are not limited to, benzothiophene, furyl, thienyl, pyrrolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazolyl, thieno[3,2-b]thiophene, triaryl, ... Zolyl, triazinyl, imidazo[1,2-b]pyrazolyl, furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl, pyrazolo[3,4-c]pyridinyl, benzimidazolyl, thieno[3,2-c]pyridinyl, thieno[2,3-c]pyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl , indolyl, indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxanyl, quinolinyl, isoquinolinyl, 1,6-naphthyridinyl, benzo[de]isoquinolinyl, pyrido[4,3-b][1,6]naphthyridinyl, thieno[2,3-b]pyridinyl, Razinyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1λ 2-pyrrolo[2,1-b]pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, 1H-pyrido[3,4-b][1,4]thiazinyl, benzoxazolyl, benzisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl, [1,2,4]triazolyl Examples of heteroaryl groups include azolo[4,3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl, and derivatives thereof. Furthermore, when containing two fused rings, the heteroaryl groups defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring.

[0039] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated monocyclic fused or spiro polycyclic carbocycle having 3 to 18 carbon atoms per ring. The cycloalkyl ring or carbocycle may be optionally substituted at any attachment point with one or more substituents (e.g., 1 to 5 substituents). The substituents may themselves be optionally substituted. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, bicyclo[2.2.2]octenyl, decahydronaphthalenyl, octahydro-1H-indenyl, cyclopentenyl, cyclohexenyl, cyclohexa-1,4-dienyl, cyclohexa-1,3-dienyl, 1,2,3,4-tetra ...hexa-1,4-dienyl, cyclohexa-1,3-dienyl, cyclohexa-1,4-dienyl, cyclohexa-1,3-dienyl, cyclohexa- dropentalenyl, 3a,4,5,6,7,7a-hexahydro-1H-indenyl, 1,2,3,3a-tetrahydropentalenyl, bicyclo[3.1.0]hexanyl, bicyclo[2.1.0]pentanyl, spiro[3.3]heptanyl, bicyclo[2.2.1]heptanyl, bicyclo[2.2.1]hept-2-enyl, bicyclo[2.2.2]octanyl, 6-methylbicyclo[3.1.1]heptanyl, 2,6,6-trimethylbicyclo[3.1.1]heptanyl, and derivatives thereof.

[0040] As used herein, the term "cycloalkenyl" refers to a partially saturated, monocyclic fused or spiro polycyclic carbocycle having 3 to 18 carbon atoms per ring and having at least one double bond. The cycloalkenyl ring may be optionally substituted at any attachment point with one or more substituents (e.g., 1 to 5 substituents). The substituents may themselves be optionally substituted.

[0041] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially unsaturated and non-aromatic monocyclic, or fused or spiro polycyclic ring structure of 4 to 18 atoms containing carbon atoms and heteroatoms derived from oxygen, nitrogen, or sulfur, where there are no delocalized π electrons (aromaticity) shared between the ring carbons or heteroatoms. The heterocycloalkyl or heterocyclyl ring structure may be substituted by one or more substituents. The substituents may themselves be optionally substituted. Examples of heterocycloalkyl or heterocyclyl rings include, but are not limited to, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide. , piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, homotropanyl, dihydrothiophen-2(3H)-onyl, tetrahydrothiophene 1,1-dioxide, 2,5-dihydro-1H-pyrrolyl, imidazolidin-2-one, pyrrolidin-2-one, dihydrofuran-2(3H)-one, 1,3-dioxolan-2-one, isothiazolidine 1,1-dioxide, 4,5-dihydro-1H-imidazolidin-2-one, pyrrolidin-2-one, dihydrofuran-2(3H)-one, 1,3-dioxolan-2-one, isothiazolidine 1,1-dioxide, Zolyl, 4,5-dihydrooxazolyl, oxiranyl, pyrazolidinyl, 4H-1,4-thiazinyl, thiomorpholinyl, 1,2,3,4-tetrahydropyridinyl, 1,2,3,4-tetrahydropyrazinyl, 1,3-oxazinan-2-one, tetrahydro-2H-thiopyran 1,1-dioxide, 7-oxabicyclo[2.2.1]heptanyl, 1,2-thiazepane 1,1-dioxide, octahydro-2H-ky noridinyl, 1,3-diazabicyclo[2.2.2]octanyl, 2,3-dihydrobenzo[b][1,4]dioxin, 3-azabicyclo[3.2.1]octanyl, 8-azaspiro[4.5]decane, 8-oxa-3-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.1]heptane, 2,8-diazaspiro[5.5]undecanyl, 2-azaspiro[5.5]undecanyl, 3-azaspiro[5.5]undecanyl, decahydroisoquinolinyl, 1-oxa-8-azaspiro[4.5]decanyl, 8-azabicyclo[3.2.1]octanyl, 1,4'-bipiperidinyl, azepanyl, 8-oxa-3-azabicyclo[3.2.1]octanyl, 3,4-dihydro-2H-benzo[b][1,4]oxazinyl, 5,6,7,8-tetrahydroimidazo[1,2-a]pyridinyl, 1,4-diazepanyl, phenoxathiinyl, benzo[d][1 ,3]dioxolyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, 4-(piperidin-4-yl)morpholinyl, 3-azaspiro[5.5]undecanyl, decahydroquinolinyl, piperazin-2-one, 1-(pyrrolidin-2-ylmethyl)pyrrolidinyl, 1,3'-bipyrrolidinyl, and 6,7,8,9-tetrahydro-1H,5H-pyrazolo[1,2-a][1,2]diazepinyl.

[0042] As used herein, numerical ranges are intended to include consecutive integers unless otherwise stated. For example, a range expressed as "from 0 to 5" would include 0, 1, 2, 3, 4, and 5.

[0043] As used herein, the term "substituted" means that the specified group or moiety has one or more suitable substituents that may be attached to the specified group or moiety at one or more positions. For example, an aryl substituted with a cycloalkyl may indicate that the cycloalkyl is attached to an atom of the aryl by a bond or by being fused to the aryl and sharing two or more common atoms.

[0044] As used herein, the term "unsubstituted" means that the specified group bears no substituents.

[0045] The term "optionally substituted" should be understood to mean that a given chemical moiety (e.g., an alkyl group) may (but need not) be bound to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group may be a fully saturated alkyl chain (i.e., pure hydrocarbon). Alternatively, the same optionally substituted alkyl group may have a substituent other than hydrogen. For example, an alkyl group may be bound at any point along the chain to a halogen atom, a hydroxyl group, or any other substituent described herein. Thus, the term "optionally substituted" means that a given chemical moiety may include other functional groups, but does not necessarily have any additional functional groups. Unless otherwise specified, suitable substituents used in the optional replacement of a described group include, but are not limited to, oxo, -halogen, C-C alkyl, C-C alkoxy, C-C haloalkyl, C-C haloalkoxy, -OC-C alkenyl, -OC-C alkynyl, -C-C alkenyl, -C-C alkynyl, -OH, CN(cyano), -CHCN, -OP(O)(OH), -C(O)OH, -OC(O)C-C alkyl, -C(O)C-C alkyl, -C(O)-C-C alkylenyl-cycloalkyl, -C(O)-C-C alkylenyl-heterocycloalkyl, -C(O)-C-C alkylenyl-aryl, -C(O)-C-C alkylenyl-hetero aryl, -OC(O)OC1-C6 alkyl, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, -C(O)NH2, -C(O)NH(C1-C6 alkyl), -C(O)N(C1-C6 alkyl)2, -C(O)NHcycloalkyl, -C(O)N(C1-C6 alkyl)cycloalkyl, -C(O)NHheterocycloalkyl, -C(O)N(C1-C6 alkyl)heterocycloalkyl, -C(O)NHaryl, -C(O)N(C1-C6 alkyl)aryl, -C(O)NHheteroaryl, -C(O)N(C1-C6 alkyl)heteroaryl, -S(O)2-C1-C6 alkyl, -S(O)2-C1-C6 haloalkyl, -S(O)2-cycloalkyl, -S(O)2-heterocycloalkyl,-S(O)2-aryl, -S(O)2-heteroaryl-C0-C6 alkylenyl-S(O)2NH2, -S(O)2NHC1-C6 alkyl, -S(O)2N(C1-C6 alkyl)2, -S(O)2NH cycloalkyl, -S(O)2NH heterocycloalkyl, -S(O)2NH aryl, -S(O)2NH heteroaryl, -NHS(O)2C1-C6 alkyl, -N(C1-C6 alkyl)S(O)2(C1-C6 alkyl), -NHS(O)2 aryl, -N(C1-C6 alkyl)S(O)2 aryl, -NHS(O)2 heteroaryl, -N(C1-C Examples of the substituents include -C-C alkyl)S(O)2 heteroaryl, -NHS(O)2 cycloalkyl, -N(C1-C6 alkyl)S(O)2 cycloalkyl, -NHS(O)2 heterocycloalkyl, -N(C1-C6 alkyl)S(O)2 heterocycloalkyl, -N(C1-C6 alkyl)S(O)2 aryl, -C0-C6 alkylenyl-aryl, -C0-C6 alkylenyl-heteroaryl, -C0-C6 alkylenyl-cycloalkyl, -C0-C6 alkylenyl-heterocycloalkyl, -O-aryl, -NH-aryl, and N(C1-C6 alkyl)aryl. The substituents may themselves be optionally substituted. When multifunctional moieties are depicted, the point of attachment to the core is indicated with a line. For example, (cycloalkyloxy)alkyl- means that the alkyl is the point of attachment to the core, while the cycloalkyl is attached to the alkyl via an oxy group. "Optionally substituted" can also mean "substituted" or "unsubstituted," the meanings of which are explained above.

[0046] As used herein, the term "linker" or "linking moiety" means a group that connects two groups and has a backbone of between 0 and 100 atoms. The linker or bond can be a covalent bond (i.e., a backbone of 0 atoms) connecting two groups or a chain between 1 and 100 atoms in length, for example, about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 carbon atoms in length, where the linker can be linear, branched, cyclic, or a single atom. In certain cases, one or more carbon atoms of the linker backbone may be optionally substituted with a sulfur, nitrogen, or oxygen heteroatom. The bonds between the backbone atoms may be saturated or unsaturated. The linker may include one or more substituents, such as an alkyl, aryl, or alkenyl group. The linker may include, but is not limited to, an oligo(ethylene glycol), an ether, a thioether, a tertiary amine, an alkyl that may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocyclic, or cycloalkyl group, where the backbone includes two or more atoms of the cyclic group, such as 2, 3, or 4 atoms. The linker may be cleavable or non-cleavable.

[0047] Unless otherwise specified, the term molecular weight refers to the weight average molecular weight (M W )

[0048] The term "heteroalkylene" refers to any group that is heterocyclic, such as sulfur, oxygen, or NR d (In the formula, R dmeans a divalent alkylene having one or more carbon atoms replaced with a heteroaryl group (wherein R is hydrogen or alkyl). The heteroalkylene can be linear, branched, cyclic, or combinations thereof.

[0049] The term "heteroalkenylene" means a divalent straight or branched chain hydrocarbyl group having at least one carbon-carbon double bond and one or more heteroatoms (e.g., N, S, or O) in its backbone.

[0050] The term "heteroalkynylene" means a divalent straight or branched chain hydrocarbyl group having at least one carbon-carbon triple bond and one or more heteroatoms (e.g., N, S, or O) in its backbone.

[0051] As used herein, the term "polyplex" refers to a complex between a nucleic acid and a polymer, where the nucleic acid is bound to the polymer via non-covalent bonds, particularly electrostatic bonds.

[0052] The term "plasmid" refers to an extrachromosomal element that often carries genes that are not part of the central metabolism of the cell, and usually takes the form of a circular double-stranded DNA molecule. Such elements can be linear, circular, or supercoiled, autonomously replicating, genomic integrating, phage, or nucleotide sequences of single- or double-stranded DNA or RNA from any source, in which multiple nucleotide sequences are joined or resynthesized into a unique structure that allows the introduction of a promoter fragment and DNA sequence of a selected gene product, along with appropriate 3' untranslated sequences, into a cell. As used herein, the term "plasmid" refers to a construct made up of genetic material (i.e., nucleic acid). Typically, a plasmid contains an origin of replication that is functional in a bacterial host cell (e.g., Escherichia coli) and a selection marker for detecting bacterial host cells that contain the plasmid.

[0053] The term "Nanoplasmid" refers to a circular DNA sequence with reduced bacterial sequences that provides a desired gene insert in a smaller plasmid. For example, Nanoplasmids generated by the antibiotic-free RNA-OUT selection system and methods for their production are described in U.S. Patent No. 9,109,012 (patented by Nature Technology), which is incorporated herein by reference in its entirety.

[0054] The term "nucleic acid" refers to a biological polymer of nucleotide bases, including, but not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), microRNA (miRNA), and peptide nucleic acid (PNA).

[0055] The term "minicircle" refers to a small, minimally sized circular DNA derived from a parent plasmid by intramolecular recombination to remove bacterial replication sequences.

[0056] The term "gene editing system" refers to a system capable of modifying a target nucleic acid by one of a number of DNA repair pathways.

[0057] The present disclosure is directed to a novel class of branched polymers, including polymers synthesized by a combinatorial strategy of linear oligomers. In some embodiments, linear poly(β-amino ester) oligomers are connected by branching units to form multifunctional linear-branched hybrid poly(β-amino ester)s (LBPAEs). The polymers of the present disclosure are designed and prepared for a variety of applications, including but not limited to, high performance fibroblast gene transfection. Ultra-high transgene expression was achieved with LBPAEs in human primary dermal fibroblasts (HPDFs) and mouse embryonic fibroblasts (3T3), with 3292-fold enhancement of Gluciferase (Gluc) expression and nearly 100% green fluorescent protein (GFP) expression detected. Detailed mechanistic studies revealed that LBPAEs can cross multiple extracellular and intracellular barriers involved in fibroblast gene transfection. More importantly, LBPAE can effectively deliver various genes (e.g., COL7A1) to substantially upregulate desired expression (e.g., type VII collagen protein (C7) in HPDF), showing great potential in treating diseases (e.g., C7-deficient genodermatoses such as recessive dystrophic epidermolysis bullosa (RDEB)).

[0058] polymer In some embodiments, the present disclosure provides a method for producing a method for the production of a pharmaceutical composition comprising the steps of: (a) A compound of formula (A) [ka] with a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1; (b) reacting the product of step (a) with a second amine having the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2; (c) reacting the product of step (b) with a compound of formula (B), [ka] A polymer made by the formula: each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; Each Q is H or C1-C 10 is a linear or branched alkyl group, Each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl), and R1 is unsubstituted or is halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 ) aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; wherein each n is at least 1.

[0059] In some embodiments, the present disclosure provides a method for producing a method for the production of a pharmaceutical composition comprising the steps of: (a) A compound of formula (A) [ka] and a compound of formula (B): [ka] with a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1; (b) reacting the product of step (a) with a second amine having the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2; A polymer made by the formula: each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; Each Q is H or C1-C 10 is a linear or branched alkyl group, Each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl), and R1 is unsubstituted or is halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 ) aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; wherein each n is at least 1.

[0060] In some embodiments, Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms. Z may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. In some embodiments, Z is a linear carbon chain of 1 to 30 carbon atoms. For example, Z can be, but is not limited to, C1-C6 alkyl. 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 It can be an alkylene group, including alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, and C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. In some embodiments, Z is a linear or branched carbon chain of 1-30 carbon atoms, or a linear or branched heteroatom-containing carbon chain of 1-30 atoms. In some embodiments, Z is a linear or branched carbon chain of 1-10 carbon atoms. For example, in some embodiments, Z is [ka] In some embodiments, Z is a branched carbon chain of 1-30 carbon atoms. In some embodiments, Z is a linear or branched heteroatom-containing carbon chain of 1-30 atoms. For example, Z can be a linear or branched carbon chain having one or more carbon atoms substituted with a heteroatom (including, but not limited to, O, N, S, or P). In some embodiments, Z is a carbocycle containing 3-30 carbon atoms. In some embodiments, Z is an alkylene-carbocycle containing 3-30 carbon atoms. For example, in some embodiments, Z is [ka] wherein x is 1 to 1000. In some embodiments, Z is a heterocycle containing 3 to 30 atoms. In some embodiments, Z is an alkylene-heterocycle containing 3 to 30 atoms. In some embodiments, Z is unsubstituted. In some embodiments, Z is substituted. In some embodiments, Z is selected from the following: [ka] It is one of them.

[0061] In some embodiments, Z' is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms. Z' may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. In some embodiments, Z' is a linear carbon chain of 1 to 30 carbon atoms. For example, Z' can be, but is not limited to, C1-C6 alkyl. 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 It can be an alkylene group including alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. In some embodiments, Z' is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms.

[0062] In some embodiments, Z'' is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms. Z'' may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. In some embodiments, Z'' is a linear carbon chain of 1 to 30 carbon atoms. For example, Z'' can be, but is not limited to, C1-C6 alkyl. 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 It can be an alkylene group including alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, and C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. In some embodiments, Z'' is a straight or branched carbon chain of 1 to 30 carbon atoms or a straight or branched heteroatom-containing carbon chain of 1 to 30 atoms.

[0063] According to certain embodiments of the present disclosure, G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-, thereby forming a carbonyl, sulfoxide, sulfone, and phosphono group, respectively. Thus, in some embodiments, G is -C-. In some embodiments, G is -S-. In some embodiments, G is -S(O)-.

[0064] In some embodiments, the compound of formula (B) is [ka] wherein R is a linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched carbon chain containing a heteroatom of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms, and R is unsubstituted or is selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C 6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; ’’is an unsubstituted or substituted linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms. In some embodiments, R is 1 carbon atom. In some embodiments, R'' is a straight or branched carbon chain, e.g., methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl. For example, in some embodiments, the compound of formula (B) is [ka] In some embodiments, R is a carbocycle containing 3 to 10 carbon atoms. For example, R can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, phenyl, or naphthyl. In some embodiments, R is a heterocycle containing 3 to 10 atoms.

[0065] In certain embodiments, the first amine has the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1. In some embodiments, the first amine has the formula R1-NH2. In some embodiments, the first amine has the formula R1-N(H)-Z'-N(H)-R1. In some embodiments, the first amine has the formula R1-N(H)-Z'-N(H)-R1. In some embodiments, the first amine having the formula R1-N(H)-Z'-N(H)-R1 is [ka] In some embodiments, the first amine has the formula R1-N(H)-Z'-N-(R1)2. In some embodiments, the first amine has the formula R1-N(H)-Z'-N-(R1)2: [ka] It is.

[0066] In certain embodiments, the second amine has the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2. In some embodiments, the second amine has the formula R2-NH2. In some embodiments, the second amine has the formula R2-N(H)-Z''-N(H)-R2. In some embodiments, the second amine having the formula R2-N(H)-Z''-N(H)-R2 is [ka] In some embodiments, the first amine has the formula R2-N(H)-Z''-N-(R2). In some embodiments, the first amine has the formula R2-N(H)-Z''-N-(R2)2: [ka] It is.

[0067] In certain embodiments, R is C-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 Heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2. R1 may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 In some embodiments, R is substituted with at least one of C1-C6 alkyl, C1-C6 aryl, or C1-C6 aryl. 20 For example, R1 is C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20Alkyl groups, for example, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- It can be 1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, or n-icosyl. In some embodiments, R1 is unsubstituted. In some embodiments, R1 is substituted. In some embodiments, R1 is [ka] In some embodiments, R is selected from the group consisting of: [ka] In some embodiments, R is [ka] It is.

[0068] In certain embodiments, R2 is C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40Heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2. R2 may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 In some embodiments, R2 is substituted with at least one of C1-C6 alkyl, C1-C6 aryl, or C1-C6 aryl. 20 For example, R2 is C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20Alkyl groups, for example, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- It can be 1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, or n-icosyl. In some embodiments, R2 is unsubstituted. In some embodiments, R2 is substituted. In some embodiments, R2 is [ka] In some embodiments, R2 is selected from the group consisting of: [ka] In some embodiments, R2 is [ka] It is.

[0069] In some embodiments, each Q is H or C 10 A straight chain or branched alkyl group. Thus, in some embodiments, each Q is H. In other embodiments, each Q is C-C 10It is a straight chain or branched alkyl group.For example, each Q can be methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.In some embodiments, each Q is methyl.

[0070] In some embodiments, each J is -O-. In some embodiments, each J is -NH-.

[0071] In some embodiments, each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene. In some embodiments, each E1 is heteroalkylene. In some embodiments, each E1 is -CH2-O-. In various embodiments, each n is at least 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 1.

[0072] In some embodiments, A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms. For example, in some embodiments, A is [ka] It is.

[0073] In some embodiments, the polymers of the present disclosure have the general structure [ka] wherein the wavy bond [ka] represents a bond to the remainder of the polymer, and R1, R2, and R4 have any of the definitions provided herein. Due to the highly controlled growth and branching of the continuous linear oligomers, the resulting polymer has a more uniform distribution of linear segments and branching units, as shown in the structure above. As described in the following sections and examples, the polymer has strong DNA binding affinity and can condense DNA to formulate nano-sized polyplexes with nearly 100% cellular uptake efficiency. In some embodiments, the polymer of the present disclosure is [ka] wherein the R2 group has any definition given herein.

[0074] In some embodiments, the present disclosure provides: [ka] wherein R1, R2, A, E1, G, J, Q, Z, Z'', and n have any of the definitions provided herein. In some further embodiments, the M of the polymer W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. WIn some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0075] In some embodiments, the present disclosure provides: [ka] wherein R1, R2, A, E1, G, J, Q, Z, and n have any of the definitions provided herein. W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. Wis about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0076] In some embodiments, the present disclosure provides: [ka] wherein R1, R2, A, E1, G, J, Q, Z, Z'', and n have any of the definitions provided herein. In some further embodiments, the M of the polymer W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. Wis about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0077] In some further embodiments, the polymer is [ka] Includes.

[0078] In some further embodiments, the polymer is [ka] wherein J is O and Z is [ka] In the formula, x is 1 to 1000.

[0079] In some further embodiments, the polymer is [ka] Includes.

[0080] In some further embodiments, R1 is [ka] is selected from.

[0081] In some further embodiments, R1 is [ka] It is.

[0082] In some further embodiments, R1 is [ka] It is.

[0083] In some further embodiments, R2 is [ka] is selected from.

[0084] In some further embodiments, R2 is [ka] It is.

[0085] In some further embodiments, R2 is [ka] It is.

[0086] In some further embodiments, R1 is [ka] and R2 is [ka] It is.

[0087] In some further embodiments, R1 is [ka] and R2 is [ka] It is.

[0088] In some embodiments, the polymer is [ka] wherein R1 is [ka] and R2 is [ka] In some further embodiments, the M of the polymer is selected from W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0089] In some embodiments, the polymer is [ka] wherein J is O and Z is [ka] wherein x is 1 to 1000; R1 is [ka] and R2 is [ka] It is. In some further embodiments, the M of the polymer W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0090] In some embodiments, certain polymers of the present disclosure can be described as a linear polymer (oligomer) of a compound of formula (A) and a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1 (as described herein) crosslinked with a compound of formula (B) (as described herein). When the linear oligomer is prepared under conditions in which the compound of formula (A) is present in molar excess over the first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1, the resulting oligomeric species terminates with a Michael acceptor group (e.g., acrylate, methacrylate, acrylamide, or other such group) and may also be subsequently endcapped with a second amine having the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2 (as described herein) under appropriate conditions. The resulting end-capped oligomers can then be reacted with a trifunctional Michael acceptor compound of formula (B) (as described herein) to give branched structures. Alternatively, such crosslinked polymers can be prepared by reacting the oligomer segments with a molecular weight distribution (e.g., M ranging from about 3 to about 200 as disclosed herein). w value) and the molar or weight percentage of crosslinks derived from the incorporation of a Michael acceptor compound of formula (B).

[0091] In some embodiments, a molar excess of the compound of formula (A) is reacted with the first amine. For example, the stoichiometric ratio of the compound of formula (A):first amine can range from about 1.1:1 to about 10:1, including about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, including all ranges therebetween.

[0092] In some embodiments, the stoichiometric ratio of the compound of formula (A): the first amine is about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the stoichiometric ratio of the compound of formula (A): the first amine can range from about 1.1:1 to about 2:1. In some embodiments, the stoichiometric ratio of the compound of formula (A): the first amine is about 1.2:1. In some embodiments, the compound of formula (A) is reacted with the first amine at a molar equivalent (i.e., about 1:1).

[0093] In some embodiments, step (a) is carried out in an organic solvent. A wide variety of organic solvents can be used in the context of the present disclosure, including but not limited to dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), etc.; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.; ethers, such as tetrahydrofuran (THF), diethyl ether, methyl tertiary butyl ether, etc.; hydrocarbons, such as toluene, xylene, cyclohexane, etc. In some embodiments, step (a) is carried out in DMSO.

[0094] In some embodiments, step (a) is carried out at a temperature ranging from about 40° C. to about 120° C., including about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79 , about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, or 120° C. (including all ranges therebetween).

[0095] In some embodiments, step (a) comprises 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133 1, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, or 120° C. In some embodiments, step (a) is performed at about 90° C.

[0096] In some embodiments, the product of step (a) is not purified prior to step (b). In other embodiments, the product of step (a) is purified prior to step (b). The product of step (a) can be purified by a variety of methods and techniques apparent to those of skill in the art.

[0097] In some embodiments, a molar excess of the second amine is added to the product of step (a). For example, the stoichiometric ratio of the second amine:product of step (a) can range from about 1.1:1 to about 10:1, including about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, including all ranges therebetween.

[0098] In some embodiments, the stoichiometric ratio of the second amine:product of step (a) is about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the stoichiometric ratio of the second amine:product of step (a) is about 5:1. In some embodiments, the second amine is reacted with the product of step (a) in molar equivalents (i.e., about 1:1).

[0099] In some embodiments, step (b) is carried out at a temperature ranging from about 16° C. to about 40° C. For example, step (b) is carried out at a temperature ranging from about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39 to about 40° C., including all ranges therebetween.

[0100] In some embodiments, step (b) is carried out at a temperature of about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40° C.

[0101] In some embodiments, the product of step (b) is not purified prior to step (c). In other embodiments, the product of step (b) is purified prior to step (c). The product of step (b) can be purified by a variety of methods and techniques apparent to one of skill in the art. For example, the product of step (b) can be purified by dialysis.

[0102] In some embodiments, step (c) is carried out at a higher temperature than step (b), for example, step (c) is carried out at a temperature in the range of about 21°C to about 200°C. For example, step (c) can be performed using any of the following methods: about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 1 1, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 20, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, about 199 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200° C. (including all ranges therebetween).

[0103] In some embodiments, step (c) is about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159 , about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200° C. In some embodiments, step (c) is performed at about 90° C.

[0104] In some embodiments, the present disclosure provides a polymer of formula (I): [ka] (I) wherein each A is independently a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; Each B independently: [ka] and G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-, and n is at least 1; Each E1 is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; each E2 is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; Each X is independently [ka] and Each Y is independently [ka] and each L is independently a second linking moiety; Each R, R, and R, independently in each occurrence, is H, C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C6 alkyl, C2-C8 alkenyl, C4-C8 cycloalkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2; or R2 and R3 together with the atom to which they are attached may form a heterocyclyl or heteroaryl containing 1 to 3 heteroatoms selected from the group consisting of N, S, P, and O; a is 1 to 1000; b is 3 or 4, c is 1 to 3; z is 1 to 100; provided that when at least one of R2 and R3 is not H, and G is C, E1 is not -CH2-O-.

[0105] In certain embodiments, the present disclosure provides a polymer of formula (II): [ka] wherein Each E1 is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; each E2 is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; n is at least 1, provided that when G is C, E1 is not -CH2-O-.

[0106] In some embodiments, each E1 and E2 is independently selected from the group consisting of a covalent bond, -N-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene. In some embodiments, each E1 is heteroalkylene. In some embodiments, each E1 is -CH2-N-. In some embodiments, each E2 is alkylene. In some embodiments, each E2 is [ka] In some embodiments, each E2 is [ka] In some embodiments, each n is at least 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 1. In some embodiments, G is -C-. In some embodiments, each B is [ka] be.

[0107] In some embodiments, each L is [ka] wherein x is 1 to 1000. In some embodiments, a is at least 2, b is 3, and each X is [ka] In some embodiments, each A is [ka] In some embodiments, each L is [ka] It is.

[0108] In some embodiments, each R2 and / or R3 is [ka] It is.

[0109] In some embodiments, each R is [ka] It is.

[0110] In some embodiments, the present disclosure provides a polymer of formula (III)-(VIIe): [ka] [ka] [ka] wherein each R, R, and R is independently at each occurrence H, C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C6 alkyl, C2-C8 alkenyl, C4-C8 cycloalkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2, and the remaining variables are as defined above.

[0111] In some embodiments, z is 1, 2, or 3. In some embodiments, z is 1.

[0112] The alpha parameter defined from the Mark-Hwink equation refers to the Mark-Hwink plot. The Mark-Hwink plot is a powerful tool for investigating polymer structures in solution because it reveals structure-molecular weight relationships with high sensitivity. The plot is generated by plotting molecular weight (MW) against intrinsic viscosity (IV) on a log-log graph. Molecular weight, of course, indicates the length (or degree of polymerization) of the polymer chain, but cannot by itself indicate any structure. Intrinsic viscosity (expressed in dL / g) is a measure of the molecular density of polymer chains in solution. The more tightly the chains fold or coil in the solution, the higher the density and the lower the intrinsic viscosity. This measurement is independent of molecular weight, so two different structures with the same molecular weight may have different intrinsic viscosities, for example, a linear (unbranched) polymer and a branched polymer of the same molecular weight will have different intrinsic viscosities. Furthermore, if a polymer changes structure (e.g., becomes more substituted) across its molecular weight distribution, the change in intrinsic viscosity is easily detected. This makes the Mark-Hwink plot very useful and powerful. The raw data for the Mark-Hwink plot is conveniently and simply obtained from high quality multi-detector GPC / SEC data by combining molecular weight from a light scattering detector and intrinsic viscosity from a viscosity detector. Both data sets are measured at each point across the elution profile of the sample. The resulting plot can be used in many ways, ranging from simply assessing how close two structures are to performing complex quantitative measurements of polymer branching. In general: α<0.5: compact / globular chains, 0.5<α<0.8: random coil / flexible chains, 0.5<α<0.8: rigid rods / stiff chains.

[0113] In some embodiments, the alpha parameter as defined by the Mark-Hwink equation for the polymers of the present disclosure is less than about 0.5. For example, the alpha parameter as defined by the Mark-Hwink equation for the polymers of the present disclosure ranges from about 0.01 to about 0.49. For example, the alpha parameter as defined by the Mark-Hwink equation for the polymers of the present disclosure is less than about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49 ... 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48 to about 0.49. In some embodiments, the alpha parameter as defined by the Mark-Hwink equation for the polymers of the present disclosure is from about 0.2 to about 0.5.

[0114] In some embodiments, the alpha parameter, as defined by the Mark-Hwink equation, for the polymers of the present disclosure is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 5 21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, or about 0.49.

[0115] The term "polydispersity index" (PDI) refers to a measure of molecular weight distribution in a given polymer sample. The polydispersity index is calculated by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn). As used herein, the term "weight average molecular weight" generally refers to a molecular weight measurement that depends on the contribution of polymer molecules according to their size. As used herein, the term "number average molecular weight" generally refers to a molecular weight measurement that is calculated by dividing the total weight of all polymer molecules in a sample by the total number of polymer molecules in the sample. These terms are well known to those skilled in the art.

[0116] In some embodiments, the PDI of the polymers of the present disclosure is about 1.01 to about 8.0. For example, the PDI can be about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4 ... The range can be from about 0.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 to about 8.0 (including all ranges therebetween).

[0117] In some embodiments, the PDI, as defined by the Mark-Hwink equation, of the polymers of the present disclosure is about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, 6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the PDI of the polymer of the present disclosure is about 2.5.

[0118] In some embodiments, the M of the polymer of the present disclosure W In some embodiments, the M of the polymer of the present disclosure is at least 3 kDa. W is about 3 kDa to about 200 kDa. Therefore, the M Wis about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 11 4, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about In some embodiments, the M of the polymer ranges from about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200 kDa, including all ranges therebetween. W In some embodiments, the M of the polymer is between about 5 kDa and 50 kDa. Wis between approximately 10 kDa and 50 kDa.

[0119] In some embodiments, the M of the polymer of the present disclosure Wis about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112 , about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 1 57, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200 kDa. W In some embodiments, the M of the polymer is between about 5 kDa and 50 kDa. WIn some embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some embodiments, the M of the polymer is about 10 kDa. W In some embodiments, the M of the polymer is about 20 kDa. W In some embodiments, the M of the polymer is about 30 kDa. W is approximately 40 kDa.

[0120] In some embodiments, the M of the product after step (b) W In some embodiments, the M of the product after step (b) is about 3 kDa. W In some embodiments, the M of the product after step (b) is about 10 kDa. W In some embodiments, the M of the product after step (b) is about 20 kDa. W In some embodiments, the M of the product after step (b) is about 30 kDa. W is approximately 40 kDa.

[0121] Method of preparation In some embodiments, the present disclosure provides a method of making a polymer, comprising: (a) A compound of formula (A) [ka] (A) with a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1; (b) reacting the product of step (a) with a second amine having the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2; (c) reacting the product of step (b) with a compound of formula (B); [ka] wherein each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; Each Q is H or C1-C 10 is a linear or branched alkyl group, Each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl), and R1 is unsubstituted or is halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 ) aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; wherein each n is at least 1.

[0122] In some embodiments, the present disclosure provides a method of making a polymer, comprising: (a) A compound of formula (A) [ka] and a compound of formula (B): [ka] with a first amine having the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1; (b) reacting the product of step (a) with a second amine having the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2; wherein each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10 substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; Each Q is H or C1-C 10 is a linear or branched alkyl group, Each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R1 and R2 are each independently C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl), and R1 is unsubstituted or is halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 ) aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; wherein each n is at least 1.

[0123] In some embodiments, Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms. Z may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. In some embodiments, Z is a linear carbon chain of 1 to 30 carbon atoms. For example, Z can be, but is not limited to, C1-C6 alkyl. 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 It can be an alkylene group, including alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, and C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. In some embodiments, Z is a linear or branched carbon chain of 1-30 carbon atoms, or a linear or branched heteroatom-containing carbon chain of 1-30 atoms. In some embodiments, Z is a linear or branched carbon chain of 1-10 carbon atoms. For example, in some embodiments, Z is [ka] In some embodiments, Z is a branched carbon chain of 1-30 carbon atoms. In some embodiments, Z is a linear or branched heteroatom-containing carbon chain of 1-30 atoms. For example, Z can be a linear or branched carbon chain having one or more carbon atoms substituted with a heteroatom (including, but not limited to, O, N, S, or P). In some embodiments, Z is a carbocycle containing 3-30 carbon atoms. In some embodiments, Z is an alkylene-carbocycle containing 3-30 carbon atoms. For example, in some embodiments, Z is [ka] wherein x is 1 to 1000. In some embodiments, Z is a heterocycle containing 3 to 30 atoms. In some embodiments, Z is an alkylene-heterocycle containing 3 to 30 atoms. In some embodiments, Z is unsubstituted. In some embodiments, Z is substituted. In some embodiments, Z is selected from the following: [ka] It is one of them.

[0124] In some embodiments, Z' is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms. Z' may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. In some embodiments, Z' is a linear carbon chain of 1 to 30 carbon atoms. For example, Z' can be, but is not limited to, C1-C6 alkyl. 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 It can be an alkylene group including alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. In some embodiments, Z' is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms.

[0125] In some embodiments, Z'' is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms. Z'' may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. In some embodiments, Z'' is a linear carbon chain of 1 to 30 carbon atoms. For example, Z'' can be, but is not limited to, C1-C6 alkyl. 24 Alkylene, C1-C 20 Alkylene, C1-C 16 Alkylene, C1-C 12 It can be an alkylene group including alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene, and C1 alkylene. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. In some embodiments, Z'' is a straight or branched carbon chain of 1 to 30 carbon atoms or a straight or branched heteroatom-containing carbon chain of 1 to 30 atoms.

[0126] According to certain embodiments of the present disclosure, G can be -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-, thereby forming a carbonyl, sulfoxide, sulfone, and phosphono group, respectively. Thus, in some embodiments, G is -C-. In some embodiments, G is -S-. In some embodiments, G is -S(O)-.

[0127] In some embodiments, the compound of formula (B) is [ka] where: R is a linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched carbon chain containing a heteroatom of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms, and R is unsubstituted or is selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C 6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halo C1-C6 alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 aryl, where each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; ’’is an unsubstituted or substituted linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms. In some embodiments, R is 1 carbon atom. In some embodiments, R'' is a straight or branched carbon chain, e.g., methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl. For example, in some embodiments, the compound of formula (B) is [ka] In some embodiments, R is a carbocycle containing 3 to 10 carbon atoms. For example, R can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, phenyl, or naphthyl. In some embodiments, R is a heterocycle containing 3 to 10 atoms.

[0128] In certain embodiments, the first amine has the formula R1-NH2 or R1-N(H)-Z'-N(H)-R1. In some embodiments, the first amine has the formula R1-NH2. In some embodiments, the first amine has the formula R1-N(H)-Z'-N(H)-R1. In some embodiments, the first amine has the formula R1-N(H)-Z'-N(H)-R1. In some embodiments, the first amine having the formula R1-N(H)-Z'-N(H)-R1 is [ka] In some embodiments, the first amine has the formula R1-N(H)-Z'-N-(R1)2. In some embodiments, the first amine has the formula R1-N(H)-Z'-N-(R1)2: [ka] It is.

[0129] In certain embodiments, the second amine has the formula R2-NH2 or R2-N(H)-Z''-N(H)-R2. In some embodiments, the second amine has the formula R2-NH2. In some embodiments, the second amine has the formula R2-N(H)-Z''-N(H)-R2. In some embodiments, the second amine having the formula R2-N(H)-Z''-N(H)-R2 is [ka] In some embodiments, the first amine has the formula R2-N(H)-Z''-N-(R2). In some embodiments, the first amine has the formula R2-N(H)-Z''-N-(R2)2: [ka] It is.

[0130] In certain embodiments, R is C-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 Heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2. R1 may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 In some embodiments, R is substituted with at least one of C1-C6 alkyl, C1-C6 aryl, or C1-C6 aryl. 20 For example, R1 is C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20Alkyl groups, for example, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- It can be 1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, or n-icosyl. In some embodiments, R1 is unsubstituted. In some embodiments, R1 is substituted. In some embodiments, R1 is [ka] In some embodiments, R is selected from the group consisting of: [ka] In some embodiments, R is [ka] It is.

[0131] In certain embodiments, R2 is C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40Heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C 40 Alkyl, C2-C 40 Alkenyl, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2. R2 may be unsubstituted or may be selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, and C6-C 10 In some embodiments, R2 is substituted with at least one of C1-C6 alkyl, C1-C6 aryl, or C1-C6 aryl. 20 For example, R2 is C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20Alkyl groups, for example, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- It can be 1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, or n-icosyl. In some embodiments, R2 is unsubstituted. In some embodiments, R2 is substituted. In some embodiments, R2 is [ka] In some embodiments, R2 is selected from the group consisting of: [ka] In some embodiments, R2 is [ka] It is.

[0132] In some embodiments, each Q is H or C 10 A straight chain or branched alkyl group. Thus, in some embodiments, each Q is H. In other embodiments, each Q is C-C 10It is a straight chain or branched alkyl group.For example, each Q can be methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.In some embodiments, each Q is methyl.

[0133] In some embodiments, each J is -O-. In some embodiments, each J is -NH-.

[0134] In some embodiments, each E1 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene. In some embodiments, each E1 is heteroalkylene. In some embodiments, each E1 is -CH2-O-. In some embodiments, each n is at least 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 1.

[0135] In some embodiments, A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms. For example, in some embodiments, A is [ka] It is.

[0136] In some embodiments, the polymers of the present disclosure have the general structure [ka] where the wavy bond represents the bond to the rest of the polymer. Due to the highly controlled growth and branching of the continuous linear oligomers, the resulting polymer has a more uniform distribution of linear segments and branching units, as shown in the structure above. As described in the following sections and examples, the polymer has strong DNA binding affinity and can compact DNA to formulate nano-sized polyplexes with nearly 100% cellular uptake efficiency. In some embodiments, the polymer of the present disclosure has [ka] It is.

[0137] In some embodiments, a molar excess of the compound of formula (A) is reacted with the first amine. For example, the stoichiometric ratio of the compound of formula (A):first amine can range from about 1.1:1 to about 10:1, including about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, including all ranges therebetween.

[0138] In some embodiments, the stoichiometric ratio of the compound of formula (A): the first amine is about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the stoichiometric ratio of the compound of formula (A): the first amine can range from about 1.1:1 to about 2:1. In some embodiments, the stoichiometric ratio of the compound of formula (A): the first amine is about 1.2:1. In some embodiments, the compound of formula (A) is reacted with the first amine at a molar equivalent (i.e., about 1:1).

[0139] In some embodiments, step (a) is carried out in an organic solvent. A wide variety of organic solvents can be used in the context of the present disclosure, including but not limited to dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), etc.; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.; ethers, such as tetrahydrofuran (THF), diethyl ether, methyl tertiary butyl ether, etc.; hydrocarbons, such as toluene, xylene, cyclohexane, etc. In some embodiments, step (a) is carried out in DMSO.

[0140] In some embodiments, step (a) is carried out at a temperature ranging from about 40° C. to about 120° C., including about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79 , about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, or 120° C. (including all ranges therebetween).

[0141] In some embodiments, step (a) comprises 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133 1, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, or 120° C. In some embodiments, step (a) is performed at about 90° C.

[0142] In some embodiments, the product of step (a) is not purified prior to step (b). In other embodiments, the product of step (a) is purified prior to step (b). The product of step (a) can be purified by a variety of methods and techniques apparent to those of skill in the art.

[0143] In some embodiments, a molar excess of the second amine is added to the product of step (a). For example, the stoichiometric ratio of the second amine:product of step (a) can range from about 1.1:1 to about 10:1, including about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, including all ranges therebetween.

[0144] In some embodiments, the stoichiometric ratio of the second amine to the product of step (a) is about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments, the stoichiometric ratio of the second amine to the product of step (a) is about 5:1. In some embodiments, the second amine is reacted with the product of step (a) at a molar equivalent (i.e., about 1:1).

[0145] In some embodiments, step (b) is carried out at a temperature ranging from about 16° C. to about 40° C. For example, step (b) is carried out at a temperature ranging from about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39 to about 40° C., including all ranges therebetween.

[0146] In some embodiments, step (b) is carried out at a temperature of about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40° C.

[0147] In some embodiments, the product of step (b) is not purified prior to step (c). In other embodiments, the product of step (b) is purified prior to step (c). The product of step (b) can be purified by a variety of methods and techniques apparent to one of skill in the art. For example, the product of step (b) can be purified by dialysis.

[0148] In some embodiments, step (c) is carried out at a higher temperature than step (b), for example, step (c) is carried out at a temperature in the range of about 21°C to about 200°C. For example, step (c) can be performed using any of the following methods: about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 1 1, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 20, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, about 199 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200° C. (including all ranges therebetween).

[0149] In some embodiments, step (c) is about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159 , about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200° C. In some embodiments, step (c) is performed at about 90° C.

[0150] In some embodiments, the alpha parameter, as defined by the Mark-Hwink equation, of the polymers produced by the methods of the present disclosure is less than about 0.5. For example, the alpha parameter, as defined by the Mark-Hwink equation, of the polymers of the present disclosure is less than about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 10.30, about 10.31, about 10.32, about 10.33, about 10.34, about 10.35, about 10.36, about 10.37, about 10.38, about 10.39, about 11.40, about 11.50, about 11.60, about 11.70, about 11.80, about 11.90, about 12.11, about 12.12, about 12.13, about 12.14, about 12.15, about 12.16, about 12.17, about 12.18, about 12.19, about 13.18, about 13.20, about 13.21, about 13.19, about 14. 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48 to about 0.49. In some embodiments, the alpha parameter, as defined by the Mark-Hwink equation, for polymers produced by the methods of the present disclosure is from about 0.2 to about 0.5.

[0151] In some embodiments, the alpha parameter, as defined by the Mark-Hwink equation, for polymers produced by the methods of the present disclosure is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, 0, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, or about 0.49.

[0152] In some embodiments, the PDI of the polymers produced by the methods of the present disclosure is about 1.01 to about 8.0. For example, the PDI can be about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4 ... The range can be from about 0.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 to about 8.0 (including all ranges therebetween).

[0153] In some embodiments, the PDI, as defined by the Mark-Hwink equation, of the polymers produced by the methods of the present disclosure is about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 0.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the PDI of the polymer of the present disclosure is about 2.5. In some embodiments, the PDI of the polymer of the present disclosure is about 3.5. In some embodiments, the PDI of the polymer of the present disclosure is about 6.5. In some embodiments, the PDI of the polymer of the present disclosure is about 8.5.

[0154] In some embodiments, the M of the polymer produced by the method of the present disclosure. W In some embodiments, the M of the polymer produced by the methods of the present disclosure is at least 3 kDa. W is about 3 kDa to about 200 kDa. Therefore, the M of the polymer of the present disclosure Wis about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157 , about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200 kDa. W In some embodiments, the M of the polymer is between about 5 kDa and 50 kDa. Wis between approximately 10 kDa and 50 kDa.

[0155] In some embodiments, the M of the polymer produced by the method of the present disclosure. Wis about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112 , about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 1 57, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199 to about 200 kDa. W In some embodiments, the M of the polymer produced by the methods of the present disclosure is between about 5 kDa and 50 kDa.W In some embodiments, the M of the polymer produced by the methods of the present disclosure is about 10 kDa. W In some embodiments, the M of the polymer produced by the methods of the present disclosure is about 20 kDa. W In some embodiments, the M of the polymer produced by the methods of the present disclosure is about 30 kDa. W is approximately 40 kDa.

[0156] In some embodiments, the M of the product after step (b) W is approximately 3 kDa.

[0157] Polyplex In some embodiments, the disclosure provides a method for the preparation of a branched polymer comprising the steps of: combining a nucleic acid moiety as described herein with any of the branched polymers disclosed herein, e.g., a polymer made by a process described herein, or a polymer of formula (I): [ka] and a polyplex comprising: each A is independently a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, haloC1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl, or C6-C 10substituted with aryl, wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; each B is independently a first binding moiety; Each X is independently [ka] and Each Y is independently [ka] and each L is independently a second linking moiety; Each R, R, and R, in each occurrence, is independently H, C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C6 alkyl, C2-C8 alkenyl, C4-C8 cycloalkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2; or R2 and R3 together with the atom to which they are attached may form a heterocyclyl or heteroaryl containing 1 to 3 heteroatoms selected from the group consisting of N, S, P, and O; a is 1 to 1000; b is 1 to 4; c is 1 to 3; z is 1 to 100; provided that at least one of R2 and R3 is not H.

[0158] In certain embodiments, a polyplex comprises a nucleic acid component and a polymer of formula (II): [ka] and wherein Each E1 is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; each E2 is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; G is -C-, -S-, -S(O)-, -P(OR1)-, or -P(OH)-; n is at least 1.

[0159] In some embodiments, each B is independently: [ka] In some embodiments, each E1 and E2 is independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene. In some embodiments, each E1 is heteroalkylene. In some embodiments, each E1 is -CH2-O-. In some embodiments, each E2 is alkylene. In some embodiments, each E2 is [ka] In some embodiments, each E2 is [ka] In some embodiments, each n is at least 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 1. In some embodiments, G is -C-. In some embodiments, each B is [ka] It is.

[0160] In some embodiments, B and A are combined to form [ka] Form.

[0161] In some embodiments, each L is [ka] wherein x is 1 to 1000. In some embodiments, a is at least 2, b is 3, and each X is [ka] In some embodiments, each A is [ka] In some embodiments, each L is [ka] It is.

[0162] In some embodiments, Y is [ka] It is.

[0163] In some embodiments, each R2 and / or R3 is [ka] It is.

[0164] In some embodiments, each R is [ka] It is.

[0165] In some embodiments, a polyplex comprises a nucleic acid component and a polymer of Formulae (III)-(VIIe): [ka] [ka] [ka] wherein each R, R, and R, in each occurrence, is independently selected from H, C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C 40 Alkenyl, C2-C 40 Heteroalkenylene, C4-C8 cycloalkenyl, C2-C 40 Alkynyl, C2-C 40 heteroalkynylene, C3-C8 cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C1-C6 alkyl, C2-C8 alkenyl, C4-C8 cycloalkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with D, halogen, C1-C6 alkyl, -OH, -O-C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2, and the remaining variables are as defined above.

[0166] In some embodiments, z is 1, 2, or 3. In some embodiments, z is 1.

[0167] In some embodiments, the polyplex comprises a nucleic acid component and [ka]

[0168] wherein R1, R2, A, E1, G, J, Q, Z, Z'', and n have any of the definitions provided herein. In some further embodiments, the M of the polymer W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0169] In some embodiments, the polyplex comprises a nucleic acid component and [ka] wherein R1, R2, A, E1, G, J, Q, Z, and n have any of the definitions provided herein. In some further embodiments, the M W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0170] In some embodiments, the polyplex comprises a nucleic acid component and [ka] wherein R1, R2, A, E1, G, J, Q, Z, Z'', and n have any of the definitions provided herein. In some further embodiments, the M of the polymer W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa.W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0171] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer comprising:

[0172] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer comprising the formula: J is O and Z is [ka] In the formula, x is 1 to 1000.

[0173] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer comprising:

[0174] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and [ka] and a polymer selected from the group consisting of

[0175] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer in which

[0176] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer in which

[0177] In some further embodiments, the polyplex comprises a nucleic acid component and R [ka] and [ka] and a polymer selected from the group consisting of

[0178] In some further embodiments, the polyplex comprises a nucleic acid component and R [ka] and a polymer in which

[0179] In some further embodiments, the polyplex comprises a nucleic acid component and R [ka] and a polymer in which

[0180] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and R2 is [ka] and a polymer in which

[0181] In some further embodiments, the polyplex comprises a nucleic acid component and [ka] and R2 is [ka] and a polymer in which

[0182] In some embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer comprising the formula: R1 is [ka] and R2 is [ka] In some further embodiments, the M of the polymer is selected from W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0183] In some embodiments, the polyplex comprises a nucleic acid component and [ka] and a polymer comprising the formula: J is O and Z is [ka] wherein x is 1 to 1000; R1 is [ka] and R2 is [ka] It is. In some further embodiments, the M of the polymer W In some further embodiments, the M of the polymer is from about 3 kDa to about 200 kDa. W In some further embodiments, the M of the polymer is about 5 kDa to about 50 kDa. W In some further embodiments, the M of the polymer is between about 10 kDa and 50 kDa. W In some further embodiments, the M of the polymer is from about 5 kDa to about 15 kDa. W In some further embodiments, the M of the polymer is about 10 kDa. W In some further embodiments, the M of the polymer is about 20 kDa. W In some further embodiments, the M of the polymer is about 30 kDa. W is about 40 kDa. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is less than about 0.5. In some further embodiments, the alpha parameter as defined by the Mark-Hwink equation of the polymer is in the range of about 0.3 to about 0.5. In some further embodiments, the PDI of the polymer is about 1.0 to about 8.0. In some further embodiments, the PDI of the polymer is about 2.5.

[0184] In some embodiments, the polymer and nucleic acid components are present in a ratio of about 0.1:1 to about 200:1 (w / w). For example, the polymer and nucleic acid components may be at about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28: 1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67:1, about 68:1, about 69:1, about 70:1, about 71:1, about 72:1, about 73:1, about 74:1, about 75:1, about 76:1, about 77:1, about 78:1, about 79:1, about 80:1, about 81:1, about 82:1, about 83:1, about 84:1, about 85:1, about 86:1, about 87:1, about 88:1, about 89:1, about 90:1, about 91:1, about 92:1, about 93:1, about 94:1, about 95:1, about 96:1, about 97:1, about 98:1, about 99:1, about 100:1, about 101:1, about 102:1, about 103:1, about 104:1, about 105:1, about 1 06:1, about 107:1, about 108:1, about 109:1, about 110:1, about 111:1, about 112:1, about 113:1, about 114:1, about 115:1, about 116:1, about 117:1, about 118:1, about 119:1, about 120:1, about 121:1, about 122:1, about 123:1, about 124:1, about 125:1, about 126:1, about 127:1, about 128:1, about 129:1, about 130:1, about 131:1, about 132:1, about 133:1, about 134:1, about 135:1, about 136:1, about 137:1, about 138:1, about 139:1,about 140:1, about 141:1, about 142:1, about 143:1, about 144:1, about 145:1, about 146:1, about 147:1, about 148:1, about 149:1, about 150:1, about 151:1, about 152:1, about 153:1, about 154:1, about 155:1, about 156:1, about 157:1, about 158:1, about 159:1, about 160:1, about 161:1, about 162:1, about 163:1, about 164:1, about 165:1, about 166:1, about 167:1, about 168:1, about 169:1, about 170:1, about 171:1, about 17 In some embodiments, the polymer and nucleic acid components are present in a ratio ranging from about 2:1, about 173:1, about 174:1, about 175:1, about 176:1, about 177:1, about 178:1, about 179:1, about 180:1, about 181:1, about 182:1, about 183:1, about 184:1, about 185:1, about 186:1, about 187:1, about 188:1, about 189:1, about 190:1, about 191:1, about 192:1, about 193:1, about 194:1, about 195:1, about 196:1, about 197:1, about 198:1, about 199:1 to about 200:1, including all ranges therebetween. In some embodiments, the polymer and nucleic acid components are present in a ratio of about 20:1 to about 80:1 (w / w). ,

[0185] In some embodiments, the polymer and nucleic acid components are at about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1 , about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67:1, about 68:1, about 69:1, about 70: 1, about 71:1, about 72:1, about 73:1, about 74:1, about 75:1, about 76:1, about 77:1, about 78:1, about 79:1, about 80:1, about 81:1, about 82:1, about 83:1, about 84:1, about 85:1, about 86:1, about 87:1, about 88:1, about 89:1, about 90:1, about 9 1:1, about 92:1, about 93:1, about 94:1, about 95:1, about 96:1, about 97:1, about 98:1, about 99:1, about 100:1, about 101:1, about 102:1, about 103:1, about 104:1, about 105:1, about 106:1, about 107:1, about 108:1, about 109:1, about 11 0:1, approx. 111:1, approx. 112:1, approx. 113:1, approx. 114:1, approx. 115:1, approx. 116:1, approx. 117:1, approx. 118:1, approx. 119:1, approx. 120:1, approx. 121:1, approx. 122:1, approx. 123:1, approx. 124:1, approx. 125:1, approx. 126:1, approx. 127:1, approx. 128:1, about 129:1, about 130:1, about 131:1, about 132:1, about 133:1, about 134:1, about 135:1, about 136:1, about 137:1, about 138:1, about 139:1, about 140:1, about 141:1, about 142:1, about 143:1, about 144:1, about 145:1,About 146:1, about 147:1, about 148:1, about 149:1, about 150:1, about 151:1, about 152:1, about 153:1, about 154:1, about 155:1, about 156:1, about 157:1, about 158:1, about 159:1, about 160:1, about 161:1, about 162:1, about 163:1, about 164:1, about 165:1, about 166:1, about 167:1, about 168:1, about 169:1, about 170:1, about 171:1, about 172:1, about 173:1, about In some embodiments, the polymer and nucleic acid components are present in a ratio of about 30:1 (w / w).

[0186] In some embodiments, the particle size is less than 2 μm. In some embodiments, the particle size of the polyplex is less than about 300 nm. For example, the particle size of the polyplexes may be about 50, 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102 , about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, about 200, about 201, about 202, about 203, about 204, about 205, about 206, about 207, about 208, about 209, about 210, about 211, about 212, about 213, about 214, about 215, about 216, about 217, about 218, about 219, about 220, about 221, about 222, about 223, about 224, about 225, about 226, about 227, about 228, about 229, about 230, about 231, about 232, about 233, about 5, about 196, about 197, about 198, about 199, about 200, about 201, about 202, about 203, about 204, about 205, about 206, about 207, about 208, about 209, about 210, about 211, about 212, about 213, about 214, about 215, about 216, about 217, about 218, about 219, about 220, about 221, about 222, about 223, about 224, about 225, about 226, about 227, about 228, about 229, about 230, about 231, about 232, about 233, about 234, about 235, about 236, about 237, about 238, about 239, about 240, about 241,about 242, about 243, about 244, about 245, about 246, about 247, about 248, about 249, about 250, about 251, about 252, about 253, about 254, about 255, about 256, about 257, about 258, about 259, about 260, about 261, about 262, about 263, about 264, about 265, about 266, about 267, about 268, about 269, about 270, about 271, about 272 , about 273, about 274, about 275, about 276, about 277, about 278, about 279, about 280, about 281, about 282, about 283, about 284, about 285, about 286, about 287, about 288, about 289, about 290, about 291, about 292, about 293, about 294, about 295, about 296, about 297, about 298, about 299, or about 300 nm. In some embodiments, the particle size of the polyplexes of the present disclosure is about 60 nm to about 250 nm. In some embodiments, the particle size of the polyplexes of the present disclosure is about 175 nm to about 250 nm.

[0187] In some embodiments, the zeta potential of the polyplexes of the present disclosure is about 0 mV to about 100 mV. For example, the polyplexes of the present disclosure may have a zeta potential of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 89, about 90, about 91, about 92 The zeta potential can range from about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99 to about 100 mV. In some embodiments, the zeta potential is from about 30 mV to about 34 mV.

[0188] In some embodiments, the zeta potential of a polyplex of the disclosure is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 mV.

[0189] In some embodiments, the nucleic acid component of the polyplex is a plasmid, a nanoplasmid, a nucleic acid, a minicircle, or a gene editing system. In some embodiments, the nucleic acid component of the polyplex is a plasmid. In some embodiments, the nucleic acid component of the polyplex is a nanoplasmid. In some embodiments, the nanoplasmid comprises a eukaryotic transgene and a bacterial backbone less than 0.5 kb in size. In some embodiments, the plasmid or nanoplasmid is a plasmid without an antibiotic resistance marker or a nanoplasmid without an antibiotic resistance marker. In some embodiments, the plasmid or nanoplasmid comprises a sucrose selection marker or a nonsense suppressor marker.

[0190] In some embodiments, the nucleic acid component of the polyplex is a gene editing system. In some embodiments, the gene editing system is (i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) system, (ii) a transcription activator-like effector nuclease (TALEN) system, or (iii) a zinc finger nuclease (ZFN) system.

[0191] In some embodiments, the nucleic acid is an RNAi-inducing molecule. The RNAi-inducing molecule may be selected from the group consisting of siRNA, dsRNA, shRNA, and microRNA.

[0192] In some embodiments, the nucleic acid component comprises a tissue-specific promoter.

[0193] In some embodiments, the nucleic acid component comprises a gene associated with a genetic disease or disorder. The genetic disease or disorder may be caused by a mutation in one or more genes that results in reduced, absent, or dysfunctional protein expression. The gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5, and FLG. In some embodiments, the gene is COL7A1 and the genetic disease or disorder is a form of epidermolysis bullosa. Epidermolysis bullosa includes dystrophic epidermolysis bullosa (autosomal recessive), dystrophic epidermolysis bullosa (localized variant), prurigomatous epidermolysis bullosa, epidermolysis bullosa (pretibial), epidermolysis bullosa simplex (Dowling-Meara), epidermolysis bullosa simplex (Koebner), epidermolysis bullosa simplex (recessive 1), epidermolysis bullosa simplex (Weber-Cockayne), and epidermolysis bullosa (thalatacantholytic). In some embodiments, the genetic disorder or disease is adenosine deaminase (ADA) deficiency, alpha-1 antitrypsin deficiency, cystic fibrosis, Huntington's disease, type 1 neurofibromatosis, phenylketonuria, sickle cell disease, sporadic inclusion body myositis, Duchenne muscular dystrophy, Kindler syndrome, junctional epidermolysis bullosa, dermatosis pigmentosa reticularis, Naegeli-Franceschetti-Jadassohn syndrome, Netherton syndrome, ichthyosis vulgaris, atopic dermatitis, Usher syndrome, Ehlers-Danlos syndrome, homozygous familial hypercholesterolemia (HoFH), or Crohn's disease.

[0194] In some embodiments, the sequence of the gene is optimized to maximize protein expression upon delivery of the polyplex to a cell.

[0195] Pharmaceutical Compositions In some embodiments, the present disclosure provides pharmaceutical compositions comprising an effective amount of one or more polyplexes according to certain embodiments of the present disclosure in combination with a pharma- ceutically acceptable carrier.

[0196] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a combination of an effective amount of one or more polyplexes described herein and a pharma- ceutically acceptable excipient. In some embodiments, the pharma- ceutically acceptable excipient is selected from the group consisting of one or more bulking agents, buffers, tonicity agents, and cryoprotectants. In some embodiments, the bulking agent is selected from the group consisting of hydroxyethyl starch, trehalose, mannitol, lactose, and glycine. In some embodiments, the buffer is selected from the group consisting of phosphate buffer, Tris-HCl buffer, citrate buffer, and histidine. In some embodiments, the tonicity agent is selected from the group consisting of mannitol, sucrose, glycine, glycerol, and sodium chloride.

[0197] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of one or more polyplexes described herein in combination with a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glucose, sucrose, trehalose, lactose, mannitol, sorbitol, aerosil (colloidal silicon dioxide), maltose, poly(vinylpyrrolidone), fructose, dextran, glycerol, poly(vinyl alcohol), glycine, hydroxypropyl-β-cyclodextrin, and gelatin. In certain embodiments, the cryoprotectant is selected from the group consisting of trehalose, sucrose, glucose, and mannitol. In some embodiments, the cryoprotectant is sucrose.

[0198] In some embodiments, the pharma- ceutically acceptable carrier is suitable for oral, parenteral, inhalation, topical, subcutaneous, intramuscular, intravenous, intraocular, or intradermal administration. In some embodiments, the pharmaceutical composition is formulated as a lotion selected from the group consisting of a non-aqueous lotion, a water-in-oil lotion, and an oil-in-water lotion. In some embodiments, the pharmaceutical composition is lyophilized for future use. In some embodiments, the pharmaceutical composition is frozen in an aqueous solution.

[0199] In some embodiments, the pharmaceutical composition is a lyophilized material. In some embodiments, the lyophilized material comprises an effective amount of one or more polyplexes described herein in combination with a pharma- ceutically acceptable excipient. In certain embodiments, the pharma-ceutically acceptable excipient comprises a cryoprotectant. In certain embodiments, the cryoprotectant is selected from the group consisting of trehalose, sucrose, glucose, and mannitol. In some embodiments, the cryoprotectant is sucrose.

[0200] In some embodiments, the present disclosure provides a method of making a pharmaceutical composition comprising a combination of an effective amount of one or more polyplexes described herein and a pharma- ceutically acceptable carrier. In some embodiments, the method comprises combining one or more polyplexes described herein with a suitable solvent. In some embodiments, the suitable solvent is selected from the group consisting of water, dimethyl sulfoxide, and mixtures thereof. In certain embodiments, the suitable solvent comprises water.

[0201] In some embodiments, the method comprises: (a) combining one or more polyplexes described herein with a suitable solvent; (b) adding one or more pharma- ceutically acceptable excipients to the mixture of step (a); (c) lyophilizing the mixture of step (b) to obtain a lyophilisate.

[0202] In some embodiments, the one or more pharma- ceutically acceptable excipients in step (b) include a cryoprotectant. In certain embodiments, the concentration of the cryoprotectant is from about 1% to about 20% by weight of the mixture in step (b), including about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, and about 19% by weight, including all ranges therebetween. In certain embodiments, the concentration of the cryoprotectant is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight of the mixture of step (b). In certain embodiments, the concentration of the cryoprotectant is about 1% by weight of the mixture of step (b). In certain embodiments, the concentration of the cryoprotectant is about 3% by weight of the mixture of step (b). In certain embodiments, the concentration of the cryoprotectant is about 5% by weight of the mixture of step (b).

[0203] In some embodiments, the present disclosure provides pharmaceutical compositions prepared according to the methods described herein.

[0204] In some embodiments, the present disclosure provides a method for the preparation of a composition comprising: (a) combining one or more polyplexes described herein with a suitable solvent; (b) adding one or more pharma- ceutically acceptable excipients to the mixture of step (a); (c) lyophilizing the mixture of step (b) to obtain a lyophilizate.

[0205] In some embodiments, the one or more pharma- ceutically acceptable excipients in step (b) include a cryoprotectant. In certain embodiments, the concentration of the cryoprotectant is from about 1% to about 20% by weight of the mixture in step (b), including about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, and about 19% by weight, including all ranges therebetween. In certain embodiments, the concentration of the cryoprotectant is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight of the mixture of step (b). In certain embodiments, the concentration of the cryoprotectant is about 1% by weight of the mixture of step (b). In certain embodiments, the concentration of the cryoprotectant is about 3% by weight of the mixture of step (b). In certain embodiments, the concentration of the cryoprotectant is about 5% by weight of the mixture of step (b).

[0206] Methods for cell transfection In some embodiments, the present disclosure provides a method of cell transfection, comprising contacting one or more target cells with a pharmaceutical composition according to certain embodiments of the present disclosure under conditions suitable for transfecting the target cells with polyplexes. In some embodiments, the one or more target cells are eukaryotic cells. In some embodiments, the one or more target cells are one or more of T cells, B cells, blood cells, alveolar cells, lung cells, brain neurons, skin neurons, epithelial cells, keratinocytes, iPS cells, fibroblasts, and sweat gland cells.

[0207] Treatment method In some embodiments, the present disclosure provides a method of treating a disease in a patient in need of such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition according to certain embodiments of the present disclosure, such that one or more of the patient's cells are transfected with a polyplex nucleic acid component.

[0208] In some embodiments, the present disclosure provides a method of treating a disease in a patient in need of such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition according to certain embodiments of the present disclosure, wherein administration of the composition corrects defective translation of a target gene in the subject.

[0209] In some embodiments, the gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5, and FLG. In some embodiments, the gene is COL7A1 and the genetic disease or disorder is a form of epidermolysis bullosa. Epidermolysis bullosa includes dystrophic epidermolysis bullosa (autosomal recessive), dystrophic epidermolysis bullosa (localized variant), prurigomatous epidermolysis bullosa, epidermolysis bullosa (pretibial), epidermolysis bullosa simplex (Dowling-Meara), epidermolysis bullosa simplex (Koebner), epidermolysis bullosa simplex (recessive 1), epidermolysis bullosa simplex (Weber-Cockayne), epidermolysis bullosa (lethal acantholytic). In some embodiments, the genetic disorder or disease is adenosine deaminase (ADA) deficiency, alpha-1 antitrypsin deficiency, cystic fibrosis, Huntington's disease, type 1 neurofibromatosis, phenylketonuria, sickle cell disease, sporadic inclusion body myositis, Duchenne muscular dystrophy, Kindler syndrome, junctional epidermolysis bullosa, dermatosis pigmentosa reticularis, Naegeli-Franceschetti-Jadassohn syndrome, Netherton syndrome, ichthyosis vulgaris, atopic dermatitis, Usher syndrome, Ehlers-Danlos syndrome, homozygous familial hypercholesterolemia (HoFH), or Crohn's disease. EXAMPLES

[0210] The following examples are provided to illustrate the present disclosure and should not be construed as limiting thereof. In these examples, all parts and percentages are by weight unless otherwise stated. The abbreviations in the examples are as follows:

[0211] Example 1: LBPAE prepared by combination of linear oligomers Fibroblast gene delivery has not yet demonstrated the necessary efficiency for therapeutic applications. To overcome this limitation, as described herein, a novel linear oligomer combination strategy was used to prepare a novel multifunctional LBPAE gene delivery material according to certain embodiments of the present disclosure. The LBPAE according to certain embodiments of the present disclosure achieves superior transfection efficiency and reduced cytotoxicity in the difficult-to-transfect fibroblast HPDF and the commonly used 3T3, and substantially outperforms the commercially available reagents branched PEI and SuperFect. The high LC of LBPAE polyplexes 50 The values ​​demonstrate favorable biocompatibility in fibroblast transfection. Mechanistic studies have shown that LBPAE with sufficient amounts of primary, secondary, and tertiary amines can condense DNA into nano-sized particles with uniform spherical morphology, promote cellular uptake, and mediate strong buffering capacity to meet efficient endosomal escape. Hydrolysis of the ester bonds on LBPAE can facilitate DNA release, significantly enhance biocompatibility, and allow flexible design and preparation of polymer / DNA w / w ratios. Together with the high performance of LBPAE in reporter gene delivery, LBPAE can efficiently deliver minicircle COL7A1 gene into HPDF and notably improve the expression of C7, which is important for maintaining skin integrity. These results demonstrate that LBPAE is a high-performance non-viral vector in fibroblast-based gene delivery and highlight its enormous potential in genetic skin disease treatment and regenerative medicine.

[0212] The continuous growth and branching of linear oligomers results in a more uniform distribution of linear segments and branching units in the resulting LBPAE. Surprisingly, in difficult-to-transfect HPDFs and commonly used mouse embryonic fibroblasts (3T3), the newly developed LBPAE exhibited robust gene transfection ability, Gluc expression was up to three orders of magnitude higher than that of the commercially available gene transfection reagents PEI and SuperFect, and nearly 100% green fluorescent protein (GFP) expression was achieved without inducing obvious cytotoxicity. To decipher the possible mechanisms behind the extremely strong gene transfection ability of LBPAE in fibroblasts, multiple extracellular and intracellular barriers related to the gene transfection process were explored. The results show that LBPAE exhibits strong DNA-binding affinity and can condense DNA to formulate nano-sized polyplexes with nearly 100% cellular uptake efficiency. The strong proton buffering capacity and biodegradability of LBPAE may also facilitate endo / lysosomal escape of LBPAE / DNA polyplexes and DNA release into the cytoplasm. Furthermore, LBPAE was used to deliver a minicircle plasmid encoding the COL7A1 gene (MCC7) to HPDFs, and a significant upregulation of C7 expression was detected, indicating that LBPAE is highly promising for the treatment of C7-deficient genodermatological diseases (e.g., the devastating and debilitating genodermatological disorder RDEB).

[0213] This example illustrates a linear oligomer combinatorial strategy for synthesizing LBPAE. As shown in FIG. 8, this strategy involves two consecutive steps: linear oligomer formation and branching. In the first step, A2-type amine reacts with C2-type diacrylate to form acrylate-terminated base oligomer, forming linear A2-C2 base oligomer, which is further end-capped by a second amine. After purification to remove unreacted monomer and excess end-capping agent, linear A2-C2 oligomer is formed. In the second step, B3-type triacrylate is introduced to combine linear A2-C2 oligomer to obtain LBPAE. The advantages of LBPAE are twofold: 1) the length of the linear segment of the resulting LBPAE can be predetermined and therefore easily adjusted, and 2) the branching units of LBPAE are more uniformly distributed among the linear segments.

[0214] To test this hypothesis, 5-amino-1-pentanol (AP), trimethylolpropane triacrylate (TMPTA), 1,4-butanediol diacrylate (BDA), and 1,11-diamino-3,6,9-trioxaundecane (DATOU) were used as A2, B3, and C2 type monomers and end-capping agents, respectively, in the synthesis of LBPAE. AP and BDA were reacted in a stoichiometric ratio of 1.2:1 at 90 °C in dimethyl sulfoxide (DMSO), and the weight average molecular weights (M w ) was measured by gel permeation chromatography (GPC). After 24 hours, the M w When the molecular weight approached 3000 Da, the reaction was stopped by cooling to room temperature, diluting with DMSO, and then adding excess DATOU to end-cap the acrylate-terminated base oligomer at 25 °C for 48 h. Unreacted monomers and end-capping agent were removed in acetone with M w After removal by dialysis along with oligomers of <3000 Da, the M wA linear A2-C2 oligomer with a molecular weight of 1.69 and a polydispersity index (PDI) of 1.69 was obtained (FIG. 10). To produce LBPAE, the linear A2-C2 oligomer and TMPTA were dissolved in DMSO (A2-C2:TMPTA molar ratio was set at 3:1) and reacted at 90° C. w The reaction was stopped when the M was approximately 10 kDa, and excess DATOU was incorporated to consume all unreacted vinyl groups. The polymer was then precipitated in diethyl ether and dried in a vacuum oven to obtain the final LBPAE product. GPC measurements showed that the M of the LBPAE was 0.01 to 0.01. w It has been shown to have a molecular mass of 9.4 kDa and a PDI of 2.5 (Figure 19). The Mark-Hwink (MH) plot alpha value of 0.36 confirms the highly branched structure (Figure 10). The chemical composition of LBPAE was 1 This was confirmed by H-NMR (Figure 11).

[0215] Example 2: LBPAE achieves strong gene transfection efficiency and excellent cell viability in fibroblast cells Potent gene delivery vectors can not only achieve high gene transfection efficiency but also induce minimal cytotoxicity. Nevertheless, in practice, improving the transfection efficiency of a gene vector usually comes at the expense of its biocompatibility and vice versa. To evaluate the gene transfection ability of the synthesized LBPAE and identify the most optimal parameters for fibroblast transfection, a series of LBPAE / DNA polyplexes with different w / w ratios were first evaluated for transfection of HPDF and 3T3. Gluc DNA was used as a reporter gene, and the gene transfection efficiency was quantified by Gluc activity measurements after transfection. Alamar Blue assay and lethal concentration 50 (LC 50 ) assay was used to measure the cytotoxicity and toxicological profile of fibroblasts after transfection. The transfection efficacy of LBPAE was then further verified by flow cytometry using GFP DNA as a reporter gene.

[0216] Gluc expression and cell viability of fibroblasts after transfection with LBPAE

[0217] For cationic polymer-based gene delivery vectors, the polymer / DNA weight ratio (w / w) is a useful parameter for quantifying both transfection efficiency and cytotoxicity. [5、11] Therefore, we first systematically optimized the w / w ratio. Considering that primary cells (e.g., HPDFs) are usually vulnerable to cationic polymers, the LBPAE / DNA w / w ratio used for HPDF transfection was gradually increased from 10:1 to 50:1. To set a strong benchmark for comparison, the w / w ratios used for two commercially available dendritic gene transfection reagents, PEI and SuperFect, were also optimized according to the manufacturer's protocols and previous publications. [6、13]Figure 1a shows a summary of Gluc activity and cell viability of HPDF after transfection. It clearly shows that the optimal w / w ratios for PEI and SuperFect gene transfection are 1:1 and 3:1, respectively. Further increase in the w / w ratio not only obviously reduces Gluc activity but also substantially increases cytotoxicity. For example, compared with the w / w ratio of 3:1, the Gluc activity of HPDF after transfection with SuperFect / DNA polyplexes is 3.4-fold lower and the cell viability decreased from more than 89% to less than 44% at the w / w ratio of 9:1. In stark contrast, the Gluc expression of HPDF after transfection with LBPAE / DNA polyplexes is still stronger than that mediated by PEI / DNA polyplexes and SuperFect / DNA polyplexes at the optimal w / w ratios, even at the lowest w / w ratio of 10:1, in the range of w / w ratios tested. Notably, the Gluc activity of HPDFs transfected by LBPAE / DNA polyplexes at a w / w ratio of 40:1 is up to 103-fold higher than that mediated by PEI / DNA polyplexes. Importantly, LBPAE did not induce obvious cytotoxicity. Even at the highest w / w ratio of 50:1, over 95% cell viability was still retained. In 3T3, PEI / DNA polyplexes showed similar trends in gene transfection efficiency and cytotoxicity as those of HPDFs. At w / w ratios of 6:1 and 9:1, SuperFect / DNA polyplexes showed higher gene transfection efficiency but only retained 62% and 49% cell viability (Figure 1b). Again, at all tested w / w ratios, LBPAE / DNA polyplexes show both strong gene transfection ability and high cell viability. The Gluc activity of 3T3 cells after transfection with LBPAE / DNA polyplexes is an order of magnitude higher than that mediated by PEI / DNA and SuperFect / DNA polyplexes at their optimal w / w ratios. Surprisingly, at a w / w ratio of 70:1, LBPAE / DNA polyplexes mediated up to 3292-fold higher Gluc activity than PEI / DNA polyplexes, while still maintaining over 90% cell viability. Note that the same amount of DNA was used between groups.The very high w / w ratio used in the LBPAE / DNA polyplexes meant that significantly more LBPAE was used than either PEI or SuperFect, further demonstrating the excellent biocompatibility of LBPAE.

[0218] Toxicological profile of LBPAE

[0219] Over 2500 candidates have been developed and screened for gene transfection.

[28] To date, no toxicological studies have been conducted on any PAE polymers in fibroblasts. To further verify the biocompatibility of LBPAE in gene transfection, we determined the toxicological profile of LBPAE and investigated its LC 50 For this purpose, BPAE / DNA polyplexes with the same w / w ratio of 40:1 were used, and the polyplex concentration was set at 355 μg mL -1 to 755 μg mL -1 For comparison, SuperFect / DNA polyplex was used at a concentration of 15 μg mL -1 to 55 μg mL -1 Twenty-four hours after transfection, cells were simultaneously stained with green fluorescent calcein-AM (C-AM, for live cells) and red fluorescent ethidium homodimer-1 (EthD-1, for dead cells). -1 LBPAE / DNA polyplexes at concentrations of 35 μg / mL -1 Representative fluorescence images of cells treated with SuperFect / DNA polyplexes at a concentration of 100 μg / ml are shown in Figure 2a. It can be seen that HPDF and 3T3 treated with LBPAE / DNA polyplexes showed similar cell viability to those treated with SuperFect / DNA polyplexes, despite the concentration being one order of magnitude higher. Polyplex concentration-dependent cell viability was quantified by Alamar Blue assay, and the results are shown in Figure 2b and Figure 2c. From these, it can be seen that the LC of SuperFect / DNA polyplexes in HPDF and 3T3 cells was significantly higher than that in HPDF cells. 50 The values ​​were 35.2 μg mL-1 and 39.5 μg mL -1 In contrast, the LC of LBPAE / DNA polyplex is 50 The value was 538.4 μg mL -1 and 552.3 μg mL -1 which correspond to a 14-fold and 13-fold increase over their SuperFect / DNA counterparts. SuperFect has been widely used for gene transfection due to its outstanding biocompatibility. [29、30] LBPAEs exhibit very low cell killing and have been demonstrated to be extremely biocompatible, which is of great importance in gene transfection, especially for difficult-to-transfect cell types, since much higher doses of polyplexes or multiple repeated transfections can be used to enhance transfection efficiency.

[0220] GFP expression quantified by flow cytometry

[0221] Gluc DNA was used to quantify the overall transgene expression levels mediated by LBPAE, and GFP DNA was used to quantify the percentage of transfected cells. HPDFs and 3T3 were transfected with LBPAE / DNA polyplexes at the same w / w ratios as above. As evidenced by the fluorescence images shown in Figure 3a, at all w / w ratios, much more HPDFs were transfected by LBPAE / DNA polyplexes than by their PEI / DNA and SuperFect / DNA counterparts at optimal w / w ratios. Flow cytometry measurements indicate that the percentage of GFP-positive HPDFs achieved by PEI / DNA and SuperFect / DNA polyplexes was only 50% and 44%, respectively. In contrast, LBPAE / DNA polyplexes achieved a much higher level of the GFP-positive population, as reflected by the shift of the cell populations responding to the GFP spectral channel away in the histogram distribution (Figure 3b). The lowest GFP-positive population mediated by LBPAE / DNA polyplexes is 68% at a w / w ratio of 10:1. At w / w ratios above 40:1, more than 93% of HPDFs are GFP-positive. Moreover, the median fluorescence intensity (MFI) of HPDFs transfected by LBPAE / DNA polyplexes is up to 140-fold higher than that of their PEI / DNA and SuperFect / DNA counterparts (Figure 3c). In the case of 3T3, the percentage of GFP-positive cells achieved by LBPAE / DNA polyplexes increased from 35% at a w / w ratio of 30:1 to more than 91% at a w / w ratio of 70:1, in contrast to 5% and 11% achieved by PEI / DNA and SuperFect / DNA polyplexes, respectively (Figures 3d and 3e). In addition, at a w / w ratio of 70:1, the MFI of 3T3 mediated by LBPAE / DNA polyplexes was 272- and 230-fold higher than that of the PEI / DNA and SuperFect / DNA counterparts (Figure 3f). These results indicate that LBPAE not only transfects a higher number of cells but also significantly enhances protein expression levels in the individually transfected cells.Primary fibroblasts are a difficult cell type to transfect, and the fact that LBPAE can mediate over 90% gene transfection efficiency in primary HPDFs demonstrates the extremely strong gene transfection ability of LBPAE. Considering that LBPAE has been proven to have high biocompatibility over a wide range of w / w ratios with excellent gene transfection ability, it can be envisioned that LBPAE will have broad applicability in fibroblast gene transfection.

[0222] Example 3: Possible mechanism in LBPAE to achieve highly potent gene transfection efficiency and excellent biocompatibility To decipher the possible mechanisms behind the high performance of LBPAE in fibroblast transfection, a series of studies were performed on multiple extracellular and intracellular gene delivery barriers, including DNA condensation and binding affinity, polyplex size, zeta potential, morphology, proton buffering capacity, degradation rate, and DNA release from polyplexes.

[0223] DNA condensation and binding affinity of LBPAE

[0224] Effective DNA condensation not only protects DNA from endonuclease-induced DNA degradation but also favors polyplex cellular uptake, a prerequisite for successful gene transfection.

[30] For cationic polymers, DNA condensation is mainly driven by electrostatic interactions. There are various amines that can be partially or fully protonated to generate positive charges. For example, amines that can be partially or fully protonated are the multiple terminal primary amines derived from the end-capping agent DATOU, or the numerous backbone tertiary amines derived from AP. The DNA condensation ability of LBPAE was quantified by agarose gel electrophoresis. As shown in Figure 4a, no DNA shift bands were observed at all w / w ratios. This indicates that the negatively charged DNA is effectively shielded by the positively charged LBPAE and thus retained in the agarose well without migration. Both the commercially available gene transfection reagents PEI and SuperFect show high DNA condensation ability, and in particular, SuperFect strongly condenses DNA, making it difficult for the DNA staining dye to approach the DNA, resulting in a pale color of the DNA band. The binding affinity of DNA with LBPAE was further quantified by PicoGreen assay. As shown in Figure 4b, LBPAE shows strong DNA binding affinity at all w / w ratios. Generally, the binding affinity increases with the w / w ratio, for example, from 86% at a w / w ratio of 10:1 to 96% at a w / w ratio of 70:1. This demonstrates that the more LBPAE, the stronger the electrostatic interaction between LBPAE and DNA. In comparison, PEI and SuperFect show much stronger binding affinity than the nearly 100% binding affinity of LBPAE, and PEI and SuperFect correlate very well with their condensation ability. However, it should be noted that a moderate DNA binding affinity is preferred for gene transfection, since an excessively strong interaction impairs DNA release from polyplexes. [31、32]

[0225] Size, zeta potential, and morphology of LBPAE / DNA polyplexes

[0226] The nanometer size and positive surface charge may facilitate cellular uptake of the particles via the endocytic pathway. [33、34]As shown in Figure 4c, in physiological solutions, the average size of LBPAE / DNA polyplexes measured by dynamic light scattering (DLS) is all less than 250 nm across all tested w / w ratios. In the w / w ratio range of 10:1 to 60:1, the particle size of the polyplexes is between 228 nm and 188 nm. However, when the w / w ratio is further increased to 70:1, the polyplex size decreases to 97 nm. Correspondingly, all polyplexes exhibit positive zeta potentials. At the lowest w / w ratio of 10:1, the LBPAE / DNA polyplexes have a very low zeta potential of 6 mV. At w / w ratios higher than 10:1, the zeta potential increases significantly to above 30 mV, achieving the highest value of 34 mV at a w / w ratio of 40:1. Under the same test conditions, the SuperFect / DNA polyplexes have a very small size of approximately 92 nm and a high zeta potential of approximately 37 mV. These observations are consistent with the DNA condensation ability and binding affinity of the polymer. In contrast, PEI / DNA polyplexes have high DNA condensation and binding abilities but substantially larger sizes of over 500 nm. Transmission electron microscopy (TEM) was further used to observe the size and morphology of the polyplexes. As shown in Figure 4d, all LBPAE / DNA and SuperFect / DNA polyplexes exhibit uniform spherical morphology with sizes between 60 nm and 250 nm, similar to those measured by DLS. Importantly, no obvious polyplex aggregation was observed, demonstrating the high stability of the polyplexes. Meanwhile, PEI / DNA polyplexes exhibit ellipsoidal morphology and are much larger in size than other polyplexes. It is widely accepted that polyplexes with sizes below 250 nm and moderately positive surface charges are more favorable for cellular uptake while avoiding the induction of potential cytotoxicity brought by excess positive charges. [7、9]The above gene transfection studies showed that the best w / w ratios for HPDF and 3T3 transfection were 40:1 and 70:1, respectively, indicating that the most favorable polyplex size and surface charge for effective fibroblast gene transfection may vary substantially depending on the cell type. In this regard, the LBPAE / DNA polyplexes always had an average size below 250 nm and a moderate zeta potential in a wide range of w / w ratios, indicating that they are widely applicable to transfect various cell types to achieve high performance.

[0227] Example 4: Cellular uptake of LBPAE / polyplexes Further investigations were carried out on the cellular uptake of polyplexes. As shown in Figure 5a, all polyplexes show high cellular uptake efficiency at the same cell density 4 h after transfection. In comparison, much more LBPAE / DNA polyplexes are taken up by HPDF and 3T3 compared to PEI / DNA and SuperFect / DNA polyplexes, which is supported by the much stronger red fluorescence observed from Cy3-labeled DNA. Flow cytometry quantification reveals that PEI / DNA and SuperFect / DNA polyplexes achieve cellular uptake efficiencies of 96.5% and 98.4% in the case of HPDF (Figure 5b). Even so, the uptake efficiency of LBPAE / DNA polyplexes is still slightly higher, nearly 100% (99.3%). A similar trend was observed in the case of 3T3 (Figure 5c). Moreover, in HPDF, the normalized MFI of LBPAE / DNA polyplexes is 3.05, 1.39-fold higher than that achieved by PEI / DNA and SuperFect / DNA counterparts (Fig. 5d). In the case of 3T3, the performance is outperformed by 1.98-fold and 1.68-fold, respectively (Fig. 5e). All these results indicate that the cellular uptake efficiency of PEI / DNA polyplexes is the lowest, while LBPAE / DNA polyplexes significantly outperform both PEI / DNA and SuperFect / DNA counterparts. Taken together, these uptake results correlate very well with the polyplex size, zeta potential, and gene transfection performance of the different polyplexes mentioned above.

[0228] Example 5: Measurement of proton buffer capacity Cationic polymer-based polyplexes are usually taken up into cells via the endocytic pathway, and once internalized, they are mainly trapped in endo / lysosomes. If the polyplexes cannot escape from the endo / lysosomal compartment in time, the DNA condensed within the polyplexes will be degraded by digestive enzymes in the acidic compartment. Therefore, endo / lysosomal escape is another major bottleneck to overcome for efficient non-viral gene delivery. The "proton sponge effect" is widely considered to be the main mechanism of cationic polymers to facilitate polyplex escape from endo / lysosomes. Considering the mechanism of the "proton sponge effect", cationic polymers with a high content of protonatable secondary and tertiary amines with pKa close to the endosomal / lysosomal pH are more favorable for polyplex escape from endosomes / lysosomes, of which PEI and SuperFect are the most typical representatives.

[29] To verify the proton buffering capacity of LBPAE, acid-base titration was performed. As shown in Figure 6a, it is not surprising that for a given amount of polymer dissolved in NaCl solution, PEI exhibits the strongest proton buffering capacity with a relatively flat slope in the acid-base titration curve between pH 7.4 and 5.1. This is due to the fact that PEI has a very high content of primary, secondary, and tertiary amines and has a nitrogen for every third atom in the backbone. After normalization, the proton buffering capacity of PEI, SuperFect, and LBPAE was 5.1 mmol H, respectively. + g -1 , 4.6 mmol H + g -1 , and 1.6 mmol H + g -1It can be seen that (Table 1). In fact, LBPAE shows a lower proton buffering capacity than PEI and SuperFect. However, due to the much lower cytotoxicity, the w / w ratio can be significantly increased in practical applications to effectively promote the endo / lysosomal escape of LBPAE / DNA polyplexes. For example, for HPDF and 3T3 gene transfection, LBPAE / DNA polyplexes were used at w / w ratios of 40:1 and 70:1, respectively. In this condition, the proton buffering capacity of the entire LBPAE used is 12-fold and 21-fold higher than that of PEI at a w / w ratio of 1:1, and 5-fold and 8-fold higher than that of SuperFect at a w / w ratio of 3:1. Based on the "proton sponge effect" hypothesis, the high proton buffering capacity of LBPAE causes an increase in osmotic pressure, leading to the swelling and rupture of endo / lysosomes, thus releasing LBPAE / DNA polyplexes into the cytoplasm in time and efficiently. Table 1. Buffer capacity of LBPAE and commercial reagents. [Table 1]

[0229] Example 6: Degradation of LBPAE and DNA release evaluation A versatile gene delivery vector can not only effectively condense DNA and protect it from enzymatic degradation, but also release the condensed DNA from polyplexes after nuclear import. For cationic polymers, a series of strategies have been proposed to promote polymer degradation in the cytoplasm, thus facilitating DNA release and reducing accumulated cytotoxicity after gene transfection. However, a moderately long half-life is required for gene vectors for efficient gene transfection, because a half-life that is too short leads to insufficient DNA protection and premature DNA release, while a half-life that is too long makes polyplex dissociation and DNA release difficult. There are multiple ester bonds on the backbone of PAE. Under physiological conditions, the ester bonds can be decomposed by hydrolysis to yield biocompatible small molecule β-amino acids and diols. Depending on the chemical composition, the half-life of LPAEs has been reported to range from 1.5 hours to more than 6 hours in an aqueous environment. [7]For LBPAE according to certain embodiments and examples of the present disclosure, 43% degradation was observed after 2 hours of incubation at 37°C. The degradation continued to increase to 81% and 85% after 6 and 8 hours of incubation, respectively (Figure 6b). DNA release from the corresponding polyplexes is quantified by PicoGreen assay. As shown in Figure 6c, at the lowest w / w ratio of 10:1, LBPAE / DNA polyplexes had the fastest DNA release rate, with over 60% of DNA released after 2 hours of incubation. In comparison, at the medium w / w ratio of 40:1 and the high w / w ratio of 70:1, condensed DNA was released from the polyplexes at a slower but similar rate. However, over 60% of DNA was released after 6 hours. The DNA release profile is consistent with the LBPAE degradation profile, indicating that LBPAE can spontaneously release condensed DNA via hydrolysis under physiological conditions without the need for any additional external trigger. All results from DNA condensation, binding affinity, polyplex size, zeta potential, cellular uptake, degradation, and DNA release correlated very well with the gene transfection efficiency and biocompatibility of LBPAE / DNA polyplexes, highlighting the manipulation of LBPAE composition, structure, and functionality to achieve favorable polyplex properties for high-performance gene transfection in fibroblast cells.

[0230] Example 7: LBPAE delivers functional COL7A1 to manipulate C7 expression in HPDFs It has been demonstrated that the multifunctional LBPAE according to certain embodiments and examples of the present disclosure can deliver Gluc DNA and GFP DNA to transfect HPDF and 3T3 with extremely high efficiency and excellent biocompatibility. However, many gene delivery vectors show high levels of reporter gene expression, and it is much more difficult to translate such success to functional protein expression. Therefore, the efficacy of LBPAE was further evaluated by delivering a functional COL7A1 gene that promotes the expression of C7 in HPDF. Currently, there is no effective treatment for RDEB other than symptomatic care. Both keratinocytes and dermal fibroblasts can produce and secrete C7.

[35] However, the latter is a more robust target cell type than the former for gene therapy of genocutaneous diseases. [21、36] Minicircle (MC) DNA cassettes have demonstrated stable, non-integrative transgene expression that is 10-1000 times higher than standard plasmids, without the risk of immunogenic responses from the bacterial backbone of standard plasmids. [37、38] Considering that the COL7A1 gene is quite large with a cDNA / mRNA transcript of approximately 9 kb, MCC7, encoding a full-length COL7A1 cDNA of 8.9 kb with a cytomegalovirus promoter, was used for transfection into HPDF. As shown in Figure 12, MCC7 contains 8.9 kb of COL7A1 cDNA and 3 kb of backbone, which is 2 kb less than the pcDNA3.1COL7A1 parental plasmid. The maximum cargo size of retroviral and adeno-associated viral (AAV) vectors is usually less than 8 kb,

[39] Because the sizes of both pcDNA3.1COL7A1 and MCC7 exceed the gene packaging capacity of most viral vectors, efficient COL7A1 gene transfection by LBPAE is crucial for gene therapy of RDEB. Hoping to enhance the usefulness of gene therapy of RDEB, we combined LBPAE and MC DNA to deliver MCC7 to manipulate C7 expression in HPDFs. Figure 7a shows an overview of the cell immunofluorescence staining images of HPDFs 4 days after transfection with LBPAE / MCC7 polyplexes. As expected, no obvious C7 expression (red fluorescence) was observed in the untreated group and the group incubated only with anti-C7 secondary antibody. The wild-type HPDFs and the SuperFect-treated group showed moderate fluorescence, which indicates the generation of C7. In contrast, HPDFs transfected with LBPAE / MCC7 polyplexes showed the strongest fluorescence, demonstrating the more recombinant C7 expression obtained by LBPAE. Flow cytometry was further used to quantify the C7 expression efficiency. Consistent with previous reports,

[40] Wild-type HPDFs showed approximately 41% C7 expression. After transfection with LBPAE / MCC7 polyplexes, the C7 expression efficiency was significantly increased to 74.4%, compared to 44.9% achieved by SuperFect / MCC7 polyplexes (Fig. 7b). In addition, a 40% enhancement of MFI was also achieved by LBPAE / MCC7 polyplexes, compared to 10% achieved by the SuperFect / MCC7 counterpart (Fig. 7c). All these results demonstrate that LBPAE can not only effectively deliver MCC7 to increase the overall population of C7-expressing HPDFs, but also enhance the C7 levels of individual HPDFs. In addition, our preliminary studies have shown that the C7 expression efficiency was restored to approximately 40% after transfection with LBPAE / MCC7 in C7-null RDEB fibroblasts (RDEBFs), and further optimization of transfection and quantification of C7 expression in RDEBFs are still ongoing. These findings indicate that LBPAE has a strong payload capacity to deliver large cDNAs to primary skin cells. Primary skin fibroblasts can be further engineered with this polymer vector to secrete potent cellular C7, which is important for strengthening the dermal-epidermal junction. Although non-viral gene therapy requires repeated application, for inherited C7 dysfunction skin disease, multiple local administrations are much safer than systemic gene delivery, considering that the wound site is obvious and the drug accessibility is good, so LBPAE is very promising for fibroblast-based gene therapy to restore or enhance C7 expression and reverse the disease phenotype of RDEB.

[0231] Example 8: Experimental Materials: Trimethylolpropane triacrylate (TMPTA), 5-amino-1-pentanol (AP), 1,11-diamino-3,6,9-trioxaundecane (DATOU), sodium chloride (NaCl), sodium hydroxide (NaOH), branched polyethyleneimine (PEI, M w= 25 kDa), lithium bromide (LiBr), dimethyl sulfoxide (DMSO), diethyl ether, deuterated chloroform (CDCl3), hydrochloric acid solution (HCl), Hank's balanced salt solution (HBSS), Tris-EDTA acetate buffer (TAE), trypsin-EDTA solution (0.25%), Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin (P / S), agarose, paraformaldehyde (PFA), 0.1% Triton X-100, and monoclonal anti-collagen VII antibody produced in mouse and goat serum were purchased from Sigma-Aldrich. Sodium acetate (3.0 M, Sigma-Aldrich) was diluted to 0.025 M before use. 1,4-butanediol diacrylate (BDA) was purchased from VWR and used as received. Dimethylformamide (DMF) was purchased from Fisher Scientific. Fetal bovine serum (FBS) purchased from Gibco was filtered through a 0.2 μm filter before use. HPDF and 3T3 cells were purchased from Lonza and ATCC, respectively. For the culture and subculture of HPDF, fibroblast basal medium, FGM-2 SingleQuots, Clonetics reagent pack (including HEPES-buffered saline, trypsin-EDTA solution (0.25%), and trypsin neutralizing solution) were purchased from Lonza. Cell-secreted Gaussia princeps luciferase plasmid (GLuc DNA) and BioLux™ Gaussia luciferase assay kit were purchased from New England Biolabs UK. Gluc-Prep kit (Qiagen) was used to perform the extension and purification of Gluc DNA according to the protocol. Green fluorescent protein plasmid (GFP DNA) was purchased from Aldevron. pcDNA3.1COL7A1 plasmid was kindly provided by Dr. Andrew South, University of Dundee (UK). MCC7 was constructed by inserting the COL7A1 sequence generated from pcDNA3.1COL7A1 into the MN511A-1 cassette provided by System Biosciences, and the generation of minicircle DNA was according to the System Bioscience user manual.SuperFect gene transfection reagent was purchased from Qiagen. LIVE / DEAD viability / cytotoxicity kit, goat anti-mouse IgG (H+L) highly cross-adsorbing secondary antibody, Alexa Fluor 568, and Alexa Fluor 647 were purchased from Thermo Fisher Scientific. Alamar Blue assay kit, SYBR safe DNA gel stain, IC fixation buffer, and 10x Bioscience permeabilization buffer were purchased from Invitrogen. Cy3 DNA labeling kit was purchased from Mirus and used according to the protocol. 4',6-diamidino-2-phenylindole (DAPI) and PicoGreen assay kit were purchased from Life Technologies and used according to the manufacturer's protocol. 1x Dulbecco's phosphate-buffered saline (PBS) was purchased from Life Technologies. Mounting medium with DAPI was purchased from Abcam.

[0232] Synthesis and characterization of LBPAE: LBPAE was synthesized by a linear oligomer combinatorial strategy. First, BDA and AP with a stoichiometric ratio of 1.2:1 were mixed at 100 mg mL -1 The compounds were dissolved in DMSO and then reacted at 90° C. The weight average molecular weight (M) was determined using an Agilent 1260 Infinite gel permeation chromatography (GPC) equipped with a triple detector (refractive index detector (RI), viscosity detector (VS DP), and dual light scattering detector (LS 15° and LS 90°)). w ), number average molecular weight (M n The growth of the dispersity index (DPI) and polydispersity index (PDI) were monitored. For GPC measurements, 20 μL of the reaction mixture was taken, diluted with 1 mL DMF, and then filtered through a 0.45 μm filter. The reaction mixture was heated at 60 °C for 1 mL min using DMF containing 0.1% LiBr. -1 The GPC columns (Polar Gel-M, 7.5 × 300 mm, two in series) were eluted at a flow rate of 100 μg / ml. Linear poly(methyl methacrylate) (PMMA) standards were used to calibrate the GPC columns. wWhen M approached 3000 Da, the reaction was stopped by cooling to room temperature, diluting with DMSO, then adding excess endcapping agent DATOU, and continuing the reaction for another 48 h to obtain the DATOU endcapped linear A2-C2 oligomer, which was purified by dialysis against acetone for 3 days and then dried in a vacuum oven to remove the solvent. The linear A2-C2 oligomer was then dissolved in DMSO and reacted with the branched monomer TMPTA at 90 °C. w When the molecular weight (M) of the final product was approximately 10 kDa, the reaction mixture was cooled to room temperature and excess DATOU was added to consume all unreacted vinyl groups for another 48 h. The polymer was then purified by precipitation with diethyl ether three times, lyophilized for 2 days, and stored at -20 °C for further testing. w To measure the PDI, PDI, and Mark-Hwink (MH) plot alpha value (α), 10 mg LBPAE was dissolved in 2 mL DMF and subjected to GPC measurement as described above. The chemical composition and purity of LBPAE were analyzed using a 400 MHz Varian Inova spectrometer. 1 Quantitation was performed by H NMR. Samples are reported in parts per million (ppm) relative to the solvent CDCl3 (7.24 ppm) or an internal reference (tetramethylsilane 0.00 ppm).

[0233] Determination of Mark-Hwink Alpha Parameter: Determination of Mark-Hwink Alpha Parameter of polymers was performed on a 1260 Infinite GPC system equipped with a refractive index detector (RI), a viscosity detector (VS DP), and a dual angle light scattering detector (LS 15° and LS 90°). To prepare the polymer for analysis, 10.0 mg of sample was dissolved in 2 mL DMF and then filtered through a 0.45 μm filter. GPC columns (30 cm PLgel Mixed-C, two in series) were eluted with DMF and 0.1% LiBr at 60°C and a flow rate of 1 mL / min, and the columns were calibrated with linear poly(methyl methacrylate) standards (PMMA). Universal calibration was used to analyze the GPC data.

[0234] LBPAE / DNA polyplex preparation: For polyplex preparation, LBPAE was first dissolved in DMSO to give 100 mg mL -1 According to the LBPAE / DNA weight ratio (w / w), the required amount of LBPAE stock solution and DNA solution were diluted with sodium acetate buffer (0.025 M, pH=5.2) to equal volumes, respectively. Then, LBPAE solution was added, mixed by vortexing for 10 seconds, and left at room temperature for 10 minutes to form polyplexes.

[0235] DNA condensation by LBPAE: Agarose gel electrophoresis was used to measure the DNA condensation ability of LBPAE. 1 μg of DNA was used for each sample preparation, and polyplexes with a range of w / w ratios were prepared as described above. 20 μL of polyplex solution was then loaded onto an agarose gel (1% in 1× TAE buffer) containing 10 μL of SYBR safe DNA gel stain in a well, and naked DNA was used as a control. Gel electrophoresis was performed at 120 V for 40 min in 1× TAE buffer, and images were captured using Syngene's G:BOX.

[0236] DNA binding affinity of LBPAE: The DNA binding affinity of LBPAE was quantified using PicoGreen assay. 0.25 μg of DNA was used for each sample preparation. Polyplexes with different w / w ratios were prepared in 15 μL of sodium acetate buffer, then mixed with 15 μL of PicoGreen working solution and incubated for 5 min. After that, 220 μL of 1× PBS buffer was added to dilute the polyplexes in a black 96-well plate. The fluorescence intensity (F) of the polyplex solution was measured in quadruplicate by a SpectraMax M3 plate reader at an excitation of 490 nm and an emission of 535 nm. The DNA binding affinity of LBPAE is defined by the following equation:

number

[0237] Proton buffering capacity of LBPAE: The proton buffering capacity of LBPAE was quantified by acid-base titration. 0.1 M NaCl solution was used as background control, and PEI and SuperFect were used as positive controls. The pH value was measured using a Mettler Toledo S20 pH meter. 10 mg LBPAE, 5 mg PEI, or 0.8 mg SuperFect was dissolved in 20 mL 0.1 M NaCl solution. The pH value of the solution was adjusted to 3.0 with 1.0 M HCl solution and then titrated to 10.5 with 0.1 M NaOH solution. The proton buffering capacity of LBPAE (mmol g-1) was calculated using the following formula:

[0238]

number

[0239] Decomposition profile of LBPAE: To measure the decomposition profile, LBPAE was added at 10 mg mL -1 The samples were dissolved in PBS at a concentration of 0.01 mg / mL and kept shaking at 180 rpm at 37°C. At 0, 2, 4, 6, and 8 hours, 1 mL of the solution was taken and immediately frozen. After freeze-drying, the samples were dissolved in 1 mL DMF. w was measured in triplicate by GPC as previously described. The percentage of LBPAE decomposition was defined as

number

[0240] Quantification of polyplex size and zeta potential: Polyplex size and zeta potential were measured using a Malvern Instruments Zetasizer (Nano-ZS90, scattering angle 173°, 633 nm laser). 4 μg of DNA was used for each sample preparation, and polyplexes with a range of w / w ratios were prepared as above, diluted with 800 μL of deionized water, and then transferred to a Zetasizer cell or cuvette. Size and zeta potential measurements were performed in quadruplicate at 25°C.

[0241] Polyplex morphology characterization by transmission electron microscopy (TEM): The morphology of LBPAE / DNA, PEI / DNA, and SuperFect / DNA polyplexes was characterized by TEM. 80 μL of polyplex solution containing 2 μg of DNA was prepared as described above, washed twice with deionized water to remove salts, and then resuspended in 10 μL of deionized water. 2.5 μL of the resuspended polyplex solution was cast onto a Formvar support film on a 200 mesh copper grid and immediately lyophilized. TEM images were captured on an FEI Tecnai 120 TEM at 120 kV at the UCD Conway Imaging Core Centre.

[0242] DNA release from polyplexes: DNA release from polyplexes can be quantified by measuring the decrease in binding affinity by LBPAE using the PicoGreen assay. LBPAE / DNA polyplexes with w / w ratios of 10:1, 40:1, and 70:1 were prepared as above and kept shaking at 180 rpm and 37°C. At 0, 2, 4, 6, and 8 hours, 100 μL of polyplex solution was removed and DNA binding affinity was immediately measured as before in quadruplicate. DNA release rate from polyplexes was quantified as follows:

[0243]

number

[0244] Cell culture: HPDFs were cultured in fibroblast basal medium supplemented with FGM-2 SingleQuots containing 2% FBS. 3T3 were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin (P / S). Both types of cells were cultured under standard cell culture conditions in a humidified incubator at 37°C with 5% CO2.

[0245] Gene transfection ability of LBPAE quantified by Gluc expression: The gene transfection ability of LBPAE in HPDF and 3T3 was first evaluated by Gluc expression using Gluc DNA as a reporter gene. 1 × 10 cells / well for 3T3 were transfected in 100 μL medium in a 96-well plate. 4 Cell density: 2 x 10 per well for HPDF 4 Cells were seeded at a density of 100 μL and incubated for 1 day before transfection. Commercial gene transfection reagents PEI and SuperFect were optimized according to the manufacturer's protocol. For this purpose, 0.5 μg Gluc DNA was used in each well, and the w / w ratios in PEI / DNA polyplexes were varied from 1:1, 2:1 to 3:1, and the w / w ratios in SuperFect / DNA polyplexes were varied from 3:1, 6:1 to 9:1. For LBPAE gene transfection, the same amount of 0.5 μg Gluc DNA was used in each well, and LBPAE / DNA polyplexes with different w / w ratios were prepared in 20 μL sodium acetate buffer as described above and then diluted with 80 μL cell culture medium. The cell culture medium in the 96-well plate was removed and 100 μL of polyplex-containing medium was added. After 4 h, the polyplex-containing medium in the plate was replaced with 100 μL of fresh medium, and the cells were incubated for another 44 h. Gluc activity of transfected cells was measured in quadruplicates using the Gluc assay according to standard protocols. Briefly, 20 μL of supernatant in a 96-well plate was removed and 50 μL of Gluc assay solution was added. Luminescence intensity was measured using a SpectraMax M3 plate reader, and Gluc activity was directly plotted in terms of relative light units (RLU).

[0246] Quantification of gene transfection capacity of LBPAE by GFP expression: The gene transfection capacity of LBPAE was further evaluated by GFP expression. HPDF and 3T3 cells were cultured at 5 × 10 per well in 500 μL medium in a 24-well plate. 4Cells were seeded at a density of 100 μg / well and incubated for 1 day before transfection. 2 μg GFP DNA was used for each well, and polyplexes with different w / w ratios were prepared in 100 μL sodium acetate buffer and then mixed with 400 μL cell culture medium. The medium inside the cells was removed and polyplex-containing medium was added. After 4 h, the polyplex-containing medium was replaced with 500 μL of fresh medium, and the cells were incubated for another 44 h. The cells were then washed with HBSS and imaged under a fluorescent microscope (Olympus IX81). To quantify GFP expression by flow cytometry, the transfected cells were digested with trypsin-EDTA, washed twice with HBSS, and then resuspended in PBS containing 2% FBS. Flow cytometry measurements were performed in triplicate on an Acculi C6 system, and at least 10,000 cells were counted for each sample. The median fluorescence intensity (MFI) of the cells was quantified with Flowjo software. Cells transfected with PEI and SuperFect were used as positive controls, and untreated cells were used as negative controls.

[0247] Cell viability measured by Alamar Blue assay: The viability of HPDF and 3T3 cells after transfection was measured by Alamar Blue assay. For this purpose, the cell supernatant was removed 48 h after transfection and the cells were washed twice with HBSS. Then, 10% Alamar Blue solution in HBSS was added and the cells were incubated for another 1–3 h. After that, the Alamar Blue solution in the wells was transferred to a flat-bottom 96-well plate and the fluorescence intensity at 590 nm was measured in quadruplicate using a SpectraMax M3 plate reader. Cells without polyplex treatment were used as a positive control and the fluorescence intensity was normalized as 100% cell viability.

[0248] Toxicological profile of LBPAE: The toxicological profile of LBPAE was determined at lethal concentration 50 (LC 50 ) assessment. Live and dead cells were stained using the LIVE / DEAD viability / cytotoxicity kit. For HPDF, 2 × 10 cells were cultured per well in 100 μL medium in a 96-well plate. 4for 3T3, 1 x 10 per well at a density of 4 Cells were seeded at a density of 1000 x 1000 and 1000 x 1000. The next day, LBPAE / DNA polyplexes and SuperFect / DNA polyplexes were prepared and five different doses of polyplexes were used to transfect cells as described above. 24 hours after transfection, the cell culture medium was removed and replaced with HBSS containing calcein-AM (C-AM) (1:5000) and ethidium homodimer-1 (EthD-1) (1:500), and cells were incubated for an additional 20 min. Cells were then washed with HBSS and imaged under a fluorescent microscope (Olympus IX81). Cell viability quantified by alamar blue assay in quadruplicates was used to determine LC. 50 was calculated.

[0249] Polyplex cellular uptake: For polyplex cellular uptake tests, Gluc DNA was labeled with a Cy3 (red fluorescent dye) labeling kit according to the recommended protocol. Fibroblasts were plated in a 24-well plate at 5 × 10 per well. 4 Cells were seeded at a density of 1000 x 1000 in 1000 x 1000 cells / well. 0.5 μg labeled DNA was used per well, and gene transfection was performed the next day. Four hours after transfection, cells were washed with HBSS, fixed with 4% PFA, permeabilized with 0.1% Triton-100, stained with DAPI, and then imaged under a fluorescent microscope (Olympus IX81). To quantify cellular uptake efficiency by flow cytometry, after transfection, cells were digested with trypsin-EDTA, washed twice with HBSS, and then resuspended in PBS containing 2% FBS, and the percentage and MFI of Cy3-positive cells were quantified in triplicate on an Accuri C6 flow cytometry system.

[0250] Detection of C7 expression in HPDFs by cytoimmunofluorescence staining: To detect C7 expression by cytoimmunofluorescence staining, HPDFs were cultured at 1.5 × 10 per well in an 8-well chamber (Ibidi). 4Cells were seeded at a density of 100x100 / mL. The next day, 1 μg MCC7 was used per well and gene transfection was performed as described above using LBPAE / DNA polyplexes at a w / w ratio of 40:1 and SuperFect / DNA polyplexes at a w / w ratio of 3:1. 48 hours after transfection, cells were fixed with 4% PFA, permeabilized with 0.1% Triton-100, blocked with 5% goat serum in 1× PBS for 1 hour at room temperature, and then incubated overnight at 4°C with primary antibodies of monoclonal anti-collagen and type VII antibodies generated in mouse in blocking buffer (antibody dilution: 1:200). The next day, cells were incubated with Alexa-568 goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody at a dilution of 1:800 for 1 hour at room temperature in the dark and DAPI. Immunofluorescence images were taken using a fluorescence microscope (Olympus IX81). Cells treated with secondary antibodies only without antibody treatment were used as the control group.

[0251] C7 expression in HPDFs quantified by flow cytometry: To quantify C7 expression by flow cytometry, HPDFs were cultured at 5 × 10 per well in a 24-well plate. 4 Cells were seeded at a density of 100x and transfected 24 hours later. 2 μg MCC7 was used per well, and transfection was performed as described above with LBPAE / DNA polyplexes at a w / w ratio of 40:1 and SuperFect / DNA polyplexes at a w / w ratio of 3:1. Four days after transfection, cells were digested with trypsin-EDTA, fixed with IC fixation buffer (2% PFA), permeabilized with permeabilization buffer (1x PBS / 1% BSA / 0.1% saponin), blocked with goat serum (10% in permeabilization solution), and then incubated with primary antibodies of monoclonal anti-collagen and type VII antibodies generated in mouse at a dilution of 1:50 in blocking buffer for 1 h at room temperature. Afterwards, cells were further incubated with Alexa-647 goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody at a dilution of 1:3000 in permeabilization buffer in the dark. Finally, cells were resuspended in PBS and analyzed in triplicate by flow cytometry. Cells treated with secondary antibody alone without antibody treatment served as a control group.

[0252] Statistics: SPSS Statistics for windows version 24 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. All gene transfection data were analyzed using Student's t-test, and data were presented as mean ± standard deviation. LC was determined by linear regression analysis. 50 Values ​​were calculated. A P value <0.05 was considered statistically significant.

[0253] Example 9: HPAE polyplexes containing COL7A1 for gene delivery to recessive dystrophic epidermolysis bullosa keratinocytes

[0254] The following examples are based on molecular weights (M w ), and describe transfection results for polyplexes comprising minicircle COL7A1 and four highly branched poly(β-amino esters) (HPAEs) of the present disclosure using HPAE:COL7A1 DNA weight ratios of 10:1, 30:1, and 50:1.

[0255] Synthesis and Characterization of HPAE: HPAE was prepared in two steps. In step 1, the monomers 5-amino-1-pentanol, trimethylolpropane triacrylate, and 1,4-butanediol diacrylate were reacted to give the highly branched C32 (poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate)) ("HC32"). In step 2, HC32 was reacted with 1,11-diamino-3,6,9-trioxaundecane (DATOU) to give the HPAE ("HC32-DATOU").

[0256] Four HC32-DATOU polymers with different molecular weights (MW) were synthesized by the "A2+B3+C2" Michael addition strategy. First, the HC32-based polymer was synthesized. Briefly, A2 monomer AP (9.0 mmol, 0.923 g), B3 monomer TMPTA (0.5 mmol, 0.148 g), and C2 monomer C (10.0 mmol, 1.98 g) were dissolved in 3.1 mL DMSO and then reacted at 90 °C. Gel permeation chromatography (GPC) was used to monitor the growth of MW and polydispersity index (PDI). 20 μL of reaction samples were taken at different time points, then diluted in 1 mL DMF and filtered through a 0.2 μm filter, after which GPC measurements were performed on an Agilent 1260 Infinite GPC equipped with triple detectors: refractive index detector (RI), viscosity detector (VS DP), and dual light scattering detector (LS 15° and LS 90°). DMF and 0.1% LiBr were used to elute the GPC columns (PolarGel-M, 7.5 × 300 mm, two in series) at a flow rate of 1 mL / min at 60° C. The GPC columns were calibrated with linear poly(methyl methacrylate) (PMMA) standards.

[0257] Weight average molecular weight of the base polymer (M w When the NA values ​​approached the target values ​​(approximately 10, 20, 30, and 40 kDa, respectively), the reaction was stopped by diluting the reaction solution in DMSO to 100 mg / mL. The HC32-based polymer was then end-capped by Michael addition using the end-capping agent DATOU (10.0 mmol, 1.92 g) dissolved in DMSO (100 mg / mL) at room temperature (RT) for 48 h to obtain the HC32-DATOU polymer, which was purified by two precipitations with diethyl ether to remove excess monomers, oligomers, and end-capping agent.

[0258] The final HC32-DATOU product was dried in a vacuum oven for 24 hours and then freeze-dried for another 24 hours to remove residual solvent. To measure the MW and PDI of the final product, a 10 mg sample was dissolved in 1 mL DMF and GPC measurements were performed as described above. Proton nuclear magnetic resonance ( 11H NMR) was used to determine whether the compound was dissolved in CDCl3 and 1 H NMR spectra were acquired on a 400 MHz Varian Inova spectrometer to confirm the chemical composition and purity of the HC32-DATOU polymer. Samples are reported in parts per million (ppm) relative to the solvent (7.24 ppm) or internal control (tetramethylsilane 0.00 ppm).

[0259] Figure 13 shows the effect of increasing the polymerization time of the base polymer on the different M w The results show that four HC32-DATOU polymers with the following M w increased from 11 kDa to 41 kDa without gelation, demonstrating the high flexibility of the "A2+B3+C2" Michael addition strategy in controlling the HAPE MW. The MH plot alpha values ​​of all HC32-DATOU polymers were less than 0.5 (Figure 13c), indicating their highly branched structures. The table below shows the PDI and MH plot α values ​​for the HC32-DATOU polymers. [Table 2]

[0260] MCC7 biosynthesis.

[0261] The normal plasmid pcDNA3.1COL7A1 was obtained. MCC7 was biosynthesized by inserting the COL7A1 sequence from pcDNA3.1COL7A1 into a MN511A-1 cassette with a cytomegalovirus promoter provided by System Biosciences, and minicircle DNA was induced and generated according to the instructions from System Bioscience and the published phiC31 plus 1-Scel digestion system of minicircle technology (Gaspar, V.; de Melo-Diogo, D.; Costa, E.; Moreira, A.; Queiroz, J.; Pichon, C.; Correia, I.; Sousa, F. Minicircle DNA Vectors for Gene Therapy: Advances and Applications. Expert Opin. Biol. Ther. 2015, 15(3), 353-379. https: / / doi.org / 10.1517 / 14712598.2015.996544.). To confirm the biosynthesis of MCC7, a DNA digestion test was performed. For this purpose, 0.5 μg pcDNA3.1COL7A1, parental plasmid (MN511A-1-COL7A1), and MCC7 were digested with 1 μL EcoRI and then subjected to agarose gel electrophoresis for 40 min at 100 V. Images were then visualized using G:BOX from Syngene.

[0262] Polyplex Preparation and Formulation

[0263] HC32-DATOU was dissolved in DMSO to a 100 μg / μL stock solution, which was stored at −20° C. for future studies. DNA was dissolved in TE buffer, which was also stored at −20° C. SA buffer was diluted to 0.025 M before use.

[0264] For standard polyplex preparation, DNA and polymer were dissolved in equal volumes in SA buffer according to the polymer / DNA weight ratio (w / w). The polymer solution was added to the DNA solution, mixed using a vortex for 10 seconds, and incubated at room temperature for another 10 minutes to allow polyplex formation. For formulation testing, typically, 5 μg GFP plasmid DNA and 150 μg HC32-DATOU were dissolved in 200 μL SA, respectively, to formulate HC32-DATOU / DNA polyplexes (w / w=30:1). Polyplexes were either used immediately as fresh or stored at room temperature, 4°C, -20°C, and -80°C prior to transfection, or lyophilized.

[0265] For lyophilization, sucrose was added to the polyplex solutions to give final sucrose concentrations of 0%, 1%, 3%, and 5%, respectively. All samples were frozen at -80°C for 1 h and then immediately subjected to freeze-drying at -55°C for 24 h using a Christ Alpha 1-2 LDplus Freeze Dryer. Polyplexes were then reconstituted with the original volume of SA and used for transfection.

[0266] After optimization of the polyplex preparation procedure (see Example 10 below), HC32-DATOU complexed with Gluc-encoding DNA stored under different conditions was used to evaluate the feasibility of long-term storage of polyplexes prior to transfection applications, where 0.5 μg DNA at a 30:1 polymer / DNA w / w ratio was used for each well of a 96-well plate.

[0267] Assay for DNA compaction and heparin release

[0268] To evaluate the DNA condensation ability of HC32-DATOU and the physical stability of HC32-DATOU / DNA polyplexes, DNA condensation assays and heparin release assays were performed using agarose gel electrophoresis. 0.5 μg DNA (MCC7) was used for each sample to prepare polyplexes at a w / w ratio of 30:1. Aqueous heparin solutions were added to the polyplex solutions at concentrations ranging from 0.1 to 6 IU / μL. Naked DNA and HC32-DATOU / MCC7 polyplexes without heparin were used as controls. All samples were incubated at room temperature for 2 h and then loaded onto a 1% agarose gel stained with 10 μL SYBR safe DNA stain. Electrophoresis was performed at 100 V for 1 h in 1× TAE buffer.

[0269] PicoGreen Assay

[0270] The PicoGreen assay was used to quantify the DNA binding affinity and DNA release of HC32-DATOU in the presence of heparin. HC32-DATOU / MCC7 polyplexes were prepared with 0.2 μg of DNA at a w / w ratio of 30:1, and then heparin was introduced into the polyplex solution at concentrations of 0.3 IU / μL, 3 IU / μL, and 6 IU / μL, respectively. Naked DNA and HC32-DATOU / MCC7 polyplexes without heparin treatment were used as controls. After 2 h of incubation, all samples were mixed with 10 μL of PicoGreen working solution and incubated for an additional 5 min. The mixed solutions were then diluted with deionized water to a final concentration of 1 μg / mL in a black 96-well plate. Fluorescence measurements were performed in quadruplicate using a SpectraMax M3 plate reader at 490 nm excitation and 535 nm emission. DNA release efficiency was quantified by normalizing the fluorescence intensity of the samples to the naked DNA control.

[0271] Measurement of polyplex size and zeta potential

[0272] Polyplex size was measured by nanoparticle tracking analysis (NTA) using a Nanosight NS300. Polyplexes were prepared with 0.5 μg DNA in 10 μL of SA at a w / w ratio of 30:1. The polyplex solution was then diluted in 1 mL of distilled water and then subjected to NTA analysis. A 60 s movie containing Brownian motion tracking of the particles was recorded using the NTA software (version 3.2). Ten tracks were evaluated for each sample. Zeta potential measurements of polyplexes were performed using a Malvern Instruments Zetasizer (Nano-ZS90) at a 90° scattering detector angle.

[0273] Transmission electron microscope (TEM) observation

[0274] The morphology of the polyplexes was characterized by TEM. 80 μL of polyplex solution containing 2 μg MCC7 at a w / w ratio of 30:1 was centrifuged, the supernatant was discarded, and the polyplexes were then washed twice with an additional 80 μL of distilled water to remove excess salts. The polyplexes were then resuspended in a final volume of 10 μL of distilled water. 2.5 μL of the polyplex solution was then cast onto a Formvar support film on a 200 mesh copper grid and immediately lyophilized. Images were captured on an FEI Tecnai 120 TEM at 120 kV at the UCD Conway Imaging Core Center.

[0275] cell culture

[0276] RDEBK and human keratinocyte (NHK) cells were cultured under standard cell culture conditions in a humidified incubator at 37°C with 5% CO2 in keratinocyte basal medium (KBM-Gold) containing supplement pack (KGM-Gold SingleQuots) and 1% PS.

[0277] GFP expression and cell viability

[0278] First, GFP reporter gene transfection was performed to evaluate the gene transfection efficiency of the four HC32-DATOU polymers and screen the best performing candidate. RDEBK was transfected at 2 × 10 per well in a 96-well plate. 4 The cells were seeded at a density of 1000 x 1000 cells / well. The next day, 0.5 μg of plasmid DNA encoding GFP was used in each well. The cells were transfected with different M w HC32-DATOU polyplexes bearing HC32-DATOU were prepared at polymer / DNA w / w ratios of 10:1, 30:1, and 50:1. Four hours after transfection, the transfection medium was replaced with fresh medium. Forty-eight hours after transfection, cells were visualized for GFP expression under a fluorescent microscope (Olympus IX81). Cell viability was measured with an Alamar Blue assay. The cell supernatant was removed, and the cells were then incubated with 10% Alamar Blue reagent in HBSS for an additional hour at 37°C. The Alamar Blue solution was then transferred to a flat-bottom 96-well plate. The fluorescence intensity was read by a SpectraMax M3 plate reader at an excitation of 570 nm and an emission of 590 nm. The fluorescence intensity of the untreated cell group was plotted as 100% survival. Cell viability was measured in quadruplicate and calculated by normalizing the fluorescence intensity of the samples to that of the untreated group. HC32-DATOU, which showed the highest GFP expression and cell viability, was used for the next study.

[0279] For formulation testing (see Example 10 below), SuperFect / DNA polyplexes were prepared at a 3:1 w / w ratio according to published studies (Zeng, M.; Zhou, D.; Alshehri, F.; Lara-Saez, I.; Lyu, Y.; Creagh-Flynn, J.; Xu, Q.; A, S.; Zhang, J.; Wang, W. Manipulation of Transgene Expression in Fibroblast Cells by a Multifunctional Linear-Branched Hybrid Poly(β-Amino Ester) Synthesized through an Oligomer Combination Approach. Nano Lett. 2019, 19(1), 381-391. https: / / doi.org / 10.1021 / acs.nanolett.8b04098. Theoharis,S.;Krueger,U.;Tan,PH;Haskard,DO;Weber,M.;George,AJT Targeting Gene Delivery to Activated Vascular Endothelium Using Anti E / P-Selectin Antibody Linked to PAMAM Dendrimers. J.Immunol. Methods 2009,343(2),79-90.https: / / doi.org / 10.1016 / j.jim.2008.12.005.). Lipofectamine 2000 / DNA lipoplexes were prepared according to the manufacturer's protocol (2:1 volume / weight ratio). Median fluorescence intensity (MFI) and GFP positive cells were quantified by flow cytometry in triplicates on an Acculi C6 system and further analyzed with Flowjo V10 software. 1×10 4 Cells were counted for each run.

[0280] Gluc reporter gene transfection and cell viability

[0281] HC32-DATOU was further evaluated in a Gluc reporter gene transfection assay. HC32-DATOU / DNA polyplexes were prepared at w / w ratios of 20:1, 30:1, and 40:1 using 0.5 μg of plasmid DNA encoding Gluc in each well, respectively.According to past publications (Green, J.J.; Zugates, G.T.; Tedford, N.C.; Huang, Y.-H.; Griffith, L.G.; Lauffenburger, D.A.; Sawicki, J.A.; Langer, R.; Anderson, D.G. Combinatorial Modification of Degradable Polymers Enables Transfection of Human Cells Comparable to Adenovirus. Adv. Mater. 2007, 19(19), 2836 - 2842. https: / / doi.org / 10.1002 / adma.200700371. Huang, J.-Y.; Gao, Y.; Cutlar, L.; O’Keeffe-Ahern, J.; Zhao, T.; Lin, F.-H.; Zhou, D.; McMahon, S.; Greiser, U.; Wang, W.; et al. Tailoring Highly Branched Poly(Beta-Amino Ester)s: A Synthetic Platform for Epidermal Gene Therapy. Chem. Commun. (Camb). 2015, 51(40), 8473 - 8476. https: / / doi.org / 10.1039 / c5cc02193f. Zeng, M.; Zhou, D.; Ng, S.; Ahern, J.O.; Alshehri, F.; Gao, Y.; Pierucci, L.; Greiser, U.; Wang, W. Highly Branched Poly(5-Amino-1-Pentanol-Co-1,4-Butanediol Diacrylate) for High Performance Gene Transfection. Polymers (Basel). 2017, 9 (12), 161. https: / / doi.org / 10.3390 / polym9050161.), PEI / DNA polyplexes were prepared at w / w ratios of 1:1, 2:1, and 3:1, respectively.

[0282] RDEBK were seeded and gene transfection was performed as described above. 48 hours after transfection, 50 μL of cell supernatant was mixed with an equal volume of Gluc assay working solution to quantify gene transfection efficiency. The fluorescence intensity of the mixture was measured using a SpectraMax M3 plate reader at 485 nm excitation and 525 nm emission. The Gluc activity results were plotted in terms of relative light units (RLU). Cell viability was measured as described above. Both Gluc activity and cell viability experiments were measured in quadruplicate.

[0283] Cellular uptake of polyplexes

[0284] MCC7 was labeled using a Cy3 DNA labeling kit according to standard protocols. RDEBK was added to a 96-well plate at 1 × 10 per well. 4 Cells were seeded at a density of 1000 x 1000 cells / well. The next day, cells were transfected with HC32-DATOU / MCC7 polyplexes (w / w=30:1) and PEI / MCC7 (w / w=1:1) for 4 h, using 0.25 μg MCC7 in each well, then fixed with 4% PFA, permeabilized with 0.1% Triton-100, and incubated with DAPI at a working concentration of 1 μg / mL in HBSS. Fluorescent images were taken with a fluorescent microscope (Olympus IX81). The MFI and Cy3-positive percentage of cells were quantified in triplicate by Acculi C6 system. 1 × 10 cells / well ... 4 The results were further analyzed with Flowjo V10 software, which counted the cells.

[0285] Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0286] RT-qPCR was performed to quantify COL7A1 mRNA expression. One day before transfection, RDEBK was added to a 6-well plate at 2.5 × 10 5Cells were seeded at a density of 1000×1000 cells / ml. Cells were transfected with HC32-DATOU / MCC7 and PEI / MCC7 polyplexes complexed with 5 μg DNA at a w / w ratio of 30:1 and 1:1. Three days after treatment, both treated and untreated cells were harvested and subjected to total RNA purification. RNA extraction procedures were performed according to the RNeasy Mini Kit protocol. Then, 0.5 μg of total RNA from each group was used to synthesize first-strand cDNA. 50 μM Oligo(dT) was added according to the SuperScript III First-Strand Synthesis SuperMix protocol. 20 Reverse transcription was performed using primers. Then, 1 μL of the final complementary DNA (cDNA) product was added to 9 μL of reaction mixture (0.5 μL TaqMan primer, 5 μL TaqMan PCR mixture, 3.5 μL RNase-free water) loaded into one well of a 384-well plate. Each sample was measured in triplicate. For quantitative gene expression of COL7A1, GAPDH was used as an endogenous control. For comparison C values ​​and TaqMan reagents, a QuantStudio 7 Flex System was set up for the experiment. The results were analyzed with QuantStudio real-time PCR software.

[0287] C7 cell immunofluorescence staining

[0288] Cell immunofluorescence staining was used to quantify C7 recovery in RDEBK after treatment with HC32-DATOU / MCC7 and PEI / MCC7 polyplexes. 4Cells were seeded on each coverslip in an 8-well chamber (Ibidi). 1 μg MCC7 was used per well, and HC32-DATOU / MCC7 and PEI / MCC7 polyplexes were prepared at 30:1 and 1:1 w / w ratios, respectively. Three days after transfection, cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, blocked in 5% goat serum in 1×DPBS for 1 h at room temperature, and then incubated with primary antibody (monoclonal anti-collagen, type VII antibody generated in mouse) at an antibody dilution of 1:200 in blocking buffer at 4°C overnight. Afterwards, cells were incubated with secondary antibody (Alexa-568 goat anti-mouse IgG (H+L), at a dilution of 1:800 in blocking buffer). After a final wash, coverslips were mounted with Fluoroshield mounting medium containing DAPI. Finally, cell images were captured using a fluorescence microscope (Olympus IX81).

[0289] Western blotting

[0290] One day before transfection, T-75 flasks were seeded with 1.5 x 10 RDEBK cells per flask. 6Cells were seeded at a density of 1000 ng / ml. HC32-DATOU / MCC7 and PEI / MCC7 polyplexes with w / w ratios of 30:1 and 1:1, respectively, were used for transfection with 39 μg MCC7 per flask. Four days after transfection, cells were harvested and treated with RIPA buffer, which allows efficient cell lysis and solubilization of cellular proteins. 1 μL PIC was added to the cell lysate to a final volume of 50 μL and stored at -80 °C. Protein concentration was quantified by normalizing sample concentration with known BSA concentration using Bradford assay. 40 μg denatured protein samples were loaded onto SDS-Page gels (4%-10%) and electrophoresis was then performed at 75 V for 20 min followed by 120 V for 1 h. Protein samples were then transferred to nitrocellulose membranes at 80 V for 1 h at room temperature followed by 90 V for 30 min at 4 °C. Membrane blocking was performed in blocking buffer (5% BSA in TBST buffer) for 1 hour at room temperature. β-actin was used as an endogenous control. Primary antibodies (polyclonal anti-C7 rabbit antibody and anti-actin mouse antibody at 2500 dilution in blocking buffer) were then added to the membrane and incubated overnight at 4°C. After a washing step, secondary antibodies (anti-rabbit HRP and anti-mouse HRP at 5000 dilution in blocking buffer) were added to the membrane and incubated for 1 hour at room temperature. After three TBST washes, the membrane was visualized with Pierce ECL Plus substrate.

[0291] statistics

[0292] SPSS Statistics for windows version 24 (IBM Corp., Armonk, NY, USA) was used for statistics. All gene transfection data were analyzed using Student's t-test and expressed as mean ± standard deviation (SD). For all analyses, a p-value <0.05 was considered statistically significant.

[0293] result:

[0294] Reporter gene transfection in RDEBK using HPAEs of different molecular weights

[0295] To identify the most favorable MW for gene delivery, different M w Four HC32-DATOU polymers with the following structures were used to transfect RDEBK cells with GFP-encoding DNA as a reporter gene. As shown in Figures 14 and 15, no obvious cytotoxicity was observed at a polymer / DNA w / w ratio of 10:1, but the transfection efficiency from all HC32-DATOU polymers was relatively low. When the w / w ratio was increased to 30:1 or higher, among all the polymers, the 11 kDa HC32-DATOU at 30:1 achieved the highest transfection efficiency with the strongest GFP expression, while retaining a high level of cell viability of 98%. Overall, GFP expression was significantly higher than that of the HC32-DATOU polymers. w It decreased as the

[0296] On the other hand, the cytotoxicity of polymer M w For example, at a w / w ratio of 50:1, the M w As MW increases, cell viability decreases from 91% to 58%, 42%, and 15%, respectively. The transfection efficiency decreases with increasing cytotoxicity, which is attributed to the increase in polymer backbone. These results suggest that MW has a significant effect on the transfection performance of HC32-DATOU, and a MW of approximately 10 kDa is required to achieve both high transfection efficiency and low cytotoxicity. w This demonstrates that RDEBK gene transfection is more favorable.

[0297] Commercially available gene transfection reagent PEI (M wThe high gene transfection capacity of 11 kDa HC32-DATOU was verified by comparing it with that of HC32-DATOU / DNA polyplexes (=25 kDa). As shown in Figure 16a, the relative Gluc activity of RDEBK cells after transfection with HC32-DATOU / DNA polyplexes was much higher than that mediated by the PEI / DNA counterpart at all three w / w ratios tested. The highest Gluc activity achieved by HC32-DATOU / DNA polyplexes at a w / w ratio of 30:1 was 17-fold higher than that of the PEI / DNA counterpart at a w / w ratio of 2:1.

[0298] Importantly, HC32-DATOU / DNA polyplexes did not induce obvious cytotoxicity and maintained nearly 100% cell viability. In contrast, PEI showed obvious dose-independent cytotoxicity, with cell viability significantly decreasing from 90% at a 1:1 w / w ratio to 38% at a 3:1 w / w ratio (Figure 16b). Due to the considerable cytotoxicity, PEI at a 1:1 w / w ratio was used in the following studies.

[0299] To confirm the high performance of HC32-DATOU / DNA polyplexes, GFP-encoding DNA was used for transfection. HC32-DATOU / DNA polyplexes mediated a much higher level of GFP expression than PEI, as evidenced by the substantially stronger green fluorescence observed (Figure 16c). Correspondingly, flow cytometry quantitative analysis shows that more cells shift correspondingly to the GFP-determining channel (Figure 16d). 75% of RDEBK cells were GFP-positive after transfection with HC32-DATOU / DNA polyplexes, compared with 39% achieved by PEI / DNA polyplexes. Moreover, the MFI of RDEBK cells transfected by HC32-DATOU / DNA polyplexes was 13-fold higher than that mediated by the PEI / DNA counterpart (Figure 16e), indicating that much higher gene expression was achieved in individual cells. All these results demonstrate that HC32-DATOU is much more efficient and safer than PEI for gene transfection in RDEBK cells.

[0300] Biosynthesis of MCC7 and cellular uptake of HPAE / MCC7 polyplexes by RDEBK cells

[0301] Using the minicircle technology phiC31 and 1-Scel digestion system (Gaspar, V.; de Melo-Diogo, D.; Costa, E.; Moreira, A.; Queiroz, J.; Pichon, C.; Correia, I.; Sousa, F. Minicircle DNA Vectors for Gene Therapy: Advances and Applications. Expert Opin. Biol. Ther. 2015, 15 (3), 353-379. https: / / doi.org / 10.1517 / 14712598.2015.996544.), we synthesized a minicircle DNA encoding the full-length COL7A1 of approximately 9 kb (Figure 17a). Gel electrophoresis showed that among all three COL7A1-tagged DNAs, only MCC7 had a 3 kb long backbone, which is 2 kb and 5 kb shorter than the regular plasmid (RP) pcDNA3.1COL7A1 and the parental plasmid (PP) MN511A-1-COL7A1, respectively (Figure 17b), indicating that MCC7 has a compact derivative from the conventional PP vector lacking bacterial sequences.

[0302] Cellular uptake of HC32-DATOU / MCC7 polyplexes was performed in RDEBK cells. MCC7 was labeled with the red fluorescent dye Cy3, and HC32-DATOU / MCC7 polyplexes were prepared as described above. After 4 h of transfection, very strong red fluorescence was observed around the nuclei of the cells (Figure 17c). In contrast, PEI / MCC7 polyplexes show a much lower cellular uptake efficiency as evidenced by the much weaker red fluorescence. Furthermore, flow cytometry measurements demonstrated that although the percentage of Cy3-positive cells was similar (96.4% vs. 93%, Figure 17d), the MFI of cells incubated with HC32-DATOU / MCC7 polyplexes was approximately two-fold higher than those treated with their PEI / MCC7 counterparts (Figure 17e), indicating that a higher number of DNA copies were taken up by RDEBK cells. The maximum DNA size that RV and adeno-associated virus (AAV) vectors can carry is 7-8 kb and 5 kb, respectively. The fact that HC32-DATOU can efficiently deliver 12 kb-long MCC7 to RDEBK cells highlights the possibility of achieving high C7 expression for RDEB treatment.

[0303] High levels of COL7A1 mRNA and recombinant C7 expression

[0304] Challenges for vector / DNA polyplexes following internalization are intracellular barriers including endo / lysosomal escape, transport through the cytoplasm, DNA release, and nuclear entry. The cell cycle is another obstacle to the efficiency of nuclear uptake in cells. Cells undergoing mitosis have a nuclear uptake efficiency more than 10-fold higher than cells in the proliferative phase of the cell cycle.

[0305] To evaluate the transcript COL7A1 mRNA and C7 protein expression mediated by HC32-DATOU / MCC7 polyplexes, RT-qPCR, immunofluorescence staining, and Western blotting studies were performed.

[0306] Figures 18a and 18b show the summary of RT-qPCR amplification plots of endogenous control GAPDH and COL7A1 mRNA expression, respectively. After normalization with respect to the endogenous control, it is shown that HC32-DATOU / MCC7 polyplexes mediated a 4019-fold upregulation of COL7A1 mRNA expression compared to UT cells (Figure 18c), which was 2.2-fold higher than that mediated by PEI / MCC7 polyplexes. Immunofluorescence staining study (Figure 18d) further reveals that null C7 expression was detected in untreated RDEBK cells. In contrast, after transfection with HC32-DATOU / MCC7 polyplexes, much higher levels of cellular C7 expression in the nuclear periphery were observed in cell immunofluorescence images. Moreover, consistent with the results of COL7A1 mRNA expression, HC32-DATOU / DNA polyplexes mediated more efficient C7 expression than their PEI / MCC7 counterparts.

[0307] Western blotting results show that no C7 secretion was detected from untreated RDEBK cells (Figure 18e). In contrast, after transfection with HC32-DATOU / polyplexes, a very clear 290 kDa protein band of C7 was discernible, and this C7 band was as intense as that of wild-type NHK cells, which have intact C7 production function. Note that PEI / MCC7 polyplexes achieved a significantly higher level of COL7A1 mRNA expression, but limited C7 production.

[0308] Mechanistic studies have demonstrated that compact DNA structures increase cellular uptake, intracellular vector copy number, nuclear localization, and mRNA transcription levels (Kobelt, D.; Schleef, M.; Schmeer, M.; Aumann, J.; Schlag, PM; Walther, W. Performance of High Quality Minicircle DNA for in Vitro and in Vivo Gene Transfer. Mol. Biotechnol. 2013, 53(1), 80-89. https: / / doi.org / 10.1007 / s12033-012-9535-6.). Herein, HC32-DATOU utilizes optimized polymers and miniaturized gene constructs to effectively deliver the COL7A1 gene into RDEBK cells, promoting subsequent mRNA transcription and eventual recombinant C7 expression, thereby enhancing skin integrity.

[0309] Mechanistic testing of high gene transfection efficiency of HPAE / MCC7 polyplexes

[0310] DNA condensation, binding, polyplex size, zeta potential, morphology, and DNA release are related to transfection performance. To better understand the mechanism of high gene transfection efficiency mediated by HC32-DATOU / MCC7 polyplexes, these physicochemical parameters were investigated.

[0311] The cationic HC32-DATOU could condense the negatively charged MCC7 to form polyplexes via electrostatic self-assembly (Figure 19a). To confirm this, agarose gel electrophoresis was performed to evaluate the DNA condensation ability of HC32-DATOU 2 h after polyplex preparation. As shown in Figure 19b, naked MCC7 DNA shifted on the gel, while HC32-DATOU could condense DNA on the well without obvious DNA shift. Furthermore, heparin competition assay showed that HC32-DATOU polyplexes with low heparin concentrations (0.1-0.3 IU / μL) still condensed most of the DNA. This suggests the strong DNA condensation ability and highly stable property of HC32-DATOU polyplexes.

[0312] Similarly, HC32-DATOU showed a stable and high DNA binding affinity of 96.3%, as quantified by PicoGreen assay (Figure 19c), indicating only 3.7% DNA unpacking. When 3 and 6 IU / μL concentrations of heparin were applied, 68.2% and 99.6% DNA unpacking was detected, respectively. These results demonstrate that HC32-DATOU efficiently condenses, binds, and releases DNA in a controlled manner in the presence of tunable negatively charged heparin.

[0313] At the optimized w / w ratio (30:1) for efficient C7 expression, the nanoparticles showed a mean size of 110 nm and a mode size of 81 nm (Fig. 19d), respectively, with a zeta potential of +37.4 mV (Fig. 19e), indicating a compact nanoparticle structure with a positive surface charge. Polyplexes are known to be most commonly formed in spherical or toroidal shapes. In this regard, HC32-DATOU / MCC7 polyplexes showed a uniform and spherical morphology (Fig. 19f).

[0314] Enhancement of transfection efficiency has been reported to benefit from diamine end-group modification. This modification increases the cationic charge of the polymer, thereby improving the polymer / DNA binding kinetics and condensation of DNA into nanoparticles, and protecting DNA from degradation. Apart from the end-modification with diamine DATOU, multiple DNA binding / condensation moieties, including primary, secondary, and tertiary amines, are present in the HC32 backbone and end groups. Generally, LPAE / DNA particles are less than 250 nm, and particles of smaller size have been found to be more efficiently internalized by cells.

[0315] Without being bound by any theory, it is believed that the small, compact, uniform, and cationic properties of HC32 / MCC7 polyplexes enable high cellular uptake efficiency. Furthermore, the multiple ionizable secondary and tertiary amines can buffer a wide range of protons, which may facilitate endo / lysosomal escape via the "proton sponge effect". Subsequently, the stable gene packaging stability of HC32-DATOU suggests that it may aid in the intracellular transport of polyplexes through the cytoplasm to the nucleus.

[0316] An efficient vector must balance sufficient binding strength to protect DNA with the ability to release DNA. Without being bound by any theory, the moderate electrostatic interaction between HC32-DATOU and MCC7 and the biodegradable property of HC32-DATOU may facilitate gene release from polyplexes in the nucleus to initiate the transcription step.

[0317] Example 10: Lyophilized compositions comprising HPAE polyplexes of the present disclosure

[0318] The following examples describe the results of varying storage conditions and cryoprotectant concentrations in the lyophilization process for exemplary HPAE / DNA polyplex formulations.

[0319] HC32-DATOU / DNA polyplex formulations were optimized by varying the storage conditions and cryoprotectant concentrations in the lyophilization process. Figure 20a illustrates the scheme of polyplex lyophilization processing and gene transfection testing in RDEBK cells with GFP-encoding DNA. Except for the freshly prepared polyplexes, all polyplexes were used for gene transfection 1 day after preparation. As shown in Figure 20b, by varying the storage temperature, the fresh polyplexes and the polyplexes stored at 4°C, -20°C, and -80°C, except for those stored at room temperature, show comparable and high GFP expression with a significant shift of cell population in the flow cytometry histogram distribution (Figure 20c). As shown in Figures 20d and 20e, the efficiency quantified by flow cytometry was higher than 70%, and the normalized MFI was approximately 10-fold higher than the UT group. These results demonstrate that a low storage temperature is favorable for maintaining the high gene transfection capacity of HC32-DATOU / DNA polyplexes.

[0320] Next, we used sucrose as a cryoprotectant during the freeze-drying process to examine the effect of cryoprotectant concentration on the gene transfection ability of HC32-DATOU / DNA polyplexes. Freeze-dried polyplexes without cryoprotectant (0% sucrose) lost most of their transfection ability, only showing 20% ​​efficiency and 1.4-fold higher MFI compared to UT cells.

[0321] Adding 1%, 3%, and 5% sucrose to the polyplex solution before lyophilization increased the transfection efficiency to 54%, 61%, and 52%, respectively. These results demonstrate that 3% sucrose is more efficient in maintaining the high gene transfection capacity of HC32-DATOU / DNA polyplexes. Although gene transfection was somewhat lower than that of the freshly prepared counterparts, the gene transfection capacity of polyplexes stored at low temperature or freeze-dried with sucrose was still much higher than that of the commercial reagents SuperFect and Lipofectamine, which have efficiencies of 10% and 32%, respectively.

[0322] Furthermore, polyplex lyophilization has inherent advantages. First, it allows reconstitution of polyplexes at higher concentrations, which is particularly beneficial for in vivo injections where limited administration volumes are required. Second, the easily tunable solute (sucrose) can make the reconstituted polyplex solution isotonic in the formulation. Lyophilized polyplexes with sucrose are also expected to be more stable in the presence of serum compared to freshly prepared polyplexes. Finally, lyophilized polyplexes can be stored for years without loss of efficacy.

[0323] Gene transfection tests were performed on high-performance HC32-DATOU / DNA polyplexes stored for different times (4°C, -20°C, -80°C). As shown in Figure 21, after storage at 4°C for 0.5 or 1 month, the Gluc activity of the polyplexes was 2-3 orders of magnitude lower than that mediated by the freshly prepared ones. After 2 months, the efficiency of the polyplexes became negligible.

[0324] In contrast, even after 1 year, polyplexes stored at -20°C and -80°C mediated the same level of Gluc activity as freshly formulated polyplexes. These results demonstrate that HC32-DATOU / DNA polyplexes are highly stable and retain full functionality for gene transfection simply by storage at -20°C or -80°C, making them highly suitable for clinical applications.

[0325] Materials for Examples 9 and 10: Monomers: 5-amino-1-pentanol (AP, 99%), trimethylolpropane triacrylate (TMPTA, 99%), 1,11-diamino-3,6,9-trioxaundecane (DATOU, 98%) were purchased from Sigma-Aldrich, and 1,4-butanediol diacrylate (BDA, 98%) was purchased from VWR. Chemicals: Lithium bromide (LiBr, 99%), Tris-buffered saline, and Tween 20 (TBST), paraformaldehyde (PFA), and Triton X-100 were purchased from Sigma-Aldrich. Solvents: Dimethyl sulfoxide (DMSO, Sigma-Aldrich, 99%), dimethylformamide (DMF, Fisher Scientific, 99%), diethyl ether (Sigma-Aldrich, 99%), and deuterated chloroform (CDCl3, Sigma-Aldrich, 99.9%) were used as received. Branched polyethyleneimine (PEI, M w= 25 kDa, Sigma-Aldrich), SuperFect (QIAGEN), Lipofectamine 2000 (Invitrogen) were used as commercial reagent controls. Keratinocyte cell basal medium (Clonetics KBM-Gold) and supplement packs (Clonetics KGM-Gold SingleQuots) were purchased from Lonza. Cell-secreted Gaussia princeps luciferase (Gluc) plasmid and BioLux Gaussia luciferase assay kit were obtained from New England Biolabs UK. Green fluorescent protein (GFP) plasmid was purchased from Aldevron. Hanks' balanced salt solution (HBSS), sodium acetate (SA, pH 5.2 ± 0.1, 3 M) buffer, Tris-EDTA acetate (TAE) buffer and radioimmunoprecipitation assay (RIPA) buffer, agarose, bovine serum albumin (BSA), goat serum, monoclonal anti-C7 antibody produced in mouse, protease inhibitor cocktail (PIC), and Bradford reagent were purchased from Sigma-Aldrich. 1× Dulbecco's phosphate-buffered saline (PBS), Gibco OPTI-MEM I reduced serum medium, 4',6-diamidino-2-phenylindole (DAPI), and PicoGreen assay kit were purchased from Life Technologies. Penicillin-streptomycin (PS), EcoRI, Alexa-568 goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, and Pierce ECL plus Western blotting substrate were purchased from Thermo Fisher Scientific. Alamar Blue assay kit, SYBR safe DNA gel stain, and SuperScript III First-Strand Synthesis SuperMix were purchased from Invitrogen. Collagen type VII alpha 1 (Fam-MGB, primers and probe), human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control (VIC / MGB probe, limiting primers), and TaqMan gene expression master mix were purchased from Applied Biosystems.TE buffer (QIAGEN), Cy3 DNA labeling kit (Mirus), RNeasy Mini Kit (QIAGEN), and Fluoroshield mounting medium with DAPI (Abcam) were used according to the manufacturer's protocol. Polyclonal anti-C7 rabbit primary antibody (Merck Millipore), anti-beta-actin mouse primary antibody (Abcam), anti-rabbit IgG HRP-conjugated antibody (Cell Signaling), and anti-mouse IgG HRP-conjugated antibody (Cell Signaling) were used as received.

[0326] All references cited or otherwise referred to or disclosed herein are incorporated by reference in their entirety for all purposes. [References] JPEG2025024113000145.jpg225170JPEG2025024113000146.jpg244170JPEG2025024113000147.jpg232170

Claims

1. The process is as follows: (a) A compound of formula (A) 【Chemistry 1】 Formula R １ -NH ２ Or R １ -N(H)-Z'-N(H)-R １ and reacting a first amine having (b) reacting the product of step (a) with a compound of formula R ２ -NH ２ Or R ２ -N(H)-Z''-N(H)-R ２ and reacting the second amine having (c) reacting the product of step (b) with a compound of formula (B); 【Chemistry 2】 and reacting a compound represented by the formula: each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, or C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), G is -C-, -S-, -S(O)-, -P(OR １ )-, or -P(OH)-, Each Q is H or C １ -C １０ is a linear or branched alkyl group, Each E １ are independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R １ and R2 are each independently １ -C ４０ Alkyl, C １ -C ４０ Heteroalkyl, C ２ -C ４０ Alkenyl, C ２ -C ４０ Heteroalkenylene, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ２ -C ４０ Heteroalkynylene, C ３ -C ８ cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and the heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O; １ -C ４０ Alkyl, C ２ -C ４０ Alkenyl, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ３ -C ８ Cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally selected from D, halogen, C １ -C ６ Alkyl, —OH, —O—C １ -C ６ Alkyl, -NH ２ , -NH(C １ -C ６ alkyl), or -N(C １ -C ６ Alkyl) ２ is substituted with R １ is unsubstituted or is selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ ) aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), wherein each n is at least 1.

2. Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, an alkylene-carbocycle containing 3 to 30 carbon atoms, a heterocycle containing 3 to 30 atoms, or an alkylene-heterocycle containing 3 to 30 atoms; Z is unsubstituted or is a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulfonamide group, a thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ 2. The polymer of claim 1, wherein the aryl group is selected from the group consisting of alkyl.

3. The polymer of claim 1 or 2, wherein G is -C-.

4. The compound of formula (B) 【Chemistry 3】 where: R is a linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms, and R is unsubstituted or is selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), R ’’ is an unsubstituted or substituted linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms.

5. The compound of formula (B) 【Chemistry 4】 5. The polymer of claim 4,

6. 6. The polymer of any one of claims 1 to 5, wherein Z is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms.

7. 7. The polymer of claim 6, wherein Z is a linear or branched carbon chain of 1 to 10 carbon atoms.

8. Z 【Chemistry 5】 And, 7. The polymer of claim 1, wherein x is 1 to 1000.

9. Each Z 【Chemistry 6】 The polymer according to any one of claims 1 to 6,

10. R １ and R ２ But independently, 【Chemistry 7】 The polymer according to any one of claims 1 to 9, selected from the group consisting of:

11. R １ but 【Chemistry 8】 and R ２ but 【Chemistry 9】 11. The polymer of claim 10,

12. The polymer of any one of claims 1 to 11, wherein a molar excess of the compound of formula (A) is reacted with the first amine.

13. 13. The polymer of claim 12, wherein the stoichiometric ratio of the compound of formula (A) to the first amine is about 1.2:

1.

14. The polymer of any one of claims 1 to 13, wherein step (a) is carried out in an organic solvent.

15. The polymer of claim 14 , wherein the organic solvent is DMSO.

16. The polymer of any one of claims 1 to 15, wherein step (a) is carried out at a temperature of from about 40°C to about 120°C.

17. 17. The polymer of claim 16, wherein step (a) is carried out at about 90°C.

18. The polymer of any one of claims 1 to 17, wherein the product of step (a) is not purified prior to step (b).

19. The polymer of any one of claims 1 to 18, wherein a molar excess of the second amine is reacted with the product of step (a).

20. The polymer of any one of claims 1 to 19, wherein step (b) is carried out at a temperature of from about 20°C to about 25°C.

21. The polymer of any one of claims 1 to 20, wherein the product of step (b) is purified prior to step (c).

22. The polymer of any one of claims 1 to 21, wherein step (c) is carried out at a temperature higher than the temperature of step (b).

23. 23. The polymer of claim 22, wherein step (c) is carried out at about 90°C.

24. 24. The polymer of any one of claims 1 to 23, wherein the polymer has an alpha parameter, as defined by the Mark-Hwink equation, of less than about 0.

5.

25. The polymer of any one of claims 1 to 24, wherein the polymer has an alpha parameter, as defined by the Mark-Hwink equation, of from about 0.2 to about 0.

5.

26. 26. The polymer of any one of claims 1 to 25, wherein the PDI of the polymer is from about 1.01 to about 8.

0.

27. 27. The polymer of any one of claims 1 to 26, wherein the PDI of the polymer is about 2.

5.

28. M of the polymer Ｗ The polymer of any one of claims 1 to 27, wherein the molecular weight is at least 3 kDa.

29. M of the polymer Ｗ The polymer of any one of claims 1 to 28, wherein the molecular weight is between about 5 kDa and 50 kDa.

30. M of the polymer Ｗ The polymer of any one of claims 1 to 29, wherein the molecular weight is about 10 kDa.

31. The M of the product after step (b) Ｗ The polymer of any one of claims 1 to 30, wherein the molecular weight is about 3 kDa.

32. 1. A method of making a polymer, comprising: (a) A compound of formula (A) 【Chemistry 10】 Formula R １ -NH ２ Or R １ -N(H)-Z'-N(H)-R １ and reacting a first amine having (b) reacting the product of (a) with a compound of formula R ２ -NH ２ Or R ２ -N(H)-Z''-N(H)-R ２ and reacting with a second amine having (c) reacting the product of (b) with a compound of formula (B), 【Chemistry 11】 During the ceremony, each J is independently -O- or -NH-; Z, Z', and Z'' are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, or C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), G is -C-, -S-, -S(O)-, -P(OR １ )-, or -P(OH)-, Each Q is H or C １ -C １０ is a linear or branched alkyl group, Each E １ are independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R １ and R2 are each independently １ -C ４０ Alkyl, C １ -C ４０ Heteroalkyl, C ２ -C ４０ Alkenyl, C ２ -C ４０ Heteroalkenylene, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ２ -C ４０ Heteroalkynylene, C ３ -C ８ cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and the heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O; １ -C ４０ Alkyl, C ２ -C ４０ Alkenyl, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ３ -C ８ Cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally selected from D, halogen, C １ -C ６ Alkyl, —OH, —O—C １ -C ６ Alkyl, -NH ２ , -NH(C １ -C ６ alkyl), or -N(C １ -C ６ Alkyl) ２ is substituted with R １ is unsubstituted or is selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ ) aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), The method wherein each n is at least 1.

33. Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; Z is unsubstituted or is a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulfonamide group, a thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ 33. The polymer of claim 32, wherein the aryl group is selected from the group consisting of alkyl.

34. The method of claim 32 or 33, wherein G is -C-.

35. The compound of formula (B) 【Chemistry 12】 where: R is unsubstituted or is a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulfonamide group, a thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), R ’’ is an unsubstituted or substituted linear or branched carbon chain of 1 to 10 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 10 atoms, a carbocycle containing 3 to 10 carbon atoms, or a heterocycle containing 3 to 10 atoms.

36. The compound of formula (B) 【Chemistry 13】 36. The method of claim 35, wherein:

37. 37. The method of any one of claims 32 to 36, wherein Z is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms.

38. 38. The method of claim 37, wherein Z is a straight or branched carbon chain of 1 to 10 carbon atoms.

39. Z 【Chemistry 14】 and 38. The method of any one of claims 32 to 37, wherein x is 1 to 1000.

40. Z 【Chemistry 15】 The method according to any one of claims 32 to 38, wherein

41. R １ and R ２ But independently, 【Chemistry 16】 The method of any one of claims 32 to 40, selected from the group consisting of:

42. R １ but 【Chemistry 17】 and R ２ but 【Chemistry 18】 42. The method of claim 41 , wherein:

43. The method of any one of claims 32 to 42, wherein a molar excess of the compound of formula (A) is reacted with the first amine.

44. 44. The method of claim 43, wherein the stoichiometric ratio of the compound of formula (A) to the first amine is about 1.2:

1.

45. The method of any one of claims 32 to 44, wherein step (a) is carried out in an organic solvent.

46. 46. ​​The method of claim 45, wherein the organic solvent is DMSO.

47. The method of any one of claims 32 to 46, wherein step (a) is carried out at a temperature of from about 20°C to about 200°C.

48. 48. The method of claim 47, wherein step (a) is carried out at about 90°C.

49. The method of any one of claims 32 to 48, wherein the product of step (a) is not purified prior to (b).

50. 50. The method of any one of claims 32 to 49, wherein a molar excess of the second amine is reacted with the product of step (a).

51. 51. The method of any one of claims 32 to 50, wherein step (b) is carried out at a temperature of about 20°C to about 25°C.

52. The method of any one of claims 32 to 51, wherein the product of step (b) is purified prior to step (c).

53. The method of any one of claims 32 to 52, wherein step (c) is carried out at a temperature higher than the temperature of step (b).

54. 54. The method of claim 53, wherein step (c) is carried out at about 90°C.

55. 55. The method of any one of claims 32 to 54, wherein the polymer has an alpha parameter, as defined by the Mark-Hwink equation, of less than about 0.

5.

56. 56. The method of any one of claims 32 to 55, wherein the polymer has an alpha parameter, as defined by the Mark-Hwink equation, of from about 0.3 to about 0.

5.

57. 57. The method of any one of claims 32 to 56, wherein the polymer has a PDI of from about 2.0 to about 3.

0.

58. 58. The method of any one of claims 32 to 57, wherein the polymer has a PDI of about 2.

5.

59. M of the polymer Ｗ The method of any one of claims 32 to 58, wherein the molecular weight is at least 3 kDa.

60. M of the polymer Ｗ The method of any one of claims 32 to 59, wherein the molecular weight is between about 5 kDa and 50 kDa.

61. M of the polymer Ｗ The method of any one of claims 32 to 60, wherein the molecular weight is about 10 kDa.

62. The M of the product after step (b) Ｗ The method of any one of claims 32 to 61, wherein the molecular weight is about 3 kDa.

63. A polyplex comprising a nucleic acid component and any of the polymers of claims 1 to 31 or of formula (I), 【Chemistry 19】 During the ceremony, each A is independently a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, or C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), each B is independently a first binding moiety; Each X is independently 【Chemistry 20】 and Each Y is independently 【Chemistry 21】 and each L is independently a second linking moiety; Each R １ , R ２ , and R ３ independently in each occurrence: H, C １ -C ４０ Alkyl, C １ -C ４０ Heteroalkyl, C ２ -C ４０ Alkenyl, C ２ -C ４０ Heteroalkenylene, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ２ -C ４０ Heteroalkynylene, C ３ -C ８ cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and the heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C １ -C ６ Alkyl, C ２ -C ８ Alkenyl, C ４ -C ８ Cycloalkenyl, C ２ -C ６ Alkynyl, C ３ -C ８ Cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally selected from D, halogen, C １ -C ６ Alkyl, —OH, —O—C １ -C ６ Alkyl, -NH ２ , -NH(C １ -C ６ alkyl), or -N(C １ -C ６ Alkyl) ２ is replaced by, or R ２ and R ３ may be taken together with the atom to which they are attached to form a heterocyclyl or heteroaryl containing 1 to 3 heteroatoms selected from the group consisting of N, S, P, and O; a is 1 to 1000; b is 1 to 4; c is 1 to 3; z is 1 to 100; However, R ２ and R ３ wherein at least one of said polyplexes is not H.

64. The polymer of formula (I) has the structure of formula (II): 【Chemistry 22】 wherein Each E １ is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; Each E ２ is selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; G is -C-, -S-, -S(O)-, -P(OR １ )-, or -P(OH)-, 64. The polyplex of claim 63 having the structure of formula (II), wherein n is at least 1.

65. Each B 【Chemistry 23】 65. The polyplex of claim 63 or 64,

66. B 【Chemistry 24】 The polyplex according to any one of claims 63 to 65,

67. Each L 【Chemistry 25】 and The polyplex of any one of claims 63 to 66, wherein x is 1 to 1000.

68. a is at least 2; b is 3; Each X 【Chemistry 26】 The polyplex according to any one of claims 63 to 67,

69. Each A 【Chemical 27】 69. The polyplex of claim 68, wherein

70. Each L 【Chemistry 28】 70. The polyplex of claim 68 or 69,

71. Y 【Chemical formula 29】 and each B is 【Chemistry 30】 or 【Chemistry 31】 The polyplex according to any one of claims 68 to 70,

72. Each R ２ and / or R ３ but 【Chemistry 32】 The polyplex according to any one of claims 68 to 71,

73. Each R1 【Chemical 33】 The polyplex according to any one of claims 68 to 72,

74. The polymer of formula (I) has a structure of formula (III)-(VIIe): 【Chemical 34】 【Chemistry 35】 【Chemical 36】 And, Each R ５ , R ６ , and R ７ independently in each occurrence: H, C １ -C ４０ Alkyl, C １ -C ４０ Heteroalkyl, C ２ -C ４０ Alkenyl, C ２ -C ４０ Heteroalkenylene, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ２ -C ４０ Heteroalkynylene, C ３ -C ８ cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and the heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O, and the C １ -C ６ Alkyl, C ２ -C ８ Alkenyl, C ４ -C ８ Cycloalkenyl, C ２ -C ６ Alkynyl, C ３ -C ８ Cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally selected from D, halogen, C １ -C ６ Alkyl, —OH, —O—C １ -C ６ Alkyl, -NH ２ , -NH(C １ -C ６ alkyl), or -N(C １ -C ６ Alkyl) ２ and the remaining variables are as defined in claim 1, 63, or 64.

75. The polyplex of any one of claims 63 to 74, wherein z is 1 to 3.

76. 76. The polyplex of claim 75, wherein z is 1.

77. E ２ but 【Chemical 37】 and n is 1. The polyplex of any one of claims 63 to 74.

78. E ２ but 【Chemical 38】 78. The polyplex of claim 77, wherein

79. A nucleic acid component and a polymer: 【Chemical Formula 39】 A polyplex comprising: each J is independently -O- or -NH-; Z and Z″ are linking moieties; A is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 2 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; A is optionally one or more of halogen, hydroxyl, amino group, sulfonyl group, sulfonamide group, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, or C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), G is -C-, -S-, -S(O)-, -P(OR １ )-, or -P(OH)-, Each Q is H or C １ -C １０ is a linear or branched alkyl group, Each E １ are independently selected from the group consisting of a covalent bond, -N-, -O-, -S-, alkylene, heteroalkylene, alkenyl, heteroalkenylene, alkynyl, and heteroalkynylene; R １ and R2 are each independently １ -C ４０ Alkyl, C １ -C ４０ Heteroalkyl, C ２ -C ４０ Alkenyl, C ２ -C ４０ Heteroalkenylene, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ２ -C ４０ Heteroalkynylene, C ３ -C ８ cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein the heterocyclyl and the heteroaryl contain 1 to 5 heteroatoms selected from the group consisting of N, S, P, and O; １ -C ４０ Alkyl, C ２ -C ４０ Alkenyl, C ４ -C ８ Cycloalkenyl, C ２ -C ４０ Alkynyl, C ３ -C ８ Cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally selected from D, halogen, C １ -C ６ Alkyl, —OH, —O—C １ -C ６ Alkyl, -NH ２ , -NH(C １ -C ６ alkyl), or -N(C １ -C ６ Alkyl) ２ is substituted with R １ is unsubstituted or is selected from the group consisting of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ ) aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ alkyl), and n is at least 1.

80. Z is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; Z is unsubstituted or is a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulfonamide group, a thiol, C １ -C ６ Alkyl, C １ -C ６ Alkoxy, C １ -C ６ Ether, C １ -C ６ Thioether, C １ -C ６ Sulfone, C １ -C ６ Sulfoxide, C １ -C ６ Primary amide, C １ -C ６ Secondary amide, halo C １ -C ６ Alkyl, carboxyl group, cyano group, nitro group, nitroso group, -OC(O)NR'R', -N(R')C(O)NR'R', -N(R')C(O)O-C １ -C ６ Alkyl, C ３ -C ６ Cycloalkyl, C ３ -C ６ Heterocyclyl, C ２ -C ５ Heteroaryl, and C ６ -C １０ aryl, wherein each R′ is independently selected from hydrogen and C １ -C ６ 80. The polyplex of claim 79, wherein the alkyl group is selected from the group consisting of alkyl.

81. The polyplex of any one of claims 79 to 80, wherein G is -C-.

82. The polyplex of any one of claims 79 to 81, wherein J is O.

83. 83. The polyplex of any one of claims 79 to 82, wherein Z is a linear or branched carbon chain of 1 to 30 carbon atoms or a linear or branched heteroatom-containing carbon chain of 1 to 30 atoms.

84. The polyplex of any one of claims 79 to 83, wherein Z is a linear or branched carbon chain of 1 to 10 carbon atoms.

85. Z 【Chemistry 40】 and The polyplex of any one of claims 79 to 83, wherein x is 1 to 1000.

86. Z 【Chemistry 41】 The polyplex according to any one of claims 79 to 84,

87. R １ and R ２ But independently, 【Chemistry 42】 The polyplex of any one of claims 79 to 86, selected from the group consisting of:

88. R １ but 【Chemistry 43】 and R ２ but 【Chemistry 44】 88. The polyplex of claim 87, wherein

89. R １ but 【Chemistry 45】 and R ２ but 【Chemistry 46】 88. The polyplex of claim 87, wherein

90. The polymer is 【Chemistry 47】 The polyplex of any one of claims 79 to 89, comprising:

91. The polymer is 【Chemistry 48】 The polyplex of any one of claims 79 to 89, comprising:

92. The polymer is 【Chemistry 49】 The polyplex of any one of claims 79 to 84 or 86 to 91, comprising:

93. The polymer is 【Chemistry 50】 wherein J is O and Z is 【Chemistry 51】 The polyplex of any one of claims 79-83, 85, or 87-91, wherein x is 1 to 1000.

94. The polymer is 【Chemistry 52】 The polyplex of any one of claims 79 to 93, comprising:

95. The polymer is 【Chemistry 53】 wherein R １ but 【Chemical 54】 and R ２ but 【Chemistry 55】 The polyplex according to any one of claims 79 to 89, wherein the polyplex is selected from the group consisting of

96. The polymer is 【Chemistry 56】 wherein J is O and Z is 【Chemistry 57】 where x is 1 to 1000; R １ but 【Chemistry 58】 and R ２ but 【Chemistry 59】 The polyplex according to any one of claims 79 to 89,

97. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is from about 3 kDa to about 200 kDa.

98. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is from about 5 kDa to about 50 kDa.

98. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is about 10 kDa to 50 kDa.

99. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is from about 5 kDa to about 15 kDa.

100. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is about 10 kDa.

101. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is about 20 kDa.

102. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is about 30 kDa.

103. M of the polymer Ｗ The polyplex of any one of claims 79 to 96, wherein the molecular weight is about 40 kDa.

104. The polyplex of any one of claims 79 to 103, wherein the polymer has an alpha parameter defined by Mark-Hwink of less than about 0.

5.

105. The polyplex of any one of claims 79 to 104, wherein the alpha parameter of the polymer as defined by the Mark-Hwink equation ranges from about 0.3 to about 0.

5.

106. The polyplex of any one of claims 79 to 105, wherein the PDI of the polymer is from about 1.0 to about 8.

0.

107. The polyplex of any one of claims 79 to 106, wherein the PDI of the polymer is about 2.

5.

108. 108. The polyplex of claim 107, wherein the polymer and nucleic acid components are present in a ratio of about 20:1 to about 80:1 (w / w).

109. 109. The polyplex of claim 108, wherein the polymer and nucleic acid components are present in a ratio of about 30:1 (w / w).

110. 110. The polyplex of any one of claims 63 to 109, having a particle size of less than about 2 μm.

111. 111. The polyplex of claim 110, having a particle size of about 60 nm to about 250 nm.

112. 111. The polyplex of claim 110, having a particle size of about 175 nm to about 250 nm.

113. The polyplex of any one of claims 63 to 112, having a zeta potential of about 0 mV to about 100 mV.

114. The polyplex of claim 113, wherein the zeta potential is from about 30 mV to about 34 mV.

115. The polyplex of any one of claims 63 to 114, wherein the polyplex is spherical.

116. M of the polymer Ｗ The polyplex of any one of claims 63 to 87, wherein the molecular weight is about 10 kDa.

117. The polyplex of any one of claims 63 to 116, wherein the nucleic acid component is a plasmid, a nanoplasmid, a nucleic acid, a minicircle, or a gene editing system.

118. The polyplex of claim 117, wherein the nanoplasmid comprises a eukaryotic transgene and a bacterial backbone less than 0.5 kb in size.

119. The polyplex of claim 117, wherein the plasmid or nanoplasmid is a plasmid without an antibiotic resistance marker or a nanoplasmid without an antibiotic resistance marker.

120. The polyplex of claim 117, wherein the plasmid or nanoplasmid comprises a sucrose selection marker or a nonsense suppressor marker.

121. The polyplex of claim 117, wherein the gene editing system is (i) a clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) system, (ii) a transcription activator-like effector nuclease (TALEN) system, or (iii) a zinc finger nuclease (ZFN) system.

122. The polyplex of claim 117, wherein the nucleic acid is an RNAi-inducing molecule.

123. The polyplex of claim 122, wherein the RNAi-inducing molecule is selected from the group consisting of siRNA, dsRNA, shRNA, and microRNA.

124. The polyplex of any one of claims 63 to 116, wherein the nucleic acid component comprises a tissue-specific promoter.

125. The polyplex of any one of claims 63 to 116, wherein the nucleic acid component comprises a gene associated with a genetic disease or disorder.

126. The polyplex of claim 125, wherein the genetic disease or disorder is caused by a mutation in one or more genes that results in reduced, absent, or dysfunctional protein expression.

127. 127. The polyplex of claim 126, wherein the gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5, and FLG.

128. 128. The polyplex of claim 127, wherein the gene is COL7A1 and the genetic disease or disorder is a form of epidermolysis bullosa.

129. The polyplex of claim 125, wherein the sequence of the gene is optimized to maximize protein expression upon delivery of the polyplex to a cell.

130. 130. A pharmaceutical composition comprising an effective amount of the polyplex of any one of claims 63 to 129 in combination with a pharma- ceutically acceptable carrier.

131. 131. The pharmaceutical composition of claim 130, wherein the pharma- ceutically acceptable carrier is suitable for oral, parenteral, inhalation, topical, subcutaneous, intramuscular, intravenous, intraocular, or intradermal administration.

132. 132. The pharmaceutical composition of claim 131, wherein the pharmaceutical composition is formulated as a lotion selected from the group consisting of a non-aqueous lotion, a water-in-oil lotion, and an oil-in-water lotion.

133. The pharmaceutical composition of claim 130, wherein the pharmaceutical composition is lyophilized for future use.

134. 131. The pharmaceutical composition of claim 130, wherein the pharmaceutical composition is frozen in an aqueous solution.

135. 135. A method of cell transfection comprising contacting one or more target cells with the pharmaceutical composition of any one of claims 130 to 134 under conditions suitable for transfecting said target cells with the polyplex.

136. 136. The method of claim 135, wherein the one or more target cells are eukaryotic cells.

137. 137. The method of claim 136, wherein the one or more target cells are one or more of T cells, B cells, blood cells, alveolar cells, lung cells, brain neurons, skin neurons, epithelial cells, keratinocytes, iPS cells, fibroblasts, and sweat gland cells.

138. 135. A method of treating a disease in a patient in need of said treatment, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 130 to 134, such that one or more of the patient's cells are transfected with said polyplex nucleic acid components.

139. 135. A method of treating a disease in a patient in need of such treatment, comprising administering a therapeutically effective pharmaceutical composition according to any one of claims 130 to 134, wherein said administration of said composition corrects defective translation of a target gene in the subject.

140. 140. The method of claim 139, wherein the gene is selected from the group consisting of COL7A1, LAMB3, ADA, SERPINA1, CFTR, HTT, NF1, PHA, HBS, FERMT1, KRT14, DSP, SPINK5, and FLG.

141. The disease may be adenosine deaminase (ADA) deficiency, alpha-1 antitrypsin deficiency, cystic fibrosis, Huntington's disease, type 1 neurofibromatosis, phenylketonuria, sickle cell disease, sporadic inclusion body myositis, Duchenne muscular dystrophy, Kindler syndrome, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa (autosomal recessive), dystrophic epidermolysis bullosa (regional variant), prurigomatous epidermolysis bullosa, epidermolysis bullosa (pretibial type), reticular pigmented dermatosis, epidermolysis bullosa simplex (Dowling-M 140. The method of claim 139, wherein the patient is a patient with epidermolysis bullosa (eara type), epidermolysis bullosa simplex (Koebner type), epidermolysis bullosa simplex (recessive 1), epidermolysis bullosa simplex (Weber-Cockayne type), Naegeli-Franceschetti-Jadassohn syndrome, epidermolysis bullosa (fatal acantholytic type), Netherton syndrome, ichthyosis vulgaris, atopic dermatitis, Usher syndrome, Ehlers-Danlos syndrome, homozygous familial hypercholesterolemia (HoFH), or Crohn's disease.

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