Chitosan polyplex based local expression of il-12 alone or in combination with a type i IFN inducer for the treatment of mucosal cancer

Abstract

The present disclosure relates to methods and compositions for local expression of IL-12 in mucosal tissues, preferably in combination with an IFN-1 activator / inducer, for use in cancer immunotherapy.SOLUTION: The present invention solves the unmet need in the art for effective local expression of IL-12 using derivatized chitosan polyplexes reversibly coated with polyanion-containing block copolymers for potent transfection of mucosal tissues present in or proximal to tumors.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application Nos. 62 / 818,425, filed March 14, 2019, 62 / 923,403, filed October 18, 2019, and 62 / 924,131, filed October 21, 2019, which are incorporated herein by reference in their entireties and for all purposes.

[0002] FIELD OF THE INVENTION The present disclosure relates to methods and compositions for the local expression of IL-12 in mucosal tissues, preferably in combination with a type I IFN (IFN-1) activator / inducer, for use in cancer immunotherapy. [Background technology]

[0003] Th1 T cells are conventionally defined by the production of the cytokines IFN-γ, GM-CSF, IL-2, and lymphotoxin (LT, TNF-β). The critical decision to generate Th1 cells depends on the innate immune response to infection with intracellular bacteria, fungi, and viruses. Infection with these pathogens leads to the activation of TLR signaling and the subsequent production of cytokines important for Th1 development by dendritic cells and NK cells. Th1 polarization and differentiation are promoted by IL-12, IL-18, IFN-γ, and type 1 interferons, but are suppressed by IL-4, IL-10, and TGF-β.

[0004] IL-12, produced by mature dendritic cells, is considered a key factor associated with Th1 commitment. T cell activation and STAT1 signaling lead to the expression of IL-12Rβ, which, upon binding to IL-12, signals to activate STAT4 via Jak-2 and Tyk-2. STAT4 enters the nucleus and induces the expression of Th1 lineage-specific transcription factors such as Tbet. Tbet helps reinforce the Th1 phenotype by promoting the expression of IFN-γ and IL-12Rβ2. IFN-γ then promotes Th1 differentiation through STAT1 signaling downstream of the IFN-γ receptor to stimulate the production of Tbet. Various cells, such as NK cells and precommitted Th1 cells, are important early sources of IFN-γ. In contrast, IL-18 plays a dual role in Th1 function by promoting Th1 commitment and inducing IFN-γ production by fully differentiated Th1 cells.

[0005] Given its essential role in both innate and adaptive immune responses through the activation of NK cells and the differentiation of naive CD4+ cells into Th1 cells, and because it acts as a potent stimulator of IFN-γ, IL-12 has long been considered a potential candidate for tumor immunotherapy. IL-12 has been shown to exhibit antitumor activity in vitro (Kobayashi et al., J. Exp. Med. 170:827-845 (1989) and Stern et al., PNAS 87:6808-6812 (1990)), and early animal studies demonstrating IL-12-induced tumor regression and reduction in mouse tumor models appeared promising. Studies of systemic administration of IL-12 include Brunda et al. (J. Exp. Med., 178:1223-1230 (1993)), which showed that intraperitoneal administration of IL-12 to mice significantly reduced the development of experimental lung metastases or subcutaneous growth of B16F10 melanoma. The response to IL-12 administration was dose-dependent and resulted in prolonged survival. Similar activity was demonstrated in experimental liver metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors. Similarly, Teicher et al. (Int. J. Cancer, 65(1):80-84 (1996)) reported that IL-12 was an active antitumor agent in three solid mouse tissues: B16 melanoma, Lewis lung carcinoma, and renal cell carcinoma, while Kozar et al. (Clin. Can. Res., 9(8):3124-3133 (2003)) showed that mice inoculated with L1210 leukemia cells or B16F10 melanoma cells treated with daily injections or administration of IL-12 showed modest tumor reduction.

[0006] These animal studies reported little or no apparent toxicity, making IL-12 an attractive molecule for further clinical development. Unfortunately, early human clinical trials showed reduced efficacy and increased toxicity. In an initial Phase 1 dose-escalation study, intravenous administration of IL-12 to 40 patients with advanced solid tumors (including melanoma, renal carcinoma, and colon cancer) resulted in only one complete response in the melanoma patient and a partial response in the renal carcinoma patient (Atkins et al., Clin. Cancer Res., 3:409-17 (1997)). Significant systemic toxicity was observed in this study, with three of four patients treated with the highest dose of IL-12 (1000 ng / kg) developing stomatitis and / or liver dysfunction. In a pilot study conducted by Bajetta et al. (Clin. Cancer Res., 4:75-85 (1998)) in which 10 patients with metastatic melanoma received subcutaneous injections of 0.5 μg / kg IL-12, 100% developed a flu-like syndrome, 90% developed fever, 60% developed transient hypertransaminases, and 80% developed hypertriglyceridemia. Additional toxicities were observed in 10-20% of study patients. No partial or complete responses were observed, although tumor regression occurred in 3 of 10 participants. Most notably, a phase II trial conducted by the Genetics Institute found that daily intravenous IL-12 administration resulted in hospitalization of 12 of 17 enrolled patients and 2 deaths (Leonard et al., Blood, 90(7):2541-2548 (1995)).

[0007] To avoid the toxicity seen with systemic administration, studies were conducted using local delivery of IL-12. These studies showed less toxicity associated with IL-12 administration than systemic studies, but local delivery was largely ineffective in eliminating tumors. Lenzi et al. (Clin. Cancer Res., 8:3686-3695, (2002)) conducted a phase 1 trial in which IL-12 was administered via a peritoneal catheter at doses ranging from 3 ng / kg to 600 ng / kg to 29 patients with peritoneal carcinomatosis due to ovarian cancer and various abdominal cancers. No life-threatening toxicity was observed in the trial, but only two of the 29 patients enrolled experienced a complete response, eight had stable disease, and the remaining 19 had progressive disease. Similarly, Weiss et al. (J. Immunother., 26(4):343-348 (2003)) showed that intravesical administration of IL-12 to patients with superficial transitional cell carcinoma of the bladder did not result in moderate, severe, or life-threatening toxicity, but the patients also showed no clinically relevant evidence of antitumor or immunological effects.

[0008] The use of chitosan as an adjuvant for IL-12 for cancer treatment has been reported. Chitosan was used as part of a co-formulation with IL-12 for intravesical immunotherapy to treat bladder cancer in mice (Zaharoff et al., Cancer Res. 69(15):6192-6199(2009)), as well as intratumoral injection for colorectal and pancreatic tumors (Zaharoff et al., J. Immunotherapy, 33(7):697-705(2010)), and for breast cancer (Vo et al., Oncoimmunology, 3912):e968001(2015)). The use of IL-12 molecules covalently linked to chitosan as a therapeutic agent has also been explored to reduce the diffusion of IL-12 from the treatment site and avoid potential toxicity due to the diffusion of IL-12 within the system (Zaharoff et al., US20170106092). Unfortunately, clinical application of IL-12-based therapy remains problematic due to the rapid progression of lethal inflammatory syndromes, and improved strategies to overcome IL-12-mediated toxicity are still needed. (Wang et al., Nature Communications 8, Article 1395 (2017))

[0009] Thus, there remains a need for new compositions and methods for effective localized expression of IL-12, but with reduced toxicity and improved efficacy. Ideally, these could be used in combination with additional immune stimulatory strategies to enhance cancer immunotherapy in the tumor microenvironment. The current knowledge base regarding IL-12 combination therapy is quite limited and focuses primarily on the coadministration and / or coexpression of IL-12 with additional immune-modulating cytokines, such as IL-2, IL-7, IL-15, IL-18, and IL-21. See, e.g., Weiss et al., Expert Opin Biol Ther. 7:1705-1721 (2007). In particular, potential synergistic interactions between IL-12 and alternative innate and / or adaptive immune stimulatory strategies have been largely unexplored.

[0010] Pattern recognition receptors (PRRs) comprise another component of the innate immune system that recognize conserved pathogen-associated nucleic acid (NA) sequences. PRR activation by nucleotide ligands induces the production of type I interferons (IFNs) and proinflammatory cytokines, which serve as the first line of defense against viral and microbial infections. Iwasaki and Medzhitov, Science 327:291-95 (2010). These NA-sensing PRRs include the endosomal Toll-like receptor (TLR) family (Majer et al., Curr. Opin. Immunol. 44:26-33 (2017)), the cytosolic DNA sensor AIM2 and cyclic GMP-AMP synthetase (cGAS), part of the cGAS stimulator of interferon (STING) pathway (Chen et al., Nat. Immunol. 17:1142-49 (2016)), and the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I)-like receptor family (Schlee, Immunobiology, 218:1322-35 (2013)). Interestingly, there are very few references evaluating both IL-12 and RIG-I, and the few that do not demonstrate a significant interaction between the two. Jiang et al. (J. Exp. Med. 216:2854-68 (2019)) showed that eliminating endogenous IL-12 in vivo with a neutralizing antibody had no effect on induced RIG-I responses, whereas earlier work by Kong et al. (Cell Host & Microbe 6:150-61 (2009)) showed that altering RIG-I levels had no effect on IL-12 expression in cell-based assays. As such, the combination of IL-12 and RIG-I in particular seems useless. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention addresses an unmet need in the art for effective localized expression of IL-12 using derivatized chitosan polyplexes reversibly coated with polyanion-containing block copolymers for potent transfection of mucosal tissues within or proximal to tumors. Furthermore, as initially presented herein, effective localized expression of IL-12 obtained using the present compositions and methods can be advantageously combined with one or more innate and / or adaptive immune stimulatory strategies to enhance cytotoxic immune responses against tumors. In preferred embodiments, IL-12 expression according to the present invention is combined with simultaneous or sequential administration and / or expression of a type I interferon (IFN-I) activator / inducer, such as a RIG-I agonist, a STING agonist, and / or a TLR7 / 9 agonist. In some embodiments, the present methods and compositions include coexpression of IL-12 with at least one RIG-I agonist. [Means for solving the problem]

[0012] In one aspect, the present invention provides a composition comprising a derivatized chitosan nucleic acid polyplex comprising an amino-functionalized chitosan and a therapeutic nucleic acid construct encoding IL-12, wherein the derivatized chitosan nucleic acid polyplex further comprises a reversible coating comprising one or more polyanion-containing block copolymers having at least one polyanionic anchor region and at least one hydrophilic tail region.

[0013] In preferred embodiments, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer. In preferred embodiments, the amino-functionalized chitosan further comprises a hydrophilic polyol. In some embodiments, the amino-functionalized chitosan comprises arginine. In some embodiments, the hydrophilic polyol is glucose or gluconic acid.

[0014] In some embodiments, the therapeutic nucleic acid construct is contained within a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, or NTC9385R. In some embodiments, the therapeutic nucleic acid construct further comprises an expression control element selected from the group consisting of CMV, EF1a, CMV / EF1a, and CAG, and CMV / EF1α / HTLV. In some embodiments, the therapeutic nucleic acid construct comprises a synthetic beta-globin-based intron. In some embodiments, the therapeutic nucleic acid construct comprises an HTLV-IR. In some embodiments, the therapeutic nucleic acid construct comprises a kanamycin or sucrose-based selection element. In some embodiments, the therapeutic nucleic acid construct comprises a pUC or R6K origin of replication.

[0015] In some embodiments, the therapeutic nucleic acid construct further comprises a nucleic acid encoding at least one additional innate and / or adaptive immune stimulatory molecule. In some embodiments, the immune stimulatory molecule comprises a type I interferon (IFN-I) activator / inducer, such as a RIG-I agonist, a STING agonist, and / or a TLR7 / 9 agonist. In preferred embodiments, the therapeutic nucleic acid construct further comprises a nucleic acid encoding at least one RIG-I agonist, such as eRNA11a, adenovirus VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and / or SLR20, even more preferably eRNA11a or eRNA41H. In some embodiments, the immune stimulatory molecule is a modulator of an immune checkpoint inhibitor.

[0016] In another aspect, a method is provided for localized expression of IL-12 in mucosal tissue of a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an amino-functionalized chitosan and a derivatized chitosan nucleic acid polyplex comprising a therapeutic nucleic acid construct encoding IL-12, the derivatized chitosan nucleic acid polyplex further comprising a reversible coating comprising one or more polyanion-containing block copolymers having at least one polyanionic anchor region and at least one hydrophilic tail region.

[0017] In some embodiments, the method further comprises co-expression of an IFN-I activator / inducer and / or an immune checkpoint inhibitor. In some embodiments, the IFN-I activator / inducer and / or immune checkpoint inhibitor is co-expressed with IL-12 from the same or a different therapeutic nucleic acid construct. In preferred embodiments, the therapeutic nucleic acid construct comprises a nucleic acid encoding single-stranded hIL-12 and at least one RIG-I agonist, e.g., eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, even more preferably eRNA11a or eRNA41H. In an exemplary embodiment, the nucleic acid encoding single-chain hIL-12 comprises SEQ ID NO:7.

[0018] In some embodiments, the IFN-I activator / inducer and / or the immune checkpoint inhibitor are administered separately. In preferred embodiments, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, atezolizumab, and / or pembrolizumab. In some embodiments, the IFN-I activator / inducer is MK4621 / JetPEI.

[0019] In another aspect, a method is provided for treating mucosal cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an amino-functionalized chitosan and a derivatized chitosan-nucleic acid polyplex comprising a therapeutic nucleic acid encoding IL-12, the derivatized chitosan-nucleic acid polyplex further comprising a reversible coating comprising one or more polyanion-containing block copolymers having at least one polyanionic anchor region and at least one hydrophilic tail region.

[0020] In some embodiments, the method further comprises co-expression of an IFN-I activator / inducer and / or an immune checkpoint inhibitor. In some embodiments, the IFN-I activator / inducer and / or immune checkpoint inhibitor is co-expressed with IL-12 from the same or a different therapeutic nucleic acid construct. In preferred embodiments, the therapeutic nucleic acid construct comprises a nucleic acid encoding single-stranded hIL-12 and at least one RIG-I agonist, e.g., eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, even more preferably eRNA11a or eRNA41H. In an exemplary embodiment, the nucleic acid encoding single-chain hIL-12 comprises SEQ ID NO:7.

[0021] In some embodiments, the IFN-I activator / inducer and / or the immune checkpoint inhibitor are administered separately. In preferred embodiments, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, atezolizumab, and / or pembrolizumab. In some embodiments, the IFN-I activator / inducer is MK4621 / JetPEI.

[0022] In preferred embodiments, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer. In preferred embodiments, the amino-functionalized chitosan further comprises a hydrophilic polyol. In some embodiments, the amino-functionalized chitosan comprises arginine. In some embodiments, the hydrophilic polyol is glucose or gluconic acid.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid polyplex comprising a cationic polymer and a therapeutic nucleic acid construct encoding IL-12 and at least one RIG-I agonist. In a preferred embodiment, the therapeutic nucleic acid construct encodes a single-stranded hIL-12 molecule and at least one RIG-I agonist selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, and even more preferably selected from the group consisting of eRNA11a or eRNA41H. In an exemplary embodiment, the nucleic acid encoding the single-chain hIL-12 comprises SEQ ID NO:7.

[0024] In some embodiments, the cationic polymer is selected from the group including polyamines, polyorganoamines, poly(amidoamines), polyamino acids, polyethyleneimine cellulose, polysaccharides, chitosan, and derivatives thereof. In some embodiments, the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof.

[0025] In preferred embodiments, the cationic polymer comprises a derivatized chitosan. In particularly preferred embodiments exemplified herein, the derivatized chitosan is an amino-functionalized chitosan, more preferably a doubly derivatized chitosan comprising arginine and a hydrophilic polyol, such as gluconic acid or glucose. In some embodiments, the nucleic acid polyplex further comprises a reversible coating comprising one or more polyanion-containing block copolymers having at least one polyanionic anchor region and at least one hydrophilic tail region. In preferred embodiments, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer.

[0026] In some embodiments, the therapeutic nucleic acid construct is contained within a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, or NTC9385R. In some embodiments, the therapeutic nucleic acid construct further comprises an expression control element selected from the group consisting of CMV, EF1a, CMV / EF1a, and CAG, and CMV / EF1α / HTLV. In some embodiments, the therapeutic nucleic acid construct comprises a synthetic beta-globin-based intron. In some embodiments, the therapeutic nucleic acid construct comprises an HTLV-IR. In some embodiments, the therapeutic nucleic acid construct comprises a kanamycin or sucrose-based selection element. In some embodiments, the therapeutic nucleic acid construct comprises a pUC or R6K origin of replication.

[0027] In another aspect, a method for localized expression of IL-12 in mucosal tissue of a patient in need thereof is provided, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a cationic polymer and a nucleic acid polyplex comprising a therapeutic nucleic acid construct encoding IL-12 and at least one RIG-I agonist. In a preferred embodiment, the therapeutic nucleic acid construct encodes a single-stranded hIL-12 molecule and at least one RIG-I agonist selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, and even more preferably selected from the group consisting of eRNA11a or eRNA41H. In an exemplary embodiment, the nucleic acid encoding the single-chain hIL-12 comprises SEQ ID NO:7.

[0028] In another aspect, a method for treating mucosal cancer in a patient in need thereof is provided, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a cationic polymer and a nucleic acid polyplex comprising a therapeutic nucleic acid encoding IL-12 and at least one RIG-I agonist. In a preferred embodiment, the therapeutic nucleic acid construct encodes a single-stranded hIL-12 molecule and at least one RIG-I agonist selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, and even more preferably selected from the group consisting of eRNA11a or eRNA41H. In an exemplary embodiment, the nucleic acid encoding the single-chain hIL-12 comprises SEQ ID NO:7.

[0029] In some embodiments, the cationic polymer is selected from the group including polyamines, polyorganoamines, poly(amidoamines), polyamino acids, polyethyleneimine cellulose, polysaccharides, chitosan, and derivatives thereof. In some embodiments, the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof.

[0030] In preferred embodiments, the cationic polymer comprises a derivatized chitosan. In particularly preferred embodiments exemplified herein, the derivatized chitosan is an amino-functionalized chitosan, more preferably a doubly derivatized chitosan comprising arginine and a hydrophilic polyol, such as gluconic acid or glucose. In some embodiments, the nucleic acid polyplex further comprises a reversible coating comprising one or more polyanion-containing block copolymers having at least one polyanionic anchor region and at least one hydrophilic tail region. In preferred embodiments, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer.

[0031] definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which the present invention pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a difference beyond that commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those of ordinary skill in the art using conventional methodologies, e.g., widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Procedures involving the use of commercially available kits and reagents, where appropriate, are generally performed according to manufacturer-defined protocols and / or parameters unless otherwise noted.

[0032] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033] The term "about" refers to and encompasses the indicated value and a range above and below that value. In certain embodiments, the term "about" refers to the specified value ±10%, ±5%, or ±1%. In certain embodiments, when indicated, the term "about" refers to the specified value ±1 standard deviation of that value.

[0034] The term "combinations thereof" includes all possible combinations of the elements to which the term refers.

[0035] "Treating" or "treatment" of any disease or disorder, in certain embodiments, refers to ameliorating the disease or disorder present in a subject. In another embodiment, "treating" or "treatment" includes improving at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" includes alleviating the disease or disorder, either physically (e.g., stabilization of discernible symptoms) or physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, "treating" or "treatment" includes delaying or preventing the onset of the disease or disorder. For example, in exemplary embodiments, the phrase "treating cancer" refers to inhibiting cancer cell proliferation, inhibiting cancer spread (metastasis), inhibiting tumor growth, reducing cancer cell number or tumor growth, decreasing cancer aggressiveness (e.g., increasing differentiation), or ameliorating cancer-related symptoms.

[0036] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of the composition that, when administered to a subject, is effective in treating a disease or disorder. For example, in exemplary embodiments, the phrase "effective amount" is used interchangeably with "therapeutically effective amount" or "therapeutically effective dose," etc., and refers to an amount of a therapeutic agent that is effective for treating cancer. The effective amount of the compositions provided herein may vary depending on factors such as the condition, age, sex, and weight of the animal.

[0037] As used herein, the term "subject" or "individual" means a mammalian subject. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has cancer, an autoimmune disease or condition, and / or an infectious disease that can be treated with the antibodies provided herein. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infectious disease.

[0038] "Chitosan" is a partially or fully deacetylated form of chitin, a polymer of N-acetylglucosamine. Chitosan with a degree of deacetylation greater than 50% is used in the present invention.

[0039] Chitosan may be derivatized by functionalizing the free amino groups at the deacetylated sites. The derivatized chitosans described herein have many advantageous properties for nucleic acid delivery vehicles, including the ability to effectively bind and complex with negatively charged nucleic acids, to form nanoparticles of controllable size, to be taken up by cells, and to release nucleic acids within the cells at an appropriate time. Chitosans with any degree of functionalization from 1% to 50%. (Percent functionalization is determined relative to the number of free amino groups on the chitosan polymer before or without functionalization.) The degree of deacetylation and functionalization impart a specific charge density to the functionalized chitosan derivative.

[0040] Polyols according to the present invention may have a 3, 4, 5, 6, or 7 carbon backbone and may have at least two hydroxyl groups. Such polyols, or combinations thereof, may be useful for conjugation to chitosan backbones, such as chitosans functionalized with cationic moieties (e.g., amino-containing molecules such as lysine, ornithine, guanidinium groups, arginine-containing molecules, or combinations thereof).

[0041] The term "C2-C6 alkylene," as used herein, refers to a linear or branched divalent hydrocarbon radical, optionally containing one or more carbon-carbon multiple bonds. For the avoidance of doubt, the term "C2-C6 alkylene," as used herein, encompasses divalent radicals of alkanes, alkenes, and alkynes.

[0042] As used herein, unless otherwise indicated, the terms "peptide" and "polypeptide" are used interchangeably.

[0043] The term "polypeptide" is used in the broadest sense to refer to conventional polypeptides (i.e., short polypeptides containing L- or D-amino acids), as well as peptide equivalents, peptide analogs, and peptidomimetics that retain a desired functional activity. Peptide equivalents may differ from conventional peptides by replacing one or more amino acids with related organic acids, amino acids, etc., or by substituting or modifying side chains or functional groups.

[0044] Peptidomimetics may have one or more peptide bonds replaced by alternative bonds, as is known in the art. As is known in the art, part or all of the peptide backbone may also be replaced with conformationally constrained cyclic alkyl or aryl substituents to limit the flexibility of functional amino acid side chains.

[0045] Polypeptides of the present invention may be produced by recognized methods, including recombinant and synthetic methods well known in the art. Techniques for peptide synthesis are well known and include those described in Merrifield, J. Amer. Chem. Soc. 85:2149-2456 (1963), Atherton, et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989), and Merrifield, Science 232:341-347 (1986).

[0046] As used herein, a "linear polypeptide" refers to a polypeptide that lacks branching groups covalently attached to the constituent amino acid side chains. As used herein, a "branched polypeptide" refers to a polypeptide that includes branching groups covalently attached to the constituent amino acid side chains.

[0047] As used herein, the "final degree of functionalization" of a cation or polyol refers to the percentage of cationic (e.g., amino) groups on the chitosan backbone that are functionalized with the cation (e.g., amino) group or polyol, respectively. Thus, the terms "α:β ratio," "final degree of functionalization ratio" (e.g., the ratio of the final degree of functionalization of arginine to the final degree of functionalization of polyol), etc., can be used interchangeably with the terms "molar ratio" or "number ratio."

[0048] A dispersion is composed of particulate matter, known as the dispersed phase, distributed throughout a continuous medium. A chitosan-nucleic acid polyplex "dispersion" is a composition containing hydrated chitosan-nucleic acid polyplexes, with the polyplexes distributed throughout the medium.

[0049] As used herein, a "pre-concentrated" dispersion is one that has not been subjected to a concentration process to form a concentrated dispersion.

[0050] As used herein, "substantially free" of polyplex precipitates means that the composition is essentially free of particles observable by visual inspection.

[0051] As used herein, physiological pH refers to a pH between 6 and 8.

[0052] "Chitosan-nucleic acid polyplex" or grammatical equivalents thereof means a complex comprising multiple chitosan molecules and multiple nucleic acid molecules. In a preferred embodiment, a (e.g., doubly) derivatized chitosan is complexed with the nucleic acid.

[0053] As used herein, the term "polyethylene glycol" ("PEG") is intended to mean a polymer of ethylene oxide having repeating units of -(CH2CH2-O)- and the general formula HO-(CH2CH2-O)nH.

[0054] As used herein, the term "monomethoxypolyethylene glycol" ("mPEG") is intended to mean a polymer of ethylene oxide having repeating units of -(CHCH-O)- and the general formula CHO-(CHCH-O)H, e.g., PEG capped at one end with a methoxy group.

[0055] The present disclosure is disclosed with reference to the accompanying drawings. [Brief explanation of the drawings]

[0056] [Figure 1] 1 shows the dose-dependent enzymatic activity of the luciferase reporter gene (Luc2) after in vitro transfection of MB49 cells with the desired plasmid.

[0057] [Figure 2A] Ranking of vector backbones of interest based on in vivo transfection efficiency (reporter gene mRNA copy number) is shown.

[0058] [Figure 2B] Ranking of vector backbones of interest based on in vivo transfection efficiency (enzymatic activity of reporter gene) is shown.

[0059] [Figure 2C] 1 shows the ranking of vector backbones of interest in vivo based on mRNA copy number in transfected tissues.

[0060] [Figure 2D]A ranking of vector backbones of interest based on the level of protein expressed in vivo is shown.

[0061] [Figure 3A] GFP expression levels in vitro using MB49 cells transfected with a plasmid containing the promoter / enhancer sequence of interest are shown.

[0062] [Figure 3B] Ranking of promoter / enhancer sequences based on in vitro expression of GFP in MB49 cells is shown.

[0063] [Figure 4A] 1 shows the in vitro GFP fluorescence results of human primary bladder epithelial cells transfected with a plasmid containing a promoter / enhancer sequence of interest.

[0064] [Figure 4B] 1 shows a ranking of promoter-enhancer sequences based on activity in primary human bladder epithelial cells in vitro.

[0065] [Figure 5A] 1 shows the dose-response of GFP expression after in vitro transfection of human primary bladder epithelial cells with a plasmid of interest modified to remove bacterial components.

[0066] [Figure 5B] Improved plasmids are ranked based on their activity in primary human bladder epithelial cells in vitro.

[0067] [Figure 6] 1 shows the effect of PEGylation on bladder transfection in vivo.

[0068] [Figure 7]Panels A and B show the mRNA expression of human PD-L1-Fc upon + / -PEGylation in vivo at two different concentrations.

[0069] [Figure 8] 1 shows the level of protein expression mediated by transfection with PEG-DDX in vivo.

[0070] [Figure 9A] Comparing the effect of PEGylation on polyplex stability; appearance of PEGylated and non-PEGylated polyplexes voided after 1 hour incubation in the bladder.

[0071] [Figure 9B] 1 shows the results of a dynamic light scattering assay of post-voided PEGylated and non-PEGylated polyplexes after incubation in the bladder for 1 hour.

[0072] [Figure 10] Figure 1 shows the effects of DDX-1 (DDX) and DDX-II (RXG) on hPD-L1-Fc production in vivo.

[0073] [Figure 11] Figure 1 shows the levels of human PD-L1-Fc protein expression after administration of RXG (DDX-II, PEGylated and non-PEGylated) formulations to the mouse bladder.

[0074] [Figure 12A] Plasmid DNA constructs containing murine IL-12 with and without a RIG-I agonist cassette are shown. Additionally, the mIL-12 transgene containing the p40 and p35 genes in a single open reading frame joined by a short elastin linker is shown.

[0075] [Figure 12B]Figure 1 shows a plasmid DNA construct containing murine IL-12 without and with a RIG-I agonist cassette.Figure 2 shows a schematic diagram of a clinical embodiment of a pharmaceutical composition according to the present invention.

[0076] [Figure 13A] IFNγ production from splenocytes stimulated with plasmid-produced mIL-12 is shown.

[0077] [Figure 13B] 1 shows the functional activity of human IL-12p40p35 expressed from the NTC9385R plasmid backbone.

[0078] [Figure 13C] Figure 1 shows mouse-mediated SEAP production from an IL-12-responsive reporter cell line (HEKBlue).

[0079] [Figure 14A] IFNβ levels in cultured bladder cancer epithelial cells after transfection with an empty plasmid (FIG. 14A) versus a plasmid carrying the IL-12 gene with and without the RIG-I agonist cassette (FIG. 14B) are compared.

[0080] [Figure 14B] IFNβ levels in cultured bladder cancer epithelial cells after transfection with an empty plasmid (FIG. 14A) versus a plasmid carrying the IL-12 gene with and without the RIG-I agonist cassette (FIG. 14B) are compared.

[0081] [Figure 15] IL-12 mRNA levels are shown at 4, 24, 48, 72, and 96 hours after transfection in vivo.

[0082] [Figure 16A] 1 shows the structure of double-derivatized chitosan RXG (DDX II) and the process for synthesizing the DDX / DNA nanoparticles of the present invention.

[0083] [Figure 16B] 1 shows the structure of double-derivatized chitosan RXG (DDX II) and the process for synthesizing the DDX / DNA nanoparticles of the present invention.

[0084] [Figure 16C] The chemical structure of PEG-b-PLE is provided.

[0085] [Figure 17] 1 shows data on the physicochemical properties of DDX-DNA formulations.

[0086] [Figure 18A] 1 shows the results of an in vitro polymer screening assay.

[0087] [Figure 18B] 1 shows the results of an in vitro polymer screening assay.

[0088] [Figure 18C] 1 shows the results of an in vitro polymer screening assay.

[0089] [Figure 19] FIG. 1 shows in vivo protein expression after intravesical administration in mice comparing DDX versus RXG (DDX-II)-based polyplexes.

[0090] [Figure 20A] IL-12 mRNA in non-human primates is compared following intravesical administration of polyplexes containing human IL-12.

[0091] [Figure 20B] Compare IL-12 protein in non-human primates after intravesical administration of polyplexes containing human IL-12.

[0092] [Figure 21]Figure 1 shows eRNA11a expression in NHP bladders exposed to polyplexes containing 0.0625 mg / mL (c62.5), 0.25 mg / mL (c250), and 1 mg / mL (c1000) of hIL-12 and RIG-I agonist (EG-70), as well as 1 mg / mL (c1000) of non-coding control (RXG-PEG-N9).

[0093] [Figure 22] Figure 1 shows VA1 expression in NHP bladders exposed to polyplexes containing 0.0625 mg / mL (c62.5), 0.25 mg / mL (c250), and 1 mg / mL (c1000) of hIL-12 and RIG-I agonist (EG-70), as well as 1 mg / mL (c1000) of non-coding control (RXG-PEG-N9).

[0094] [Figure 23] Figure 1 shows the kinetics of codon-optimized mRNA expression of Il12p40p35 in the mouse bladder after a single administration of mEG-70 prototype nanoparticles.

[0095] [Figure 24] 1 shows mouse IL12p70 protein expression in mouse bladder after a single administration of mEG-70 prototype nanoparticles.

[0096] [Figure 25] 1 shows the dose response of mouse IL-12p70 protein expression in the mouse bladder after a single administration of mEG-70 prototype nanoparticles.

[0097] [Figure 26] Compare the expression of murine IL-12p70 protein in PEGylated nanoparticles versus non-PEGylated nanoparticles.

[0098] [Figure 27]A provides an assay protocol for evaluating the efficacy of mEG-70 (mIL-12 and RIG-I agonist) for the treatment of bladder cancer in a mouse model. B shows bladder weights of control and treated animals at the endpoint of the study. DETAILED DESCRIPTION OF THE INVENTION

[0099] The present invention contemplates local expression of IL-12 in mucosal tissues, preferably in combination with additional innate and / or adaptive immune stimulation. Parenteral and intravenous protein therapy lack sufficient bioavailability in mucosal tissues and suffers from systemic toxicity. Local gene therapy in mucosal tissues, such as intravesical administration to the bladder and oral dosage forms to the gastrointestinal tract (GIT), offers an attractive approach to promoting local protein expression while minimizing unwanted systemic side effects. Unfortunately, the clinical use of viral delivery vectors is limited by viral immunogenicity, which can reduce efficacy after repeated administration; inefficient penetration of mucosal barriers; the cost of vector production; and logistical complications associated with clinical implementation (e.g., biosafety containment and cold-chain storage). The present invention overcomes these limitations by providing a safe and efficient non-viral vector platform for mucosal tissues, such as the bladder.

[0100] Without being bound by theory, activation of the IL-12 pathway in mucosal tissues by the methods of the present invention acts on effector CD4+ and CD8+ effector cells, resulting in potent antitumor and antiangiogenic functions, while simultaneous or sequential stimulation of the RIG-I pathway results in induction of type I interferon and IFN-stimulated genes, leading to improved cross-presentation of tumor antigens to CD8+ cytotoxic T cells. In preferred embodiments described and exemplified herein, these coordinated biological mechanisms combine to generate a potent inflammatory response that couples stimulation of the innate immune system by RIG-I agonists with IL12-mediated stimulation of the adaptive immune response, resulting in a strong and durable antitumor immune response.

[0101] composition Provided herein are chitosan compositions comprising chitosan derivative nucleic acid nanoparticles (polyplexes) complexed with polyanion-containing block copolymers, e.g., diblock and / or triblock copolymer coatings, wherein individual polymer molecules comprise a negatively charged anchor region and one or more uncharged hydrophilic tail regions. An exemplary polymer molecule useful in the methods and compositions of the invention is a "PEG-PA" polymer molecule comprising a polyethylene glycol (PEG) moiety and a polyanion (PA) moiety.

[0102] 1.1.Chitosan The chitosan component of the chitosan derivative nucleic acid nanoparticles can be functionalized with cationic functional groups and / or hydrophilic moieties. Chitosan functionalized with two different functional groups is called doubly derivatized chitosan (DD-chitosan). Exemplary DD-chitosan is functionalized with both hydrophilic moieties (e.g., polyols) and cationic functional groups (e.g., amino groups). Exemplary chitosan derivatives are also described, for example, in U.S. Patent No. 2007 / 0281904 and U.S. Patent No. 2016 / 0235863 (each of which is incorporated herein by reference).

[0103] In one embodiment, the doubly derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 50%. In one embodiment, the degree of deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. In a preferred embodiment, the doubly derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 98%.

[0104] The chitosan derivatives described herein have a wide range of average molecular weights that are soluble at neutral and physiological pH, including, for purposes of the present invention, a molecular weight range of 3 to 110 kDa. The embodiments described herein feature lower average molecular weights of derivatized chitosans (less than 25 kDa, e.g., about 5 kDa to about 25 kDa), which may have desirable delivery and transfection properties, including small size and good solubility. Derivatized chitosans with low average molecular weights are generally more soluble than those with higher molecular weights, the former producing nucleic acid / chitosan complexes that more readily release nucleic acids and increase cellular transfection. A large body of literature has been devoted to optimizing all of these parameters for chitosan-based delivery systems.

[0105] Those of ordinary skill in the art will understand that chitosan refers to a plurality of molecules having the structure of Formula I, where n is any integer and each R1 is independently selected from acetyl or hydrogen, with the degree of R1 selected from hydrogen being 50% to 100%. It is also well-recognized that a chitosan referred to as having an average molecular weight of, for example, 3 kD to 110 kD generally refers to a plurality of chitosan molecules, each having a weight average molecular weight of, for example, 3 kD to 110 kD, each of which may be of a different chain length (n+2). It is also well-recognized that a chitosan referred to as an "n-mer chitosan" does not necessarily include chitosan molecules of Formula I, where each chitosan molecule has a chain length of n+2. Rather, as used herein, "n-mer chitosan" refers to a plurality of chitosan molecules, each of which may be of a different chain length, many of which have an average molecular weight substantially equivalent to or equal to a chitosan molecule having a chain length of n. For example, a 24-mer chitosan may comprise multiple chitosan molecules, each with different chain lengths ranging, for example, from 7 to 50, that have a weight average molecular weight that is substantially the same as or equal to a chitosan molecule with a chain length of 24.

[0106] The doubly derivatized chitosan of the present invention may also be functionalized with a polyol or hydrophilic functional group such as a polyol. Without wishing to be bound by theory, it is hypothesized that functionalization with a hydrophilic group such as a polyol may serve to increase the hydrophilicity of chitosan (including arginine-chitosan) and / or may provide hydroxyl groups. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticles is or includes gluconic acid. See, e.g., WO 2013 / 138930. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticles is or includes glucose. Additionally or alternatively, the hydrophilic functional group may include a polyol. See, e.g., US 2016 / 0235863. Exemplary polyols for functionalizing chitosan are further described below.

[0107] The functionalized chitosan derivatives described herein include doubly derivatized chitosan compounds, such as cationic chitosan-polyol compounds. Generally, the cationic chitosan-polyol compounds are functionalized with an amino-containing moiety, such as arginine, lysine, ornithine, or a molecule containing guanidinium, or a combination thereof. In certain embodiments, the cationic chitosan-polyol compounds have the following structure of Formula I: [ka] (I) In the formula, n is an integer of 1 to 650. α is the final degree of functionalization of the cationic moiety (e.g., an amino group-containing molecule such as lysine, ornithine, a molecule containing a guanidinium group, arginine, or a combination thereof); β is the final degree of functionalization of the polyol, Each R 1 are independently selected from hydrogen, acetyl, a cation (e.g., arginine), and a polyol.

[0108] Preferably, the doubly derivatized chitosan of the present invention may be functionalized with the cationic amino acid, arginine.

[0109] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid to a final degree of functionalization of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30%, or more. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with glucose to a final degree of functionalization of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30%, or more. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cationic moiety (e.g., arginine) to a final degree of functionalization of about 1% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cationic moiety (e.g., arginine) to a final degree of functionalization of about 10% to about 40%.

[0110] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 10% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 20% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 25% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 25% to about 30%.

[0111] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 15% to about 40%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 15% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 15% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 15% to about 28%.

[0112] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 10% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 10% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 10% to about 28%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to a cationic moiety (e.g., arginine) with a final degree of functionalization of about 28%.

[0113] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid to a final functionalization degree of about 2% to about 30%, about 5% to about 30%, about 7.5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid to a final functionalization degree of about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 15%, or about 7.5% to about 12%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid to a final functionalization degree of about 10%.

[0114] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a hydrophilic polyol to a final degree of functionalization of about 2% to about 30%, about 5% to about 30%, about 7.5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a hydrophilic polyol to a final degree of functionalization of about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 15%, or about 7.5% to about 12%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a hydrophilic polyol to a final degree of functionalization of about 10%.

[0115] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with glucose to a final functionalization degree of about 2% to about 30%, about 5% to about 30%, about 7.5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with glucose to a final functionalization degree of about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 15%, or about 7.5% to about 12%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with glucose to a final functionalization degree of about 10%.

[0116] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 2% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 5% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 7.5% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) at a final degree of functionalization of about 10% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) at a final degree of functionality of about 7.5% to about 15%, or about 10%.

[0117] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 2% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 5% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 7.5% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) at a final degree of functionalization of about 10% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) at a final degree of functionality of about 7.5% to about 15%, or about 10%.

[0118] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) at a final functionalization level of about 10% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) at a final functionality level of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) at a final functionalization level of about 12% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) at a final functionality level of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) at a final functionalization level of about 14% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) at a final functionality level of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 15% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 7.5% to about 15%, or about 10%.

[0119] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 25% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 7.5% to about 15%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 28% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 7.5% to about 15%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 25% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) at a final degree of functionalization of about 28% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) at a final degree of functionality of about 5% to about 20%.

[0120] In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 14% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 10%. In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 15% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 12%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine to a final degree of functionalization of about 14% and chitosan conjugated with glucose to a final degree of functionality of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine to a final degree of functionalization of about 15% and chitosan conjugated with glucose to a final degree of functionality of about 12%.

[0121] In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) to a final degree of functionalization of about 28% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) to a final degree of functionality of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine to a final degree of functionalization of about 28% and chitosan conjugated with glucose to a final degree of functionality of about 10%.

[0122] In some embodiments, DD-chitosan optionally includes DD-chitosan derivatives, such as DD-chitosan incorporating additional functionalization, e.g., DD-chitosan with attached ligands. "Derivatives" will be understood to include a broad class of chitosan-based polymers containing covalently modified N-acetyl-D-glucosamine and / or D-glucosamine units, as well as chitosan-based polymers incorporating other units or conjugated to other moieties. Derivatives are often based on modification of the hydroxyl or amine groups of glucosamine, as is done with arginine-functionalized chitosan. Examples of chitosan derivatives include, but are not limited to, trimethylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, PEI-incorporated chitosan, uronic acid-modified chitosan, glycol chitosan, and the like. For further teachings regarding chitosan derivatives, see, for example, pp. 63-74 of "Non-viral Gene Therapy", K. Taira, K. Kataoka, T. Niidome (editors), Springer-Verlag Tokyo, 2005, ISBN 4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007, vol. 52(23), pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55(2004) 77-93.

[0123] 1.2. Chitosan-Nucleic Acid Polyplexes Chitosan derivative nanoparticle compositions generally contain at least one nucleic acid molecule, preferably multiple such nucleic acid molecules. Representative nucleic acid molecules contain phosphorus as a component of the nucleic acid backbone, for example, in the form of multiple phosphodiesters or their derivatives (e.g., phosphorothioates). The ratio of cation-functionalized chitosan derivative to nucleic acid can be characterized by the cation (+) to phosphorus (P) molar ratio, where (+) refers to the cation of the cation-functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, the (+):(P) molar ratio is selected so that the chitosan-derivative-nucleic acid complex has a positive charge in the absence of a reversible coating of the polyanion-containing block copolymer. Thus, the (+):(P) molar ratio is generally greater than 1. In preferred embodiments, the (+):(P) molar ratio is greater than 1.5, at least 2, or greater than 2. In certain preferred embodiments, the (+):(P) molar ratio is greater than 2.

[0124] In some cases, the (+):(P) molar ratio is 3:1 or about 3:1. In some cases, the (+):(P) molar ratio is 4:1 or about 4:1. In some cases, the (+):(P) molar ratio is 5:1 or about 5:1. In some cases, the (+):(P) molar ratio is 6:1 or about 6:1. In some cases, the (+):(P) molar ratio is 7:1 or about 7:1. In some cases, the (+):(P) molar ratio is 8:1 or about 8:1. In some cases, the (+):(P) molar ratio is 9:1 or about 9:1. In some cases, the (+):(P) molar ratio is 10:1 or about 10:1.

[0125] In some cases, the (+):(P) molar ratio is greater than 1 to about 20:1 or less, about 2 to about 20:1 or less, or about 2 to about 10:1 or less. In some cases, the (+):(P) molar ratio is greater than 2 to about 20:1 or less, or about 2 to about 10:1 or less. In some cases, the (+):(P) molar ratio is about 3 to about 20:1 or less, about 3 to about 10:1 or less, about 3 to about 8:1 or less, or about 3 to about 7:1 or less. In some cases, the (+):(P) molar ratio is about 3 to about 20:1 or less, about 3 to 10:1 or less, about 3 to 8:1 or less, or about 3 to 7:1 or less.

[0126] In certain embodiments, the (+):(P) molar ratio is 100:1, preferably less than 100:1. For example, in certain embodiments, the (+):(P) molar ratio can be greater than 1 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio can be greater than 2 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio can be greater than 3 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio can be greater than 5 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio can be greater than 7 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio can be greater than 2 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio can be greater than 3 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio can be greater than 5 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio can be greater than or equal to 7 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio can be greater than 2 and less than or equal to 25:1. In some cases, the (+):(P) molar ratio can be greater than or equal to 3 and less than or equal to 25:1. In some cases, the (+):(P) molar ratio can be greater than or equal to 5 and less than or equal to 25:1. In some cases, the (+):(P) molar ratio can be greater than or equal to 7 and less than or equal to 25:1.

[0127] In some embodiments, the cationic functional group of the chitosan derivative nanoparticles is or includes an amino group. Examples of such amino-functionalized chitosan derivative nanoparticles include, but are not limited to, those containing chitosan functionalized with guanidinium or a molecule containing a guanidinium group, lysine, ornithine, arginine, or a combination thereof. In a preferred embodiment, the cationic functional group is arginine. The ratio of amino-functionalized chitosan derivative to nucleic acid can be characterized by the amino (N) to phosphorus (P) molar ratio, where (N) refers to the nitrogen atom of the amino group in the amino-functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, the N:P molar ratio is selected so that the chitosan derivative-nucleic acid complex has a positive charge at physiologically relevant pH in the absence of PEG-PA polymer molecules. Thus, the N:P molar ratio is generally greater than 1. In preferred embodiments, the N:P molar ratio is greater than 1.5, at least 2, or greater than 2. In certain preferred embodiments, the N:P molar ratio is greater than 2.

[0128] In some cases, the N:P molar ratio is 3:1 or about 3:1. In some cases, the N:P molar ratio is 4:1 or about 4:1. In some cases, the N:P molar ratio is 5:1 or about 5:1. In some cases, the N:P molar ratio is 6:1 or about 6:1. In some cases, the N:P molar ratio is 7:1 or about 7:1. In some cases, the N:P molar ratio is 8:1 or about 8:1. In some cases, the N:P molar ratio is 9:1 or about 9:1. In some cases, the N:P molar ratio is 10:1 or about 10:1.

[0129] In some cases, the N:P molar ratio is greater than 1 to about 20:1 or less, about 2 to about 20:1 or less, or about 2 to about 10:1 or less. In some cases, the N:P molar ratio is greater than 2 to about 20:1 or less, or about 2 to about 10:1 or less. In some cases, the N:P molar ratio is about 3 to about 20:1 or less, about 3 to about 10:1 or less, about 3 to about 8:1 or less, or about 3 to about 7:1 or less. In some cases, the N:P molar ratio is about 3 to 20:1 or less, about 3 to 10:1 or less, about 3 to 8:1 or less, or about 3 to 7:1 or less.

[0130] In certain embodiments, the N:P molar ratio is 100:1, preferably less than 100:1. For example, in certain embodiments, the N:P molar ratio can be greater than 1 and less than or equal to 100:1. In some cases, the N:P molar ratio can be greater than 2 and less than or equal to 100:1. In some cases, the N:P molar ratio can be greater than 3 and less than or equal to 100:1. In some cases, the N:P molar ratio can be greater than 5 and less than or equal to 100:1. In some cases, the N:P molar ratio can be greater than 7 and less than or equal to 100:1. In some cases, the N:P molar ratio can be greater than 2 and less than or equal to 50:1. In some cases, the N:P molar ratio can be greater than 3 and less than or equal to 50:1. In some cases, the N:P molar ratio can be greater than 5 and less than or equal to 50:1. In some cases, the N:P molar ratio can be greater than 7 and less than or equal to 50:1. In some cases, the N:P molar ratio can be greater than 2 and less than or equal to 25:1. In some cases, the N:P molar ratio can be greater than or equal to 3 and less than or equal to 25:1. In some cases, the N:P molar ratio can be greater than or equal to 5 and less than or equal to 25:1. In some cases, the N:P molar ratio can be greater than or equal to 7 and less than or equal to 25:1.

[0131] In a preferred embodiment, the polyplexes have an amine to phosphate (N / P) ratio of 2 to 100, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 5. Preferably, the N / P ratio is inversely proportional to the molecular weight of the chitosan, i.e., a lower molecular weight (e.g., doubly) derivatized chitosan requires a higher N / P ratio, and vice versa.

[0132] Nucleic acids of the present invention will generally contain phosphodiester bonds, but in some cases include nucleic acid analogs that may have alternative backbones or other modifications or moieties incorporated for various purposes, such as stability and protection. Other contemplated analog nucleic acids include those with non-ribose backbones. In addition, naturally occurring nucleic acids, analogs, and mixtures of both can be created. Nucleic acids can be single-stranded or double-stranded, or contain portions of both double-stranded and single-stranded sequence. Nucleic acids include, but are not limited to, DNA, RNA, and hybrids, and nucleic acids can contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, and the like. Nucleic acids include any form of DNA, triplex, double-stranded, or single-stranded, antisense, siRNA, ribozymes, deoxyribozymes, polynucleotides, oligonucleotides, chimeras, microRNA, and any form of RNA, including derivatives thereof. Nucleic acids include artificial nucleic acids, including, but not limited to, peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligos (PMOs), locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). For artificial nucleic acids that do not contain phosphorus, it will be understood that the equivalent measurement of the (+):P or N:P ratio can be approximated by the number of nucleotide (or nucleotide analog) bases.

[0133] In preferred embodiments, the polyplexes of the composition comprise chitosan molecules having an average molecular weight before functionalization of less than 110 kDa, more preferably less than 65 kDa, more preferably less than 50 kDa, more preferably less than 40 kDa, and most preferably less than 30 kDa. In some embodiments, the polyplexes of the composition comprise chitosan having an average molecular weight before functionalization of less than 15 kDa, less than 10 kDa, less than 7 kDa, or less than 5 kDa.

[0134] In preferred embodiments, the polyplexes comprise chitosan molecules having, on average, fewer than 680 glucosamine monomer units, more preferably fewer than 400 glucosamine monomer units, more preferably fewer than 310 glucosamine monomer units, more preferably fewer than 250 glucosamine monomer units, and most preferably fewer than 190 glucosamine monomer units. In some embodiments, the polyplexes comprise chitosan molecules having, on average, fewer than 95 glucosamine monomer units, fewer than 65 glucosamine monomer units, fewer than 45 glucosamine monomer units, or fewer than 35 glucosamine monomer units.

[0135] Chitosan and (eg, doubly) derivatized chitosan nucleic acid polyplexes may be prepared by any method known in the art, including, but not limited to, those described herein.

[0136] 1.2.1. Nucleic acids As described above, chitosan polyplexes can contain multiple nucleic acids. In one embodiment, the nucleic acid component comprises a therapeutic nucleic acid. The present (e.g., doubly) derivatized chitosan nucleic acid polyplexes are suitable for use with any therapeutic nucleic acid known in the art, including, for example, nucleic acids encoding therapeutic proteins such as hormones, enzymes, cytokines, chemokines, antibodies, mitogens, growth factors, differentiation factors, factors affecting cell apoptosis, factors affecting inflammation, and factors affecting the immune response (e.g., immunostimulatory factors).

[0137] Therapeutic nucleic acids may be used to perform gene therapy by serving as replacements or augmentations of defective genes, or to compensate for the absence of a particular gene product by encoding a therapeutic product. Therapeutic nucleic acids may also suppress the expression of endogenous genes. Therapeutic nucleic acids may encode all or part of a translation product and function by recombining with DNA already present in the cell, thereby replacing the defective portion of the gene. They may also encode part of a protein and exert their effect by co-suppressing the gene product.

[0138] In some embodiments, the nucleic acid component comprises a therapeutic nucleic acid construct. A therapeutic nucleic acid construct is a nucleic acid construct capable of exerting a therapeutic effect. A therapeutic nucleic acid construct may comprise a nucleic acid encoding a therapeutic protein as well as a nucleic acid that produces a transcript that is a therapeutic RNA.

[0139] In preferred embodiments described and exemplified herein, a therapeutic nucleic acid construct comprises a nucleic acid encoding IL-12, alone or in combination with additional immunostimulatory molecule(s). IL-12 is a heterodimeric type 1 cytokine with a four α-helical bundle structure. The active heterodimer, also known as IL-12 p70, contains two subunits encoded by two separate genes: IL-12A (encoding p35) and IL-12B (encoding p40). There are at least six splice variant transcripts of IL-12A (ENST00000305579.6, ENST00000466512.1, ENST00000480787.5, ENST00000468862.5, ENST00000496308.1, and ENST00000480088.1). Nucleic acid and peptide sequences for human IL-12A isoform 1 precursor are, for example, NM_000882.4, NM_001354582.2, NM_001354583.2, and NP_000873.2, NP_001341511.1, and NP_001341512.1, respectively. Nucleic acid and peptide sequences for mouse IL12a are, for example, NM_001159424.2 and NP_001152896.1, respectively. Human IL-12B genomic, transcript, and peptide sequences are, for example, NG_009618.1, NM_002187.3, and NP_002178.2, respectively. The nucleic acid and peptide sequences for mouse IL-12B are, for example, NM_001303244 and NP_001290173.1.

[0140] In some embodiments, single-chain IL-12 proteins can be generated by fusing the p40 subunit to the p35 subunit via a short amino acid linker sequence. The two subunits can be linked in either the p40-linker-p35 or p35-linker-p40 orientation. The protein can be secreted as a result of the inclusion of a signal peptide from the subunit 5' of the linker, but the signal peptide is removed from the subunit downstream of the linker sequence. In a preferred embodiment, the linker sequence comprises a 10-amino acid sequence derived from bovine elastin and consists of valine (V), proline (P), and glycine (G) residues (VPGVGVPGVG). In some embodiments, the linker sequence may contain G and / or serine (S) residues, such as (GGGGS)n. In other embodiments, the linker sequence may contain G, S, and additional amino acids, including, but not limited to, P, arginine (R), lysine (K), threonine (T), and glutamic acid (E). In exemplary embodiments, the linker is selected from the group consisting of GSGSSRGGSGSGGSGGGGSK, GSTSG(A / S)GKSSEGKG (SEQ ID NO: 1), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 2), GGGGGGS (SEQ ID NO: 3), or GGGGSGGGSGGGGS (SEQ ID NO: 4).

[0141] In an exemplary embodiment, the nucleic acid sequence encoding hIL-12p40p35 comprises:

[0142] In an exemplary embodiment, the hIL-12p40p35 amino acid sequence comprises: STOP (SEQ ID NO: 6).

[0143] Therapeutic nucleic acids also include therapeutic DNA in the form of circular double-stranded DNA plasmids, minicircle DNA (Science Report 6:2315, 2016) or closed-end linear double-stranded DNA (Li et al, PLoS One 8(8):e69879, 2013).

[0144] Therapeutic nucleic acids also include therapeutic RNAs, which are RNA molecules that can exert therapeutic effects in mammalian cells.Therapeutic RNAs include, but are not limited to, messenger RNA, antisense RNA, siRNA, short hairpin RNA, microRNA, and enzymatic RNA.Therapeutic nucleic acids include, but are not limited to, nucleic acids intended to form triplex molecules, protein-binding nucleic acids, ribozymes, deoxyribozymes, and small nucleotide molecules.Many types of therapeutic RNAs are known in the art. See, e.g., Meng et al., A new developing class of gene delivery: messenger RNA-based therapeutics, Biomater. Sci., 5, 2381-2392, 2017; Grimm et al., Therapeutic application of RNAi is mRNA targeting finally ready for prime time? J. Clin. Invest., 117:3633-3641, 2007; Aagaard et al., RNAi therapeutics: Principles, prospects and challenges, Adv. Drug Deliv. Rev., 59:75-86, 2007; Dorsett et al., siRNAs: Applications in functional genomics and potential as therapeutics, Nat. Rev. Drug Discov., 3:318-329, 2004. These include double-stranded small interfering RNAs (siRNAs).

[0145] 1.2.1.1. Expression control region In a preferred embodiment, the polyplexes of the present invention comprise a therapeutic nucleic acid that is a therapeutic construct comprising an expression control region operably linked to a coding region that generates a therapeutic nucleic acid, which may be therapeutic in itself or may encode a therapeutic protein.

[0146] In some embodiments, the expression control region of the therapeutic construct has constitutive activity. In many preferred embodiments, the expression control region of the therapeutic construct does not have constitutive activity. This provides dynamic expression of the therapeutic nucleic acid. "Dynamic" expression means expression that varies over time. Dynamic expression may include several periods of low or absent expression separated by periods of detectable expression. In many preferred embodiments, the therapeutic nucleic acid is operably linked to a regulatable promoter. This provides regulatable expression of the therapeutic nucleic acid.

[0147] Expression control regions include regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which affect expression of an operably linked therapeutic nucleic acid.

[0148] The expression control elements included herein can be of bacterial, yeast, plant, or animal (mammalian or non-mammalian) origin. Expression control regions include full-length promoter sequences, e.g., native promoter and enhancer elements, as well as subsequences or polynucleotide variants that retain all or part of their intact or non-mutant function (e.g., retaining some degree of nutritional regulation or cell / tissue-specific expression). As used herein, the term "functional" and grammatical variations thereof, when used with respect to a nucleic acid sequence, subsequence, or fragment, means that the sequence possesses one or more functions of the native nucleic acid sequence (e.g., a non-mutant or unmodified sequence). As used herein, the term "variant" refers to sequence substitutions, deletions, or additions, or other modifications (e.g., chemical derivatives, such as modified forms that are resistant to nucleases).

[0149] As used herein, the term "operably linked" refers to the physical juxtaposition of the described components so as to permit them to function in their intended manner. In the example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates expression of the nucleic acid. Typically, an expression control region that regulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream"). An expression control region can also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., within an intron). An expression control element can be located at a distance from the transcribed sequence (e.g., 100-500, 500-1000, 2000-5000 or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is typically located 5' from the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' from the transcribed sequence, or within the transcribed sequence.

[0150] Some expression control regions confer regulatable expression to an operably linked therapeutic nucleic acid. A signal (sometimes called a stimulus) can increase or decrease expression of a therapeutic nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as inhibitory. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0151] Numerous regulatable promoters are known in the art. Preferred inducible expression control regions include those containing inducible promoters stimulated by small molecule chemical compounds. In one embodiment, the expression control region responds to chemicals that are orally deliverable but not normally found in food. Specific examples can be found, for example, in U.S. Patent Nos. 5,989,910; 5,935,934; 6,015,709; and 6,004,941.

[0152] Promoter / enhancer sequences of particular interest include the following: [Table 1]

[0153] In some embodiments of the invention, the therapeutic construct is contained within a plasmid comprising an origin, a multiple cloning site, and a selectable marker. In some embodiments, a plasmid less than 10 kb is desirable. In some embodiments, the plasmid used is suitable for gene therapy in human patients and / or engineered for high level transient gene expression in mammalian tissues. In preferred embodiments, the plasmid is Nanoplasmid™ (e.g., NTC9385 plasmid, NTC9385R, NTC9385R-RIG-I, NTC9385R(3CpG), NTC9385R-eRNA41H-CpG, NTC8685 plasmid (Nature Technology), gWIZ plasmid (Genlantis), or pVAX1 plasmid (Thermofisher For example, the compound may be selected from the group consisting of U.S. Patent Nos. 6,027,722, 6,287,863, 6,410,220, 6,573,091, 9,012,226, 9,017,966, 9,018,012, 9,109,012, 9,487,788, 9,487,789, and 9,500. See US 6,082, US 9,550,998, US 9,725,725, US 9,737,620, US 9,950,081, US 10,047,365, US 10,144,935, and US 10,167,478. In some embodiments, the plasmids are "modified" to remove antibiotic selection agents and / or to increase expression levels.

[0154] For further teachings, see WO2008 / 020318, expressly incorporated herein by reference in its entirety. In one embodiment, the nucleic acids of the (e.g., doubly) derivatized chitosan nucleic acid polyplexes are artificial nucleic acids.

[0155] In one embodiment, the nucleic acid of the DD-chitosan nucleic acid polyplex is a therapeutic nucleic acid. In one embodiment, the therapeutic nucleic acid is a therapeutic RNA. Preferred therapeutic RNAs include, but are not limited to, antisense RNA, siRNA, short hairpin RNA, microRNA, and enzymatic RNA.

[0156] In one embodiment, the therapeutic nucleic acid is DNA.

[0157] In one embodiment, the therapeutic nucleic acid comprises a nucleic acid sequence that encodes a therapeutic protein.

[0158] 1.3. Polyols Chitosan derivative nanoparticles can be functionalized with polyols. Generally, polyols useful in the present invention are usually hydrophilic. In some cases, chitosan derivative nanoparticles are functionalized with cationic moieties such as amino groups and polyols. Such chitosan derivative nanoparticles functionalized with amino groups and cationic moieties such as polyols are called "doubly derivatized chitosan nanoparticles."

[0159] In some embodiments, the chitosan derivative nanoparticles comprise a polyol of Formula II: [ka] II (In the formula, R 2 is selected from H and hydroxyl; R 3 is selected from H and hydroxyl; X is selected from C2-C6 alkylene optionally substituted with one or more hydroxyl substituents).

[0160] In some embodiments, chitosan derivative nanoparticles are prepared by dissolving a polyol of formula II, where R 2 is selected from H and hydroxyl; R 3 is selected from H and hydroxyl; and X is selected from C2-C6 alkylene optionally substituted with one or more hydroxyl substituents.

[0161] In some embodiments, the chitosan derivative nanoparticles comprise a polyol of Formula III: [ka] III (In the formula, -----Y is =O or -H2, R 2 is selected from H and hydroxyl; R 3 is selected from H and hydroxyl; X is selected from C2-C6 alkylene optionally substituted with one or more hydroxyl substituents; [ka] indicates the bond between the polyol and the derivatized chitosan).

[0162] In one embodiment, polyols according to the present invention having 3 to 7 carbons may have one or more carbon-carbon multiple bonds. In a preferred embodiment, polyols according to the present invention comprise carboxyl groups. In a further preferred embodiment, polyols according to the present invention comprise aldehyde groups. Those skilled in the art will recognize that when polyols according to the present invention comprise aldehyde groups, such polyols encompass both open-chain conformations (aldehydes) and cyclic conformations (hemiacetals).

[0163] Non-limiting examples of polyols include gluconic acid, threonic acid, glucose, and threose. Examples of such other polyols, which may have carboxyl and / or aldehyde groups or may be sugars or their acid forms, are described in more detail in U.S. Pat. No. 10,046,066, the disclosure of which is expressly incorporated herein by reference. Those skilled in the art will recognize that polyols are not limited to a particular stereochemistry.

[0164] In a preferred embodiment, the polyol may be selected from the group consisting of 2,3-dihydroxylpropanoic acid, 2,3,4,5,6,7-hexahydroxylheptanal; 2,3,4,5,6-pentahydroxylhexanal; 2,3,4,5-tetrahydroxylhexanal; and 2,3-dihydroxylpropanal.

[0165] In a preferred embodiment, the polyol may be selected from the group consisting of D-glyceric acid, L-glyceric acid, L-glycero-D-mannoheptose, D-glycero-L-mannoheptose, D-glucose, L-glucose, D-fucose, L-fucose, D-glyceraldehyde, and L-glyceraldehyde.

[0166] In some embodiments, the polyol can be a compound of Formula IV or Formula V: [ka] (IV) (V).

[0167] In a preferred embodiment, the polyol is a compound of formula IV: In some cases, the polyol of formula IV is attached to chitosan by reductive amination.

[0168] A hydrophilic polyol bearing a carboxyl group may be conjugated to chitosan or a cationically functionalized chitosan, such as an amine-functionalized chitosan (e.g., Arg-conjugated chitosan (Arg-chitosan)). In some embodiments, the polyol is conjugated at a reaction pH of 6.0±0.3. At this pH, the carboxylic acid group of the hydrophilic polyol can be attacked by the unbound amine on the chitosan backbone according to a nucleophilic substitution reaction mechanism.

[0169] When attaching such hydrophilic polyols to Arg-chitosan, those skilled in the art will understand that the nucleophilic substitution reaction is likely to occur primarily at the amine groups of the chitosan backbone, although it is also possible that a small amount of the hydrophilic polyol may form covalent bonds with the amine groups of Arg via the same mechanism.

[0170] Hydrophilic polyols that are natural sugars may be conjugated to chitosan, cation-functionalized chitosan, e.g., amine-functionalized chitosan (e.g., Arg-conjugated chitosan (Arg-chitosan)), using reductive amination followed by reduction with NaCBH or NaBH.

[0171] 1.4. Polymer: Polyplex Composition Chitosan polyplexes can be mixed with multiple polymers, where the polymers contain a hydrophilic, uncharged portion and a negatively charged (anionic) portion. As described above, chitosan polyplexes are formulated to have no complexation with anionic portion-containing polymers or to have a positive charge prior to complexation. Thus, under suitable conditions, the polymer component will form a reversible charge:charge complex with the chitosan-derivative nucleic acid polyplex. In some embodiments, the polymers of the polymer component are unbranched. In some embodiments, the polymers are branched. In some cases, the polymer component comprises a mixture of branched and unbranched polymers.

[0172] In some embodiments, the polymer component is released from the chitosan polyplex after administration, after entry into cells, and / or after endocytosis. Without wishing to be bound by theory, it is hypothesized that by complexing a polyplex and a polymer containing an anionic moiety, the polyplex:polymer composition thus formed may provide improved in vitro, in-solution, and / or in vivo stability without substantially interfering with transfection efficiency. In some embodiments, the polyplex:polymer composition thus formed may provide, for example, reduced mucoadhesive properties compared to the same polyplex separately lacking the polymer component.

[0173] In preferred embodiments, polyplex:polymer compositions have a low net positive zeta potential, a net neutral zeta potential, or a net negative zeta potential (about +10 mV to about -20 mV) at physiological pH. Such compositions may exhibit reduced aggregation at physiological conditions and reduced nonspecific binding to ubiquitous anionic components in vivo. Such properties may enhance the transport of such compositions (e.g., enhanced diffusion through mucus) upon contact with cells, resulting in enhanced intracellular release of nucleic acids.

[0174] In preferred embodiments, the polyplex:polymer particle compositions have an average hydrodynamic diameter of less than 1000 nm, more preferably less than 500 nm, and most preferably less than 200 nm. In certain embodiments, the polyplex:polymer particle compositions have an average hydrodynamic diameter of 50 nm to 1000 nm or less, preferably 50 nm to 500 nm or less, and most preferably 50 nm to 200 nm or less. In certain embodiments, the polyplex:polymer particle compositions have an average hydrodynamic diameter of 50 nm to 175 nm or less, preferably 50 nm to 150 nm or less. In certain embodiments, the polyplex:polymer particle compositions have an average hydrodynamic diameter of 75 nm to 1000 nm or less, preferably 75 nm to 500 nm or less, and most preferably 75 nm to 200 nm or less. In certain embodiments, the polyplex:polymer particle compositions have an average hydrodynamic diameter of 75 nm to 175 nm or less, preferably 75 nm to 150 nm or less. In certain embodiments, the polyplex:polymer particle composition has an average hydrodynamic diameter greater than 100 nm and less than 175 nm.

[0175] In one embodiment, the polyplex:polymer composition has a % supercoiled DNA content of 80%, at least 80%, or preferably 90%, more preferably at least 90%.

[0176] In one embodiment, the polyplex:polymer composition has an average zeta potential of +10 mV to −10 mV at physiological pH, most preferably +5 mV to −5 mV at physiological pH.

[0177] The polyplex:polymer composition is preferably homogeneous with respect to particle size. Thus, in preferred embodiments, the composition has a low average polydispersity index ("PDI"). In particularly preferred embodiments, the dispersion of the polyplex:polymer composition has a PDI of less than 0.5, more preferably less than 0.4, more preferably less than 0.3, even more preferably less than 0.25, and most preferably less than 0.2.

[0178] In some cases, the polyplex:polymer composition dispersion exhibits one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range after one or more freeze-thaw cycles. In some cases, the polyplex:polymer composition dispersion exhibits one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range after storage in solution at 4°C for at least 48 hours. In some cases, the polyplex:polymer composition dispersion exhibits one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range after storage in solution at 4°C for at least one or two weeks or more.

[0179] In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range after lyophilization and rehydration. In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range after spray drying and rehydration. In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated (e.g., by ultrafiltration, such as tangential flow filtration) to a nucleic acid concentration of at least 250 μg / mL. In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 1,000 μg / mL. In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 25,000 μg / mL. In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 2,000 μg / mL. In some cases, dispersions of polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 5,000 μg / mL. In some cases, dispersions of the polyplex:polymer compositions exhibit one or more of the above-mentioned PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 10,000 μg / mL.

[0180] Generally, the polyplex:polymer compositions described herein exhibit favorable solution behavior (e.g., stability and / or non-aggregation) (as measured by PDI or average particle size) in the absence of excipients, such as cryoprotectants, cryoprotectants, surfactants, rehydration or wetting agents, etc. In some cases, the polyplex:polymer compositions described herein exhibit favorable solution behavior (e.g., stability and / or non-aggregation) (as measured by PDI or average particle size) in physiological or simulated physiological fluids. For example, in some embodiments, the polyplex:polymer compositions described herein are stable in simulated intestinal fluid, in mammalian urine, and / or when stored in (e.g., in contact with) the bladder of a mammal.

[0181] As noted above, the polyplex:polymer compositions described herein are preferably substantially size-stable throughout the composition. In preferred embodiments, the compositions of the invention comprise polyplex:polymer particles whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% over 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours at room temperature. In particularly preferred embodiments, the compositions of the invention comprise polyplex:polymer particles whose average diameter increases by less than 25% over at least 24 hours or at least 48 hours at room temperature.

[0182] The polyplex:polymer particles of the present compositions are preferably substantially size-stable under refrigerated conditions. In a preferred embodiment, the compositions of the present invention comprise polyplex:polymer particles whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% over 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours at 2-8°C.

[0183] The polyplex:polymer particles of the present compositions are preferably substantially size-stable under freeze-thaw conditions. In a preferred embodiment, the compositions of the present invention comprise polyplexes whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% after thawing at -20 to -80°C for 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours at room temperature.

[0184] In preferred embodiments, the composition has a nucleic acid concentration greater than 0.5 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 0.6 mg / ml, more preferably at least 0.75 mg / ml, more preferably at least 1.0 mg / ml, more preferably at least 1.2 mg / ml, and most preferably at least 1.5 mg / ml and is substantially free of precipitated polyplexes. In another preferred embodiment, the composition has a nucleic acid concentration greater than 2 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 2.5 mg / ml, more preferably at least 5 mg / ml, more preferably at least 10 mg / ml, more preferably at least 15 mg / ml, and most preferably about 25 mg / ml and is substantially free of precipitated polyplexes. In some embodiments, the composition has a nucleic acid concentration of 0.5 mg / ml to about 25 mg / ml and is substantially free of precipitated polyplexes. In some embodiments, the composition has a nucleic acid concentration of about 25 mg / ml or less and is substantially free of precipitated polyplexes. The composition can be hydrated. In a preferred embodiment, the composition is substantially free of uncomplexed nucleic acid.

[0185] In preferred embodiments, the polyplex:polymer particle compositions are isotonic. Achieving isotonicity while maintaining polyplex stability is highly desirable for formulating pharmaceutical compositions, and these preferred compositions are well suited for pharmaceutical formulation and therapeutic use.

[0186] In certain embodiments, the polyplex:polymer particle composition can be decoated by lowering the pH, e.g., to release all or a portion of the PEG. In certain embodiments, the polymer coat is released by incubating the particles under pH conditions lower than the pKa of the polyanion anchor region of the polymer. For example, if the polymer coat is a polyglutamate, the polymer coat can be released by incubating the particles at a pH lower than the pKa of the polyglutamate, e.g., a pH less than about 4.25. In certain embodiments, the polymer coat can be released by incubating the particles under pH conditions at least 0.25 pH units or at least 0.5 pH units below the pKa of the polyanion anchor region of the polymer coat.

[0187] In certain embodiments, polyplex:polymer particle compositions can be decoated to release all or part of the polymer coat, e.g., by subjecting the particles to high ionic strength.

[0188] Without wishing to be bound by theory, it is hypothesized that certain physiological conditions may promote partial (e.g., greater than 5%), substantial (e.g., greater than 50%), extensive (e.g., greater than 90%), or complete (100%) uncoating of the reversibly PEGylated chitosan-DNA polyplexes described herein. For example, the low pH conditions of certain intracellular compartments (e.g., endosomes, early endosomes, late endosomes, or lysosomes) may promote release of the polymer coat. As another example, certain extracellular conditions may promote partial (e.g., greater than 5%), substantial (e.g., greater than 50%), extensive (e.g., greater than 90%), or complete (100%) uncoating of the reversibly PEGylated chitosan-DNA polyplexes described herein. In some cases, high ionic strength and / or acidic pH conditions typically found in certain locations in the gastrointestinal tract may promote partial (e.g., greater than 5%), substantial (greater than 50%), extensive (greater than 90%), or complete (100%) uncoating of the reversibly PEGylated chitosan-DNA polyplexes described herein.

[0189] In certain embodiments, the PEGylated polyplexes described herein are formulated for delivery to cells, tissues, or body compartments (e.g., the intestine, small intestine, large intestine, colon, lung, or bladder) so that the polyplexes remain PEGylated, thereby facilitating transfection into target cells. In some embodiments, the PEGylated polyplexes described herein partially (e.g., greater than 5%), substantially (e.g., greater than 50%), extensively (e.g., greater than 90%), or completely (100%) release their polymer coat after or during entry into the intracellular environment. In certain embodiments, the PEGylated polyplexes described herein are formulated for delivery to a cell, tissue, or body compartment (e.g., the intestine, small intestine, large intestine, colon, lung, or bladder) such that the PEGylated polyplexes described herein release the polymer coat partially (e.g., greater than 5%), substantially (e.g., greater than 50%), extensively (e.g., greater than 90%), or completely (100%) upon delivery to a cell, tissue, or body compartment (e.g., the intestine, small intestine, large intestine, colon, lung, or bladder).

[0190] It will be understood that the anionic charge density and / or pKa of the anionic anchor region of the polymer can be adjusted to promote or inhibit release under the intended conditions. Similarly, it will be understood that the pH, volume, ionic strength, and other conditions of the formulation can be adjusted to promote or inhibit release under the intended conditions. For example, for delivery to the intestine via the low pH environment of the stomach, the PEGylated polyplex formulation can be enteric coated and / or delivered in a buffer to enhance the pH of the stomach environment. Optimized reversibly PEGylated particle compositions can be identified by assaying stability and transfection efficiency using the assays described herein.

[0191] Compositions comprising chitosan polyplexes complexed with anionic moiety-containing polymers can be characterized by the ratio of cationic functional groups on the (e.g., doubly) derivatized chitosan polyplexes (+) to anionic moieties on the polymer (-), referred to as the "(+):(-) molar ratio." This (+):(-) molar ratio can vary from about 1:100 to less than about 10:1.

[0192] In certain embodiments, the (+):(-) molar ratio can be greater than about 1:75 and less than about 8:1. In some cases, the (+):(-) molar ratio can be greater than 1:10 and less than 10:1. In some cases, the (+):(-) molar ratio can be 1:10, or about 1:10 to 10:1, or about 10:1. In some cases, the (+):(-) molar ratio can be 1:8, or about 1:8 to 8:1, or about 8:1. In certain embodiments, the (+):(-) molar ratio can be greater than 1:50 and less than about 10:1. In some cases, the (+):(-) molar ratio can be greater than 1:25 and less than about 10:1. In some cases, the (+):(-) molar ratio can be greater than 1:10 and less than about 7:1. In some cases, the (+):(-) molar ratio can be greater than 1:8 and less than about 7:1. In some cases, the (+):(-) molar ratio can be greater than 1:8 to less than about 6:1.

[0193] In certain embodiments in which the cationic functional (e.g., doubly) derivatized chitosan polyplexes are amino moieties, compositions comprising chitosan polyplexes complexed with anionic moiety-containing polymers can be characterized by the ratio of amino groups (N) of the (e.g., doubly) derivatized chitosan polyplexes to anionic (A) moieties of the polymer, referred to as the "N:A molar ratio." This N:A molar ratio can vary from greater than about 1:100 to less than about 10:1.

[0194] In certain embodiments, the N:A molar ratio may be greater than about 1:75 and less than about 8:1. In some cases, the N:A molar ratio may be greater than 1:10 and less than 10:1. In some cases, the N:A molar ratio may be 1:10, or about 1:10 to 10:1, or about 10:1. In some cases, the N:A molar ratio may be 1:8, or about 1:8 to 8:1, or about 8:1. In certain embodiments, the N:A molar ratio may be greater than 1:50 and less than about 10:1. In some cases, the N:A molar ratio may be greater than 1:25 and less than about 10:1. In some cases, the N:A molar ratio may be greater than 1:10 and less than about 7:1. In some cases, the N:A molar ratio may be greater than 1:8 and less than about 7:1. In some cases, the N:A molar ratio may be greater than 1:8 and less than about 6:1.

[0195] Additionally or alternatively, compositions comprising chitosan polyplexes complexed with anionic moiety-containing polymers can be characterized by a three-component ratio of cationic functional groups (+) of the (e.g., doubly) derivatized chitosan polyplexes to phosphorus atoms (P) of the nucleic acid to anionic moieties (-) of the polymer, referred to as the "(+):P:(-) molar ratio."

[0196] In certain embodiments where (+):P is at least 2:1 to no more than 20:1, the molar ratio of (+):(-) can vary from at least 1:40 to about 40:1. In certain embodiments where (+):P is at least 2:1 to no more than 20:1, the molar ratio of (+):(-) can vary from at least 1:40 to about 1:10. In some embodiments where (+):P is at least 2:1 to no more than 20:1, the molar ratio of (+):(-) can vary from at least 1:25 to about 25:1. In some embodiments where (+):P is at least 2:1 to no more than 20:1, the molar ratio of (+):(-) can vary from at least 1:25 to about 1:10. In some cases where (+):P is at least 2:1 to no more than 20:1, the molar ratio of (+):(-) can vary from at least 1:20 to about 20:1. In some cases where (+):P is at least 2:1 to no more than 20:1, the (+):(-) molar ratio may vary from at least 1:20 to about 1:10. In some cases where (+):P is at least 2:1 to no more than 20:1, the (+):(-) molar ratio may vary from at least 1:10 to about 10:1. In some cases where (+):P is at least 2:1 to no more than 20:1, the (+):(-) molar ratio may vary from at least 1:25 to about 2:1. In some cases where (+):P is at least 2:1 to no more than 20:1, the (+):(-) molar ratio may vary from at least 1:20 to about 1:1.

[0197] In certain preferred embodiments, the (+):P:(-) ratio is 3:1:3.5 to 3:1:17.5. In certain preferred embodiments, the (+):P:(-) ratio is 5:1:3.5 to 5:1:17.5. In certain preferred embodiments, the (+):P:(-) ratio is 7:1:3.5 to 7:1:17.5. In certain preferred embodiments, the (+):P:(-) ratio is about 3:1:3.5, 3:1:7, 3:1:10, 3:1:15, 3:1:17.5, or 3:1:20. In certain preferred embodiments, the (+):P:(-) ratio is about 5:1:3.5, 5:1:7, 5:1:10, 5:1:15, 5:1:17.5, or 5:1:20. In certain preferred embodiments, (+):P:(-) is about 7:1:3.5, 7:1:7, 7:1:10, 7:1:15, 7:1:17.5, or 7:1:20. In certain preferred embodiments, (+):P:(-) is about 10:1:10, 10:1:15, 10:1:20, 10:1:25, 10:1:30, or 10:1:40.

[0198] Those skilled in the art will appreciate that amino-functionalized chitosan polyplex particles complexed with anionic moiety-containing polymers can be characterized by a three-component ratio of amino functional groups of the (e.g., doubly) derivatized chitosan polyplex (N) to phosphorus atoms of the nucleic acid (P) to anionic moieties of the polymer (A), referred to as the "N:P:A molar ratio." In certain embodiments where N:P is at least 2:1 to no more than 20:1, the P:A molar ratio can vary from at least 1:40 to about 40:1.

[0199] In certain embodiments where N:P is at least 2:1 to no more than 20:1, the P:A molar ratio may vary from at least 1:40 to about 1:10. In certain embodiments where N:P is at least 2:1 to no more than 20:1, the P:A molar ratio may vary from at least 1:25 to about 25:1. In certain embodiments where N:P is at least 2:1 to no more than 20:1, the P:A molar ratio may vary from at least 1:25 to about 1:10. In some cases where N:P is at least 2:1 to no more than 20:1, the P:A molar ratio may vary from at least 1:20 to about 20:1. In some cases where N:P is at least 2:1 to no more than 20:1, the P:A molar ratio may vary from at least 1:20 to about 1:10. In some cases where N:P is at least 2:1 but not more than 20:1, the P:A molar ratio may vary from at least 1:10 to about 10:1. In some cases where N:P is at least 2:1 but not more than 20:1, the P:A molar ratio may vary from at least 1:25 to about 2:1. In some cases where N:P is at least 2:1 but not more than 20:1, the P:A molar ratio may vary from at least 1:20 to about 1:1.

[0200] In certain preferred embodiments, the N:P:A ratio is 3:1:3.5 to 3:1:17.5. In certain preferred embodiments, the N:P:A ratio is 5:1:3.5 to 5:1:17.5. In certain preferred embodiments, the N:P:A ratio is 7:1:3.5 to 7:1:17.5. In certain preferred embodiments, the N:P:A ratio is 10:1:10 to 10:1:40. In certain preferred embodiments, the N:P:A ratio is about 3:1:3.5, 3:1:7, 3:1:10, 3:1:15, 3:1:17.5, or 3:1:20. In certain preferred embodiments, the N:P:A ratio is about 5:1:3.5, 5:1:7, 5:1:10, 5:1:15, 5:1:17.5, or 5:1:20. In certain preferred embodiments, N:P:A is about 7:1:3.5, 7:1:7, 7:1:10, 7:1:15, 7:1:17.5, or 7:1:20. In certain embodiments, N:P:A is about 10:1:10, 10:1:15, 10:1:20, 10:1:25, 10:1:30, or 10:1:40.

[0201] 1.4.1.Hydrophilic uncharged moiety The hydrophilic, uncharged portion of the polymer may be or include a polyalkylene polyol or polyalkylene oxypolyol moiety, or a combination thereof. The hydrophilic, uncharged portion of the polymer may be or include a polyalkylene glycol or polyalkylene oxyglycol moiety. In certain embodiments, the polyalkylene glycol moiety is or includes a polyethylene glycol moiety and / or a monomethoxypolyethylene glycol moiety. In certain preferred embodiments, the uncharged portion of the polymer is or includes polyethylene glycol. The hydrophilic, uncharged portion of the polymer may be or include other biologically compatible polymer(s), such as polylactic acid.

[0202] In addition to PEG, several hydrophilic, uncharged entities are known in the art. See, for example, Lowe et al., Antibiofouling polymer interfaces: poly(ethyleneglycol) and other promising candidates, Polym. Chem., 6, 198-212, 2015, and Knop et al., Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives, Angewandte Chemie International Edition, 49(36), 6288-6308, 2010. Examples of hydrophilic, uncharged polymer moieties include, but are not limited to, poly(glycerol), poly(2-methacryloyloxyethyl phosphorylcholine), poly(sulfobetaine methacrylate), and poly(carboxybetaine methacrylate), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), and poly(vinylpyrrolidone).

[0203] The hydrophilic portion may have a weight average molecular weight of about 500 Da to about 50,000 Da. In some embodiments, the hydrophilic portion has a weight average molecular weight of about 1,000 Da to about 10,000 Da. In certain embodiments, the hydrophilic portion has a weight average molecular weight of about 1,500 Da to about 7,500 Da. In certain embodiments, the hydrophilic portion has a weight average molecular weight of about 3,000 Da to about 5,000 Da. In some cases, the hydrophilic portion has a weight average molecular weight of 5,000 Da or about 5,000 Da.

[0204] 1.4.2. Anionic polymer moiety The anionic polymer portion of the polymer may contain multiple functional groups that are negatively charged at physiological pH. A wide variety of anionic polymers are suitable for use in the methods and compositions described herein. However, such anionic polymers can be provided as components of polymers that have hydrophilic, uncharged polymer portions and are capable of forming (e.g., reversible) charge:charge complexes with positively charged (e.g., doubly) derivatized chitosan-nucleic acid nanoparticles.

[0205] Exemplary anionic polymers include, but are not limited to, polypeptides that have a net negative charge at physiological pH. In some cases, the polypeptide or a portion thereof is composed of amino acids with negatively charged side chains at physiological pH. For example, the anionic polymer portion of the polymer can be a polyglutamate polypeptide, a polyaspartate polypeptide, or a mixture thereof. Additional amino acids or mimetics thereof can be incorporated into the polyanionic polypeptide. For example, glycine and / or serine amino acids can be incorporated to increase flexibility or reduce secondary structure.

[0206] In some cases, the anionic polymer may be or include an anionic carbohydrate polymer. Exemplary anionic carbohydrate polymers include, but are not limited to, glycosaminoglycans that have a negative charge at physiological pH. Exemplary anionic glycosaminoglycans include, but are not limited to, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparin sulfate, hyaluronic acid, or a combination thereof. In certain embodiments, the anionic polymer portion of the polymer is or includes hyaluronic acid.

[0207] Additional or alternative anionic carbohydrate polymers may include polymers containing dextran sulfate.

[0208] In some cases, the polyanion moiety is or includes a polyanion selected from the group consisting of polymethacrylic acid and salts thereof, polyacrylic acid and salts thereof, copolymers of methacrylic acid and salts thereof, and copolymers of acrylic acid and / or methacrylic acid and salts thereof, e.g., polyalkylene oxide, polyacrylic acid copolymer.

[0209] In some cases, the polyanionic moiety is or includes a polyanion selected from the group consisting of alginate, carrageenan, furcellaran, pectin, xanthan, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, cellulose, oxidized cellulose, carboxymethylcellulose, croscarmellose, synthetic polymers and copolymers containing pendant carboxyl groups, phosphate or sulfate groups, predominantly negatively charged polyamino acids, and biocompatible polyphenolic materials.

[0210] The anionic portion of the polymer can have a weight average molecular weight of about 500 Da to about 5,000 Da. In some embodiments, the anionic portion has a weight average molecular weight of about 500 Da to about 3,000 Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 500 Da to about 2,500 Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 500 Da to about 2,000 Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 500 Da to about 1,500 Da. In some embodiments, the anionic portion has a weight average molecular weight of about 1,000 Da to about 5,000 Da. In some embodiments, the anionic portion has a weight average molecular weight of about 1,000 Da to about 3,000 Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 1,000 Da to about 2,500 Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 1,000 Da to about 2,000 Da. In some cases, the anionic portion has a weight average molecular weight of at or about 1,500 Da.

[0211] As used herein, "block copolymer," "block copolymer," and the like refer to a copolymer containing distinct homopolymer regions. A diblock copolymer contains two distinct homopolymer regions. A terpolymer contains three distinct homopolymer regions. Each of the three distinct regions can be different (e.g., AAAA-BBBB-CCCC), or two regions can be the same (e.g., AAAA-BBBB-AAAA), similar (e.g., AAAA-BBBB-AAA), and "A," "B," and "C" represent the different monomer subunits that form the copolymer. For example, "A" can represent the ethylene glycol monomer subunit of a polyethylene glycol homopolymer, and B can represent the glutamic acid subunit of a polyglutamic acid homopolymer. A block copolymer can be a linear (e.g., di- or tri-) block copolymer. Exemplary embodiments of linear diblock and triblock copolymers for use in the present invention include those listed in the following non-exhaustive list: [Table 2-1] [Table 2-2] *K: molecular weight of PEG in kDa Number of subunits

[0212] In one embodiment, the block copolymer is or comprises a PEG-polyglutamic acid polymer having the following structure: [ka]

[0213] In one embodiment, the block copolymer is or comprises a PEG-polyaspartic acid polymer having the following structure: [ka]

[0214] In one embodiment, the block copolymer is or comprises a PEG-hyaluronic acid polymer having the following structure: [ka]

[0215] 1.5. Alternative Cationic Polymers and Lipids The nucleic acid polyplexes of the present invention function to condense nucleotides and protect them from enzymatic degradation. In addition to chitosan and its derivatives, alternative materials that can also be advantageously used for this purpose include other positively charged (i.e., cationic) polymers and / or lipids.

[0216] Examples of cationic polymers that can form polyplexes with the therapeutic nucleic acid constructs of the present disclosure include polyamines; polyorganoamines (e.g., polyethyleneimine (PEI), polyethyleneimine cellulose, and their derivatives); poly(amidoamines) (PAMAM and its derivatives); polyamino acids (e.g., polylysine (PLL), polyarginine, and their derivatives); polysaccharides (e.g., cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-trialkylammonium), and Tat protein. See, e.g., Samal et al., Cationic polymers and their therapeutic potential, Chem Soc Rev. 41:7147-94 (2012).

[0217] Examples of positively charged lipids include esters of phosphatidic acid with amino alcohols, such as esters of dipalmitoylphosphatidic acid or distearoylphosphatidic acid with hydroxyethylenediamine. More specific examples of positively charged lipids include 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-chol); N,N'-dimethyl-N,N'-dioctasylammonium bromide (DDAB); N,N'-dimethyl-N,N'-dioctasylammonium chloride (DDAC); 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI); 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP); N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); dipalmitoylphosphatidylcholine (DPPC); 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP); and, for example, Martin et al., Current Pharmaceutical Design 2005, 11, 375-394.

[0218] Lipid and polymer blends of any concentration and ratio can also be used. Blending different polymer types in different ratios using different grades can result in properties borrowed from each of the contributing polymers. Different end group chemistries can also be employed.

[0219] 1.6.How to create As described above, those skilled in the art will appreciate that the polyplex:polymer particles of the present invention can be produced in a variety of ways. For example, polyplex particles can be produced and then contacted with a polymer. In an exemplary, non-limiting embodiment, polyplex particles are prepared by providing and combining functionalized chitosan and nucleotide raw materials. Raw material concentrations may be adjusted to accommodate various amino-to-phosphate (N / P) ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly for small batches (e.g., batches less than 2 mL), the functionalized chitosan and nucleotide raw materials may be mixed by gradually adding the nucleotide raw material dropwise to the functionalized chitosan raw material while vortexing the container. In other embodiments, the functionalized chitosan and nucleotide raw material may be mixed by in-line mixing of two fluid streams. In other embodiments, the resulting polyplex dispersion may be concentrated by means known in the art, such as ultrafiltration (e.g., tangential flow filtration (TFF)) or solvent evaporation (e.g., freeze-drying or spray-drying). A preferred method for polyplex formation is disclosed in WO2009 / 039657, which is expressly incorporated herein by reference in its entirety.

[0220] Similarly, a polyplex particle source (e.g., an aqueous solution containing a polyplex composition) can be provided (e.g., isolated from the reaction mixture described above) and mixed with a polymer source (e.g., an aqueous solution containing a polymer). The source concentrations can be adjusted to accommodate various amino-to-anion (N / A) ratios, amino-to-phosphorus (N:P) ratios, N:P:A ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly for small batches (e.g., batches less than 2 mL), the sources can be mixed by gradually adding a first source (e.g., polyplex) dropwise to a second source (e.g., polymer) while vortexing the vessel. In other embodiments, the sources can be mixed by in-line mixing of two fluid streams. In other embodiments, the resulting polyplex:polymer complex dispersion can be concentrated by means known in the art, such as ultrafiltration (e.g., tangential flow filtration (TFF)) or solvent evaporation (e.g., freeze-drying or spray-drying).

[0221] 2. Powder formulation The polyplex:polymer compositions of the invention comprise powders. In a preferred embodiment, the invention provides dry powder polyplex:polymer compositions. In a preferred embodiment, the dry powder polyplex:polymer compositions are produced by dehydration (e.g., spray drying or freeze drying) of chitosan-nucleic acid polyplex dispersions of the invention.

[0222] 3. Pharmaceutical preparations The present invention also provides "pharmaceutically acceptable" or "physiologically acceptable" formulations comprising the polyplex:polymer compositions of the present invention. Such formulations can be administered in vivo to a subject to carry out a method of treatment.

[0223] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients, etc., that can be administered to a subject, preferably without undue adverse side effects (e.g., nausea, abdominal pain, headache, etc.). Such preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Liquid formulations include suspensions, solutions, syrups, and elixirs. Liquid formulations may also be prepared by the reconstitution of a solid.

[0224] Pharmaceutical formulations can be made from carriers, diluents, excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with administration to a subject. Such formulations can be contained in tablets (coated or uncoated), capsules (hard or soft), microbeads, emulsions, powders, granules, crystals, suspensions, syrups, or elixirs. Supplementary active compounds and preservatives, among other additives, can also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

[0225] The excipient may include salts, isotonicity agents, serum proteins, buffers or other pH adjusters, antioxidants, viscosity-increasing agents, uncharged polymers, preservatives, or cryoprotectants. The excipients used in the compositions of the present invention may further include isotonicity agents and buffers or other pH-controlling agents. These excipients may be added to achieve a desired pH (about 6.0-8.0) and osmolality (about 50-400 mmol / L) range. Examples of suitable buffers are acetate, borate, carbonate, citrate, phosphate, and sulfonated organic molecule buffers. Such buffers may be present in the composition at a concentration of 0.01-1.0% (w / v). The isotonicity agent may be selected from those known in the art, such as mannitol, dextrose, glucose, and sodium chloride, or other electrolytes. Preferably, the isotonicity agent is glucose or sodium chloride. The isotonicity agent may be used in an amount that provides the composition with an osmotic pressure that is the same as or similar to that of the biological environment into which it is introduced. The concentration of the isotonicity agent in the composition depends on the nature of the specific agent used and may range from about 0.1 to 10%. When glucose is used, it is preferably used at a concentration of 1 to 5% w / v, more particularly 5% w / v. When the isotonicity agent is sodium chloride, it is preferably used in an amount of up to 1% w / v, particularly 0.9% w / v. The compositions of the present invention may further contain a preservative. Examples of preservatives include polyhexamethylene-biguanidine, benzalkonium chloride, stable oxychloro complexes (known as Purite®), phenylacetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and thimerosal. Typically, such preservatives are present at a concentration of about 0.001 to 1.0%. Additionally, the compositions of the present invention may also contain a cryopreservative. Preferred cryopreservatives are dextran, glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, glycerol and polyethylene glycol with a molecular weight of less than 100,000 g / mol, or mixtures thereof, most preferably glucose, trehalose, and polyethylene glycol.Typically, such cryopreservatives are present at a concentration of about 0.01-10%.

[0226] Pharmaceutical preparations can be formulated to be compatible with the intended route of administration. For example, for oral administration, the composition can be incorporated with excipients and used in the form of tablets, lozenges, capsules, e.g., gelatin capsules, or coatings, e.g., enteric coatings (Eudragit® or Sureteric®). Pharmaceutically compatible binders and / or adjuvants can be included in oral preparations. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or other stearates; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or flavorings.

[0227] The formulation may also include a carrier that protects the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone or in combination with a wax may be employed.

[0228] Suppositories and other rectally administrable formulations (e.g., those that can be administered by enema) are also contemplated. Further, regarding rectal delivery, see, e.g., Song et al., "Mucosal drug delivery: membranes, methodologies, and applications," Crit. Rev. Ther. Drug. Carrier Syst., 21:195-256, 2004; Wearley, "Recent progress in protein and peptide delivery by noninvasive routes," Crit. Rev. Ther. Drug. Carrier Syst., 8:331-394, 1991.

[0229] Additional pharmaceutical formulations suitable for administration are known in the art and are applicable to the methods and compositions of the present invention (e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)).

[0230] 4. Administration In one embodiment, the use of polyplex:polymer compositions provides long-term stability of the polyplexes at physiological pH, which provides for effective mucosal administration.

[0231] Any of a number of administration routes to contact mucosal cells or tissues are possible, and the choice of a particular route will depend in part on the target mucosal cells or tissues. Syringes, endoscopes, cannulas, intubation tubes, catheters, nebulizers, inhalers, and other items may be used for administration.

[0232] Intravesical administration of chemotherapy drugs is the standard treatment for some bladder cancers. Briefly, intravesical therapy involves instilling a therapeutic agent directly into the bladder via insertion of a urethral catheter. In some embodiments, the composition enhances stability in urine, thereby improving local expression.

[0233] A dose or "effective amount" for treating a subject is preferably sufficient to improve one, some, or all of the symptoms of a condition to a measurable or detectable extent. However, prevention or suppression of progression or worsening of a disorder or condition or symptom is a satisfactory outcome. Thus, for conditions or disorders treatable by expressing a therapeutic nucleic acid in a target tissue, the amount of therapeutic RNA or therapeutic protein produced to improve a treatable condition by the methods of the invention will depend on the condition and the desired outcome and can be readily ascertained by one of skill in the art. The appropriate amount will depend on the condition being treated, the desired therapeutic effect, and the individual subject (e.g., bioavailability within the subject, gender, age, etc.). An effective amount can be ascertained by measuring the relevant physiological effect.

[0234] Veterinary applications are also contemplated by the present invention. Accordingly, in one embodiment, the present invention provides a method of treating a non-human mammal, comprising administering a polyplex:polymer composition of the present invention to a non-human mammal in need of treatment. The compositions of the present invention may also be administered to mucosal membranes. For example, the compositions can be administered to mucosal cells or tissues of the gastrointestinal tract, including, but not limited to, mucosal cells or tissues of the small intestine and / or large intestine. Other target mucosal cells or tissues include, but are not limited to, cells or tissues of the eye, respiratory epithelium, lung, vagina, and bladder.

[0235] Representative formulations for this purpose include solutions, gels, hydrogels, solutions, creams, foams, films, implants, sponges, fibers, powders, and microemulsions.

[0236] The compounds of the invention can be administered to mucous membranes intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (alone, in a mixture, e.g., in a dry blend with lactose, or as mixed component particles), with or without the use of a suitable propellant, or as an aerosol spray mist from a pressurized container, pump, spray, atomizer, or nebulizer.

[0237] Capsules, effervescent agents, and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch, and a performance modifier such as l-leucine, mannitol, or magnesium stearate.

[0238] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release formulations.

[0239] The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, although various alternatives may be used as appropriate.

[0240] Formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0241] The compounds of the present invention may also be administered directly to the eye or ear, usually in the form of drops. Other formulations suitable for ocular and otic administration include ointments, biodegradable (e.g., absorbent gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate systems. The formulations may also be delivered by iontophoresis.

[0242] Formulations for ocular / aural administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed-release.

[0243] Mucosal administration In a preferred embodiment, the compositions of the present invention are administered to mucosal membranes. For example, the compositions can be administered to mucosal cells or tissues of the bladder and gastrointestinal tract, including, but not limited to, mucosal cells or tissues of the small intestine and / or large intestine and / or colon. Other target mucosal cells or tissues include, but are not limited to, cells or tissues of the eye, respiratory epithelium, lung, vagina, and bladder.

[0244] Representative formulations for this purpose include solutions, gels, hydrogels, solutions, creams, foams, films, implants, sponges, fibers, powders, and microemulsions.

[0245] In an exemplary embodiment of the bladder mucosa, the compounds described herein can be administered using intravesical therapy. Intravesical therapy involves instilling a therapeutic agent directly into the bladder via insertion of a urethral catheter. The agent is left in the bladder for a period of 0.5 to 6 hours. This is the standard route of administration for bladder cancer chemotherapy. It utilizes available external anatomical access for drug delivery directly to the diseased site of the bladder, thereby avoiding unwanted exposure of the instilled drug to healthy tissue elsewhere in the body.

[0246] Formulations for bladder administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed-release.

[0247] The compounds of the invention may also be administered to mucous membranes intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (alone, in a mixture, e.g., in a dry blend with lactose, or as mixed component particles) with or without the use of a suitable propellant, or as an aerosol spray mist from a pressurized container, pump, spray, atomizer, or nebulizer.

[0248] Capsules, effervescent agents, and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch, and a performance modifier such as l-leucine, mannitol, or magnesium stearate.

[0249] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release formulations.

[0250] The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, although various alternatives may be used as appropriate.

[0251] Formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0252] The compounds of the present invention may also be administered directly to the eye or ear, usually in the form of drops. Other formulations suitable for ocular and otic administration include ointments, biodegradable (e.g., absorbent gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate systems. The formulations may also be delivered by iontophoresis.

[0253] Formulations for ocular / aural administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed-release.

[0254] Therapeutic applications Therapeutic proteins contemplated for use in the present invention have a wide variety of activities and find use in treating a wide variety of disorders. The following description of the activity of the therapeutic proteins of the present invention and the indications treatable with the therapeutic proteins are exemplary and not intended to be exhaustive. The term "subject" refers to an animal, preferably a mammal, with humans being particularly preferred. Specific, non-limiting examples of therapeutic embodiments are described below. In some cases, therapeutic embodiments are intended to affect non-mucosal target tissues, cells, or organs. When the therapeutic effect is non-mucosal, it is understood that the cells or tissues contacted by the polyplex:polymer compositions described herein are mucosal, and the therapeutic effect is proximal to the mucosal target. For example, mucosal cells can be transfected to produce and secrete IL-12 and / or another immunostimulatory molecule.

[0255] In one embodiment, the polyplex:polymer compositions of the invention may be used for therapeutic treatments. Such compositions are sometimes referred to herein as therapeutic compositions. As described above, the present compositions and methods primarily employ therapeutic nucleic acids encoding IL-12, alone or in combination with additional innate and / or adaptive immune stimulatory molecules. In some embodiments, the therapeutic nucleic acid further encodes an IFN-I activator / inducer, such as, for example, a RIG-I agonist, a STING agonist, a TLR7 / 9 agonist, and / or other pattern recognition receptor agonists. See, e.g., Vasou et al., Viruses 9:186 (2017). In some embodiments, the therapeutic nucleic acid further encodes a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.

[0256] Suitable IFN-I activators / inducers include RIG-I agonists (e.g., eRNA11a, adenovirus VA RNA1, eRNA41H, MK4621 (Merck), SLR10, SLR14, and SLR20), STING (i.e., stimulators of interferon genes) agonists (e.g., CDNs, i.e., cyclic dinucleotides), PRRago (e.g., CpG, Imiquimod, or Poly I:C), and TLR agonists (e.g., CPG-1826, GS-9620, AED-1419, CYT-003-QbG10, AVE-0675, or PF-7909), including TLR7 and TLR9, and RLR stimulators (e.g., RIG-I, Mda5, or LGP2 stimulators). In some embodiments, the IFN-1 activator / inducer induces dendritic cells, T cells, B cells, and / or T follicular helper cells.

[0257] In a preferred embodiment, the IFN-I activator / inducer is a RIG-I agonist. RIG-I (retinoic acid-inducible gene I, encoded by Ddx58) is a cytosolic antiviral helicase that acts as an RNA sensor, detecting and activating viral RNA in the cytoplasm. RIG-I, a pattern recognition receptor, contains an RNA helicase domain and two N-terminal caspase recruitment domains (CARDs), which transmit signals to the downstream signaling adaptor MAVS (mitochondrial antiviral signaling protein). RIG-I signaling via MAVS leads to various responses, including the induction of type I IFN responses, including IFNα and IFNβ, via TBK1 and IRF7 / 8, and the activation of caspase-8-dependent apoptosis. They are found in most tissues, including cancer cells (Kato et al., Immunol. Rev. 243(1):91-98 (2011)).

[0258] The RIG-I induced response differs between cells. Normal healthy cells, such as melanocytes and fibroblasts, are highly resistant to RIG-I-induced apoptosis, whereas tumor cells are highly susceptible to RIG-I-induced cell death (Besch et al., 2009; Kubler et al., 2010). The natural ligand of RIG-I is a short, blunt-ended viral double-stranded RNA containing a 5' triphosphate or diphosphate (5'ppp or 5'pp). RIG-I-specific ligands are currently being developed for cancer immunotherapy (Duewell et al., 2014, 2015; Ellermeier et al., 2013; Schnurr & Duewell, Oncoimmunology, 2(5):e24170(2013) and, 2014). Part of the potent antitumor activity of RIG-I ligands is due to their binding to CD8 + The downstream ability to promote antigen cross-presentation to T cells and induce cytotoxic activity (Hochheiser et al., 2016). RIG-I ligands also show potent therapeutic activity in viral infection models such as influenza (Weber-Gerlach & Weber, 2016).

[0259] Plasmid vector backbones expressing RIG-I ligands from an RNA polymerase III promoter have been used to identify potent synthetic RIG-I ligands (Luke et al., J. Virol. 85(3):1370-1383). Triphosphate-modified stem-loop RNAs are particularly useful as agonists in the present invention. These include, but are not limited to, eRNA41H, which combines (i) eRNA11a, an immunostimulatory dsRNA expressed by convergent transcription, with (ii) adenovirus VA RNAI, SLR20, a double-stranded, triphosphorylated 20 base pair stem-loop RNA modified with a 5' triphosphate sequence (Elion et al., Cancer Res. 78(21):6183-6195 (2018)), and SLR10 and SLR14, polyphosphorylated RNAs containing a stable tetraloop at one end (Jiang et al., J. Exp. Med. 216:2854-68 (2019)).

[0260] Additional RIG-I agonists that find advantageous use in the compositions and methods described herein include SB-9200, a broad-spectrum antiviral innate sensor agonist that acts through activation of RIG-I and the nucleotide-binding oligomerization domain 2 pathway (Jones et al. J. Med. Virol. 89:1620-1628 (2017)), MK4621 (RGT100, Merck), CBS-13-BPS, a synthetic RIG-I-specific agonist that mimics the structure of the influenza virus panhandle promoter (Lee et al. Nucleic Acids Res. 46:10553 (2018)); IVT-B2 RNA (Lien et al. Molecular Therapy 24:135-45 (2016)), SeV DVGs (Xu et al., mBio 65:e01265-15(2015)), 5'ppp RNA (M8) with a uridine-rich sequence with a 99-nucleotide hairpin (Chiang et al. J. Virol. 89:8011-25(2015)), and 3pRNA.

[0261] According to the above-described embodiments, RIG-I agonists suitable for co-expression with IL-12 in the present compositions and methods include, but are not limited to, RIG-I DNA vaccines, RNA polymerase III-expressed RNA-based RIG-I agonists encoded on a plasmid, such as those disclosed in Ellermeier et al., Cancer Research (2013) 73(6), RIG-I DNA, such as eRNA11a, adenovirus VA RNA1, eRNA41H (Nature Technology Corp), GFP2, Lamin A / C and Lamin VSV, tri-GFP, SAD ΔPLp, Tri-G-AC-U, Flu vRNA, RNaseL fragment, pppRVL, pppVSVL, ppp-shRNA-luc3VA1, 5'ppp-dsRNA, 3p-hpRNA, MK4621 (Merck), SLR10, SLR14, SLR20, CBS-13-BPS, IVT-B2 RNA, SeV CVG, SB-9200, and siRNA. Similarly, STING agonists suitable for co-expression with IL-12 include, but are not limited to, DExD / H helicases, including DDX41, and TLR agonists include, but are not limited to, CpG dinucleotides, such as, for example, CpG-1826 (ODN1826, Invivogen).

[0262] According to the above-described embodiments, modulators of immune checkpoint molecules suitable for co-expression with IL-12 in the present compositions and methods include, for example, single domain antibodies (sdAbs) directed against one or more of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, and KIR (e.g., KN035 (Ablynx / Sanofi); Inhibrix 105) (e.g., Wan et al., Oncol. Rep. (2018); Hosseinzadeh et al., Rep. Biochem & Mol. Bio., (2017); Dougan et al., Can. Imm. Res. (2016); Ingram et al., PNAS (2018), and WO2017198212); dominant-negative PD-1 molecules (e.g., Atara Therapeutics), PD-1 mutants with high affinity for PD-L1 (e.g., competitive antagonists) (Maute, PNAS (2015)); and CD80 mutant(s) with increased binding to CD28 (see also, e.g., WO2017 / 181152).

[0263] In some cases, the IFN-1 agonist and / or the immune checkpoint inhibitor is encoded by: the therapeutic nucleic acid construct in the derivatized chitosan nucleic acid polyplex; - different therapeutic nucleic acid constructs in the derivatized chitosan-nucleic acid polyplexes, - therapeutic nucleic acid constructs (e.g., not including a construct encoding IL-12) in different derivatized chitosan nucleic acid polyplexes, - Therapeutic nucleic acid constructs (e.g., formulated in alternative nucleic acid delivery formulations such as PEI or cationic lipid formulations).

[0264] The therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding the IFN-I agonist and / or the immune checkpoint inhibitor can be administered simultaneously or sequentially. In some cases, the therapeutic nucleic acid constructs encoding the IFN-I agonist and / or the immune checkpoint inhibitor are co-administered in a single formulation or a combination of two different formulations, e.g., mixed together. In some cases, the therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding the IFN-I agonist and / or the immune checkpoint inhibitor are administered sequentially.

[0265] The immunostimulatory molecules of the present invention may also encode shRNA (short hairpin RNA) molecules designed to suppress a protein(s) involved in the growth or maintenance of tumor cells or other hyperproliferative cells. Plasmid DNA may simultaneously encode a therapeutic protein and one or more shRNAs. Furthermore, the nucleic acid of the composition may also be a mixture of plasmid DNA and synthetic RNA, including sense RNA, antisense RNA, or ribozymes.

[0266] Treatment method Hyperproliferative disorders The present compositions and methods are advantageously used in the treatment of hyperproliferative disorders. Of particular interest are compositions and methods for treating hyperproliferative disorders of mucosal tissues or tissues adjacent to mucosal tissues. The methods and compositions of the present invention may be used to treat gastrointestinal cancers, including, but not limited to, oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, colorectal cancer, and rectal cancer. Nasal cancer and lung cancer that can be treated by the methods and compositions of the present invention include, but are not limited to, sinonasal cancer, oropharyngeal cancer, tracheal cancer, and lung cancer. Genitourinary cancer that can be treated by the methods and compositions of the present invention include, but are not limited to, bladder cancer, urothelial cancer, urethral cancer, testicular cancer, kidney cancer, prostate cancer, penile cancer, adrenal cancer, uterine cancer, cervical cancer, and ovarian cancer.

[0267] In some embodiments according to any one of the methods provided above, the method further comprises administering (e.g., systemically or locally to the site of the tumor) a non-nucleic acid-based immune stimulatory molecule.

[0268] In some embodiments, the immunostimulatory molecule is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is a PD-L1 or PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody such as pembrolizumab or nivolumab. In some embodiments, the immunomodulatory agent is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody such as ipilimumab or tremelimumab. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody such as atezolizumab.

[0269] In some embodiments, the immunomodulatory agent is an IFN-I agonist, e.g., a RIG-I agonist, a STING agonist, or a TLR7 / 9 agonist. Suitable RIG-I agonists for co-administration include short poly I:C and poly AU compositions (e.g., poly(I:C) / LyoVec complexes (Invivogen)); RGT100 (MK4621, Merck); SLR20 (Elion et al.); SLR10 & SLR14 (Jiang et al.) al.); and US8871799, US8895608, US8927561, US9,073,946, US9458492, US9555106, US9884876, US9956285, US9775894, US9861574, US9937247, US10167476, US10350158, US10434064, US10273484, US9381208B2, US9738680B2, US9790509, US10059943, US9109012B2, US US9937247B2, US9816091B2, US9133456B2, US9409941B2, US9340789B2, US9040234B2, US20200071316, US20200063141A1, US20200061097A1, US20200055871A1, US20200016253A1, US20190076463A1, US20180195063A1, US20160287623A1.

[0270] Suitable STING agonists for co-administration with IL-12 include c-Di-AMP sodium salt, c-Di-GMP sodium salt, 2',3'-cGAMP sodium salt, 3',3'-cGAMP sodium salt, 10-carboxymethyl-9-acridanone (CMA), DMXAA (Tocris Bioscience, InvivoGen, Nimbus Therapeutics), G10, α-Mangostin, CRD100 (Curadev), cAIMP, 2'2'-c-GAMP, 2'3'-cGAM(PS)2 (Rp / Sp), 2'3'-c-di-AMP, c-di-IMP, c-di-UMP, 5,6-dimethylxanthene-4-acetic acid (DMXAA), MK-1454 (Merck), MLRR-S2 CDG, and MLRR-S2. CDA (ADU-S100), SB11285 (Springbank Pharmaceuticals), MAVU (AbbVie), DiABZI, disodium dithio-(Rp1Rp)-[cyclic[A(2'5')pA(3'5')p]][Rp,Rp]-cyclic adenosine-(2'5')-monophosphorothioate-adenosine-(3'5')-monophosphorothioate), disodium (RR-S2 CDA, ADU-S100, MIW815) (Corrales et al. al., 2016), and the compositions disclosed in US 10,176,292, US 9,724,408, US 10,011,630, US 10,435,469, US 10,414,747, US 10,413,612, US 10,131,686, US 10,106,574, US 10,047,115, US 10,045,961, US 10,011,630, US 9,994,607, US 9,937,247, US 9,840,533, US 9,770,467, US 9,724,408, US 9,718,848, and US 9,642,830.

[0271] Suitable TLR7 and TLR9 agonists for co-administration with IL-12 include imidazoquinolines and their analogs, including resiquimod and imiquimod (Aldara), hydroxychloroquine, chloroquinoline, bropirimine, loxoribine, isatoribine, CpG oligonucleotides, stabilized immunomodulatory RNA (SIMRA) AST-008 (Exicure), MEDI9197, and US 434,064, US 10,413,612, US 10,407,431, US 10,370,34 2, US 10,364,266, US 10,208,037, US 10,202,386, US 9,944,649, US 9,902,730, US 9,868,955, US 9,359,360, US 9,295,732, US 9,243,050, US 9,228,184, US 9,216,192, US 9,2206,430, US 8,735,421, US 8,728,486, US 8,399,423, and US 8,242,106.

[0272] In some embodiments, the non-nucleic acid based immunomodulatory agent and the composition are administered simultaneously, e.g., in the same composition. In some embodiments, the non-nucleic acid based immunomodulatory agent and the composition are administered sequentially.

[0273] In some embodiments, the methods for treating bladder cancer provided herein further comprise administering at least one additional therapeutic agent to the subject. In further embodiments, the additional therapeutic agent is a chemotherapy or radiation therapy agent. In some embodiments, chemotherapy agents include, but are not limited to, cisplatin, carboplatin, paclitaxel, docetaxel, 5-fluorouracil, bleomycin, methotrexate, ifosfamide, oxaliplatin, cyclophosphamide, dacarbazine, temozolomide, gemcitabine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, pentostatin, thioguanine, daunorubicin, doxorubicin, epirubicin, idarubicin, topotecan, irinotecan, etoposide, eniposide, colchicine, vincristine, vinblastine, and vinorelbine. Exemplary cancer-specific drugs and antibodies include afatinib, aldesleukin, alemtuzumab, axitinib, belimumab, bevacizumab, bortezomib, bosutinib, brentuximab vedotin, cabozantinib, canakinumab, carfilzomib, cetuximab, crizotinib, dabrafenib, dasatinib, denosumab, erlotinib, everolimus, gefitinib, ibritumomab tiuxetan, ibrutinib, imatinib, ipilimumab, lapatinib, nilotinib, and the like. Examples of additional therapeutic agents include, but are not limited to, rituximab, romidepsin, ruxolitinib, sipuleucel-T, sorafenib, temsirolimus, tocilizumab, tofacitinib, tositumomab, trametinib, trastuzumab, vandetanib, vemurafenib, vismodegib, vorinostat, zib-aflibercept, and any combination thereof. In some embodiments, the additional therapeutic agent is administered to the subject before, simultaneously with, or after administration of the immunoconjugate. In some embodiments, the additional therapeutic agent is administered systemically. For example, in some embodiments, the additional therapeutic agent is administered by intravenous injection.

[0274] Furthermore, the conventional bladder cancer treatment currently approved in the United States is the intraurethral Bacillus Calmette-Guérin vaccine. This antigenic vaccine is thought to stimulate bladder cells to express interferon, which in turn mobilizes the patient's innate immune system to better recognize cancer cell surface antigens and attack the cancer cells. However, in more than one-third of cases, the vaccine is ineffective. Similarly, intravesical instillation of exogenously produced interferon polypeptides has also been tested but has been ineffective. The present compositions and methods can also be advantageously used in combination with these more conventional approaches to enhance and improve immune responses.

[0275] The examples described herein illustrate some embodiments of the present disclosure, but should not be construed as limiting the scope of the disclosure in any way. [Example]

[0276] Example 1 Measurement of plasmid transfection efficiency in vitro.

[0277] MB49 cells were seeded in 96-well plates (35,000 cells / well) before transfection with the NTC9385-Luc2, gWiz-Luc2, or pVax-Luc2 plasmids. Transfections were performed using Lipofectamine 2000 (Thermofisher) and increasing doses of plasmid DNA (20–300 ng). 24 h after transfection, cells were lysed with Luciferase Cell Culture Lysis Reagent (IX, Promega). Immediately after adding luciferin enzyme substrate to the cell lysate, bioluminescence was measured using an Envision plate reader (Perkin-Elmer). As seen in Figure 1, cells transfected with NTC9358R showed the highest in vitro transfection efficiency.

[0278] Example 2 Measurement of plasmid transfection efficiency in vivo. JetPEI-plasmid DNA polyplexes were prepared by mixing JetPEI and 20 μg of candidate plasmid containing optimized Luc2 with an amine-to-phosphorus (NP) ratio of 6. The polyplexes were incubated at room temperature for a minimum of 15 minutes and used within 4 hours. Female mice (12-16 weeks) were anesthetized with isoflurane and administered 80 μl of JetPEI-DNA formulations via intravesical instillation with a 60-minute exposure time. 24 hours after administration, bladder tissue was harvested, and transfection efficacy was assessed using mRNA expression and luciferase enzyme activity assays. RNA was extracted from the harvested bladder tissue after homogenization in lysis buffer (Qiagen RNeasy). RT-qPCR was performed using 1 μg of input RNA and a TaqMan primer / probe recognizing Luc2. Absolute quantification was performed using a standard curve of Luc2 RNA. As seen in Figure 2A, the results of the RNA assay were consistent with those of the in vitro luciferase activity assay, with NTC9385Luc2 exhibiting the highest mRNA levels after transfection based on absolute copy number. For the luciferase assay, harvested bladder tissue was lysed by homogenization in the presence of Luciferase Cell Culture Lysis Reagent (IX, Promega). The enzyme substrate, luciferin, was added to the tissue lysate, and bioluminescence was immediately measured using an EnVision plate reader (Perkin-Elmer). As seen in Figure 2B, both the gWIZ-Luc2 and NTC9385Luc2 plasmids exhibited significantly higher luciferase enzyme activity in vivo than the pVAX-Luc2 plasmid.

[0279] To evaluate the efficacy of candidate plasmids in delivering genes of interest in vivo, transfection and post-transfection mRNA and protein expression assays were performed using the plasmid carrying hPD-L1-Fc. For RT-qPCR assays, bladder tissues were collected 24 hours after administration, homogenized in lysis buffer, and RNA was extracted (Qiagen RNeasy kit). RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes recognizing codon-optimized human PD-L1-Fc. Absolute quantification was performed using a standard curve of human PD-L1-Fc RNA. No statistically significant differences in hPD-L1-Fc mRNA expression were observed using the three different vectors 24 hours after administration (Figure 2C). For hPD-L1-Fc protein expression, bladder tissues were collected 24 hours after administration and lysed by homogenization in lysis buffer containing protease inhibitors. Human PD-L1-Fc protein was quantified using a custom-designed immunoassay (Mesoscale Discovery). Data are expressed as pg / mL of protein in lysate. As can be seen in Figure 2D, NTC9385R showed the least variable expression, with only the plasmid providing quantifiable hPDL1-Fc in all mice tested.

[0280] Example 3 In vitro promoter / enhancer screening in mouse bladder epithelial cell lines The efficacy of promoter / enhancer combinations was assessed by measuring dose-dependent expression of a green fluorescent reporter gene (GFP) after in vitro transfection of a mouse urothelial cell line with a panel of plasmids containing various promoter / enhancer sequences. The promoter / enhancer sequences used in the assay were CAG, EF1a, CMV / EF1a / HTLV, CMV / UbC, EF1a / HTLV, 2xCMV / EF1a, CMV, UbC, CMV / EF1a, CMV / UbB, PGK, UbB, and CBA. MB49 cells (mouse bladder cells) were seeded in 96-well plates (35,000 cells / well) 24 h before transfection with the indicated plasmids in the SnapFast backbone (pSF).

[0281] Transfections were performed using Lipofectamine 2000 (Thermofisher) and increasing doses of plasmid DNA. Fluorescence was measured 48 hours after transfection using an Envision plate reader. Cell viability was measured using AlamarBlue Cell Viability Reagent (Invitrogen). Data are expressed as GFP relative fluorescence units (RFU) normalized to cell viability (Figure 3A). Total fluorescence intensity was ranked for each of the plasmids across four independent experiments (Figure 3B). High levels of expression were observed with plasmids containing CAG, EF1a, and CMV-based promoters / enhancers.

[0282] Example 4 Promoter / enhancer screening in primary human in vitro cells (HBIEpC) The efficacy of promoter / enhancer combinations was evaluated by measuring the dose-dependent expression of the green fluorescent reporter gene (GFP) after in vitro transfection of human primary bladder epithelial cells. Human primary bladder epithelial cells (ATCC) were seeded in 96-well plates (25,000 cells / well) 24 h before transfection with plasmids containing alternative promoter / enhancer combinations in the SnapFast backbone (pSF). The promoter / enhancer combinations studied included CAG, UbB, EF1a, CMV-EF1a-HTLV, 2XCEF, PGK, CBA, CMV-Ubb, EF1a-HTLV, CMV, CEF, and UBC. Transfections were performed using Avalanche Transfection Reagent (EZ Biosystems) and increasing doses of plasmid DNA. Fluorescence was measured 24 h posttransfection using an Envision plate reader. Cell viability was measured using AlamarBlue Cell Viability Reagent (Invitrogen). Data are expressed as GFP relative fluorescence units (RFU) normalized to cell viability. As seen in Figure 4A, promoter / enhancer combinations that performed well in the MB49 cell line also exhibited high levels of expression in human primary cells. Similarly, low expressers in the MB49 cell line also exhibited low expression in primary cells. The total fluorescence intensity for each of the plasmids across two independent assays was ranked (Figure 4B). Plasmids containing EF1a / HTLV, CAG, and 2xCMV / EF1a exhibited the highest levels of expression, while plasmids containing the UBB and PGK promoter / enhancers exhibited the lowest expression in human bladder cells.

[0283] Example 5 Measurement of remodeled plasmids in primary human bladder epithelial cells A fluorescence-based assay was used to evaluate the transfection efficacy of modified ("improved") plasmids that were engineered to remove bacterial components. Briefly, dose-dependent expression of the green fluorescent reporter gene (GFP) was measured after in vitro transfection of human primary bladder epithelial cells with a panel of plasmids modified to remove bacterial components. Human primary bladder epithelial cells (Cell Applications) were seeded in 96-well plates (25,000 cells / well) 24 h before transfection with the indicated plasmids (Nature Technology Corp). Transfection was performed using Avalanche Transfection Reagent (EZ Biosystems) and increasing doses of plasmid DNA. Fluorescence was measured 24 h posttransfection using an Envision plate reader. Cell viability was measured using AlamarBlue Cell Viability Reagent (Invitrogen). Data are expressed as GFP relative fluorescence units (RFU) normalized to cell viability (Figure 5A). Total fluorescence intensity was ranked for each of the plasmids across two independent experiments (Figure 5B). The NP plasmid was the highest expressor in human primary bladder epithelial cells, while pVAX and gWIZ (unmodified) were the lowest expressors. The NTC9385R plasmid showed the highest expression level of the assayed plasmids.

[0284] Example 6 Effect of PEGylation on In Vivo Bladder Transfection Efficiency with and without Polyplex Urination After an Incubation Period Human PD-L1-Fc mRNA expression was assayed after administration of DDX and PEGylated DDX formulations to mouse bladders. Polyplexes were prepared at NPA ratios of 7:1:17.5 (PEG-DDX), 7:1:7 (PEG-DDX), or 7:1:0 (DDX control). Female mice (12–16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplex (20 μg of plasmid DNA) by intravesical instillation (exposure time = 60 min). After polyplex exposure and before recovery from anesthesia, the bladder was either voided (using a syringe and catheter through the urethra) or not. 24 hours after administration, bladder tissue was harvested and homogenized in lysis buffer, followed by RNA extraction (Qiagen RNeasy kit). RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes that recognize codon-optimized human PD-L1-Fc. Absolute quantification was performed using a standard curve of human PD-L1-Fc RNA. Data are expressed as RNA copy numbers (Figure 6).

[0285] Example 7 Evaluation of in vivo mRNA expression using PEG vs. non-PEG at various plasmid concentrations DDX and PEG-DDX polyplexes containing human PD-L1-Fc were prepared at an NPA ratio of 7:1:9 (PEG-DDX) or 7:1:0 (DDX). Female mice (12–16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplexes (0.25 mg DNA / mL = 20 μg plasmid DNA; 1.0 mg DNA / mL = 80 μg plasmid DNA) via intravesical instillation (exposure time = 60 min). 24 hours after administration, bladder tissue was harvested, homogenized in lysis buffer, and RNA was extracted (Qiagen RNeasy kit). RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes recognizing codon-optimized human PD-L1-Fc. Absolute quantification was performed using a human PD-L1-Fc RNA standard curve. Data are expressed as RNA copy numbers (Figure 7A). In the second experiment, the indicated polyplexes were prepared at an NPA ratio of 7:1:3.5 (PEG-DDX) or 7:1:0 (DDX control). Female mice (12–16 weeks) underwent the same regimen: under isoflurane anesthesia, 80 μL of polyplexes (0.25 mg DNA / mL = 20 μg plasmid DNA; 1.0 mg DNA / mL = 80 μg plasmid DNA) were administered via intravesical instillation (exposure time = 60 min). 24 hours after administration, bladder tissue was harvested, homogenized in lysis buffer, and RNA was extracted (Qiagen RNeasy kit). RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes recognizing codon-optimized human PD-L1-Fc. Absolute quantification was performed using a standard curve of human PD-L1-Fc RNA. Data are expressed as RNA copy numbers (Figure 7B). Higher expression was seen in animals receiving 1.0 mg / DNA / ml than in those receiving 0.25 mg / ml. PEGylation did not decrease mRNA expression in the bladder 24 hours after administration compared to the non-PEGylated formulation.

[0286] Example 8 Expression of human PD-L1-Fc in the mouse bladder after administration of a PEGylated DDX formulation PEG-DDX polyplexes were prepared at an NPA ratio of 7:1:3.5. Female mice (12-16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplexes (0.125 mg DNA / mL = 10 μg plasmid DNA) via intravesical instillation (exposure time = 60 min). Forty-eight hours after administration, bladder tissue was harvested and lysed by homogenization in the presence of protein lysis buffer and protease inhibitors. Human PD-L1-Fc protein was quantified using a custom-designed immunoassay (Mesoscale Discovery). Data are expressed as pg / mL of protein in the lysate (Figure 8). Intravesical instillation of PEG-DDX™ polyplexes resulted in robust, quantifiable protein expression in the bladder.

[0287] Example 9 Effect of PEGylation on polyplex stability in urine within the mouse bladder. 80 μL of non-PEGylated (NPA 7:1:0) and PEGylated (NPA 7:1:7 and 7:1:17.5) DDX / DNA polyplex formulations were administered to mouse bladders at 0.25 mg of DNA / mL (n=4). The formulations were incubated in the bladder for 1 hour before bladder voiding (collecting the bladder contents) for analysis. Samples were examined for appearance (Figure 9A) and nanoparticle sizing by dynamic light scattering (Figure 9B). Non-PEGylated polyplexes aggregated significantly after voiding, resulting in visible white clots, compared to PEGylated polyplexes, which showed no aggregation.

[0288] Example 10 The effect of DDX-I versus DDX-II on hPD-L1-Fc production in vivo Female mice (12–16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplex (1.0 mg DNA / mL = 80 μg plasmid DNA) via intravesical instillation (exposure time = 60 min). Forty-eight hours after administration, bladder tissue was harvested and lysed by homogenization in the presence of protein lysis buffer containing protease inhibitors. Human PD-L1-Fc protein was quantified using a custom-designed immunoassay (Mesoscale Discovery). Data are expressed as pg / mL protein in the lysate. Data are mean ± SD; *p<0.05; **p<0.005—One-way ANOVA with Kruskal-Wallis test. Polyplexes were prepared at a 10:1 or 30:1 NP ratio (as indicated) and a DNA concentration of 1.0 mg / mL. The composition of DDX-I was 14% R and 3% GA. The composition of DDX-II(RXG) was 13% R / 13% G or 28% R / 9% G (as shown). As shown in Figure 10, DDX-II(RXG) results in significantly higher protein expression in vivo than DDX-I.

[0289] Example 11 Kinetics of protein expression after administration of RXG preparations to the mouse bladder Polyplexes were prepared at an NPA ratio of 10:1:5 (PEGylated) or 20:1:0 (non-PEGylated). PEGylated formulations were made with 5% trehalose and 5% mannitol. Non-PEGylated formulations were made with 5% trehalose. The %R and %G RXG (DDX-II) compositions are shown. Female mice (12-16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplex (1.0 mg DNA / mL = 80 μg plasmid DNA) via intravesical instillation (exposure time = 60 min). Bladder tissue was collected at 24, 48, 72, and 96 hours post-administration and lysed by homogenization in protein lysis buffer containing protease inhibitors. Human PD-L1-Fc protein was quantified using a custom-designed immunoassay (Mesoscale Discovery). Data are expressed as pg / mL of protein in lysate (Figure 11). Protein expression is high (in the ng / ml range) and persists up to 96 hours after administration.

[0290] Example 12 Plasmid DNA constructs containing murine IL-12 with and without a RIG-I agonist cassette Plasmid constructs containing murine IL-12 without the RIG-I agonist cassette (Figure 12A) and with the RIG-I agonist cassette (Figure 12B). The murine IL-12 transgene contains a single open reading frame for the IL-12 p40 and p35 subunit genes, including a short elastin linker (Figure 12A). HEK293T cells were transfected with the plasmid containing the mIL-12 transgene, and supernatants were harvested 48 hours posttransfection. Murine IL-12p40p35 in the cell supernatants was quantified by immunoassay. The constructs produced bioactive IL-12 (Figure 13A). Splenocytes were seeded in 96-well plates and stimulated with anti-CD3 and anti-CD28 along with supernatants containing increasing doses of mIL-12. IFNγ was measured in splenocyte culture supernatants by ELISA 48 hours after stimulation (FIG. 13A). HEKBlue cells were seeded in 96-well plates and stimulated with increasing doses of supernatant containing mIL-12. IL-12-mediated SEAP production in HEKBlue supernatants was quantified by comparison with a standard curve of recombinant SEAP, and data were normalized to cell number.

[0291] Example 13 In vitro IFNβ production in bladder cancer epithelial cells transfected with a plasmid containing a RIG-I agonist (Figures 14A and 14B). MB49 cells were seeded in 96-well plates (35,000 cells / well) before transfection with a plasmid (NTC9385R-mIL12) containing or without a RIG-I agonist. Transfection was performed using Lipofectamine 2000 (Thermofisher) and increasing doses of plasmid DNA. 48 hours after transfection, cell culture supernatants were harvested, and IFNβ production was measured by ELISA. Data were normalized to total cellular protein and expressed as pg IFNβ / mg total protein.

[0292] Example 14 In vivo mRNA expression after administration of RXG preparations to the mouse bladder A single-chain murine IL-12p40p35 open reading frame with a codon-optimized sequence was cloned into the NTC9385R or NTC9385R-eRNA41H vector backbone, and expression was confirmed in MB49 cells in vitro (data not shown). Polyplexes were prepared using RXG polymer (NP20, non-PEGylated; 25% R, 10% G, 5% trehalose as excipient).

[0293] Female mice (12–16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplex (1.0 mg DNA / mL = 80 μg plasmid DNA) via intravesical instillation (exposure time = 60 min). Bladder tissue was collected at 4, 24, 48, 72, and 96 h post-administration. RNA was extracted (Qiagen RNeasy kit) after homogenization in lysis buffer. RT-qPCR was performed using 500 μg of input RNA and TaqMan primers / probes recognizing codon-optimized mouse IL-12p40p35. Absolute quantification was performed using a standard curve of mouse IL-12p40p35 RNA generated by in vitro transcription. Data are expressed as RNA copy numbers. As seen in Figure 15, IL-12 mRNA expression was high and sustained for 96 h. The inclusion of a RIG-I agonist did not reduce IL-12 mRNA expression.

[0294] Example 15 Polymer structure and polyplex formation Polyplexes were formed by complexing pDNA with DDX at amine-to-phosphate (N:P) ratios ranging from 3 to 30 in various stabilizers for subsequent tangential flow filtration (TFF) concentrations for freezing or lyophilization. The structure of doubly derivatized chitosan (DDX): Σ(q + p + n) = 1, q = 0.03–0.35, p = 0.12–0.28 (Figure 16).

[0295] Example 16 Physicochemical properties of DDX-DNA formulations Polyplexes were prepared at N:P ratios ranging from 3 to 30 using DDX conjugated with 12 to 28% arginine and 3 to 35% polyol. Polyplex hydrodynamic diameter (Z-average) and polydispersity index (PDI) were measured by dynamic light scattering (DLS) in 10 mM NaCl. DNA capture was determined by visual assessment of DNA release from polyplexes in a 0.8% agarose gel at pH 8 (0.5X TBE) subjected to 100 V for 1 hour. DNA supercoiling was quantified by agarose gel electrophoresis (Quantity One v4.6.7, Bio-Rad Laboratories) following DNA release from polyplexes by incubation with excess competing polyanion (poly-(α,β)-DL-aspartic acid). The zeta potential of the polyplexes was also measured by laser Doppler velocimeter (Figure 17).

[0296] Example 17 In vitro screening of polymers Using in vitro screening, a novel polymer demonstrated improved transfection efficacy compared to DDX (3% polyols, 14% arginine (R)). Mouse urothelial carcinoma cells (MB49) were seeded in 96-well plates (35,000 cells / well) 24 hours prior to transfection with the indicated formulations. Transfections were performed with increasing doses of plasmid DNA, as indicated. Fluorescence was measured 48 hours post-transfection using an Envision plate reader. Cell viability was measured using AlamarBlue Cell Viability Reagent. Data are presented as GFP relative fluorescence units normalized to cell viability (Figure 18A). A heat map depicts the fold change in maximum expression relative to DDX (3% polyols, 14% R) (Figure 18B). The percentage of GFP-positive cells was determined by high-content imaging of DDX and compared to the maximum efficacy (Figure 18C).

[0297] Example 18 In vivo protein expression after IVI administration of DDX Female mice (12-16 weeks) were anesthetized with isoflurane and administered 80 μL of polyplex (c1000 = 80 μg of plasmid DNA) via intravesical instillation (exposure time = 60 min). Forty-eight hours after administration, bladder tissue was collected and lysed by homogenization in the presence of protein lysis buffer containing protease inhibitors. Human PD-L1-Fc protein was quantified using a custom-designed immunoassay (Mesoscale Discovery). Data are expressed as pg / mL of protein in the lysate. After administration of DDX (12% polyol, 15% R) into the mouse bladder via intravenous administration, improved protein expression was observed in mice (Figure 19).

[0298] Example 19 Intravesical administration of human EG-70 in non-human primates Non-human primates were administered 0.25 mg / ml EG-70 containing IL-12p40p35 via intravesical instillation (exposure time = 60 min) under anesthesia. Animals received 10 ml or 20 mL of polyplexes containing EG-70 or 20 mL of empty vector control (n = 1 per group, female (EG-70) or male (polyplexes containing empty vector DNA)). Forty-eight hours after administration, bladder tissue was collected and lysed by homogenization in the buffer used for RNA extraction (Figure 20A) or in protein lysis buffer containing protease inhibitors (Figure 20B). RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes recognizing codon-optimized human IL-12p40p35. Absolute quantification was performed using a standard curve of human IL-12p40p35 RNA generated by in vitro transcription (Figure 20A). Human IL-12 protein was quantified using a commercially available human IL-12p70 immunoassay (Mesoscale Discovery). Data are expressed as pg / mL of protein in lysate (Figure 20B). Detectable levels of mRNA and protein were seen following both intravesical administration of 10 ml and 20 ml of treatment (corresponding to 2.5 mg or 5 mg of plasmid DNA, respectively). Individual data points represent percentages of bladder tissue within individual animals.

[0299] Example 20 Expression of eRNA11a and VA1 in non-human primates Non-human primates were administered 20 ml of C250 EG-70 polyplexes containing IL-12 / eRNA11a / VA1 or polyplexes containing empty vector control DNA via intravesical instillation (exposure time = 60 min) under anesthesia. Animals received 0.0625 mg / mL, 0.25 mg / mL, or 1 mg / mL (c62.5, c250, or c1000, respectively) of EG-70, or 1 mg / mL (c1000) of control nanoparticles (RXG-PEG-N9; n = 1 per group, females (EG-70, low and medium doses) or males (EG-70, high dose, or empty vector DNA-containing polyplexes). Forty-eight hours after administration, bladder tissue was harvested and lysed by homogenization in the presence of RNA extraction buffer. RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes recognizing eRNA11a or VA1. Absolute quantification was performed using standard curves of human eRNA11a RNA (FIG. 21) and human VA1 RNA (FIG. 22) generated by in vitro transcription (individual data points represent bladder tissue fractions within individual animals).

[0300] Example 21 Evaluation of antitumor activity in a mouse model of bladder cancer To evaluate the antitumor activity of the mEG-70 prototype nanoparticles, we used an orthotopic mouse model of bladder cancer. Briefly, disease was established by pretreating mouse bladders with poly-L-lysine to promote desquamation of the superficial urothelial layer and facilitate cancer cell implantation. Subsequently, urothelial carcinoma cells stably overexpressing the luciferase gene (MB49-Luc) were instilled into the mouse bladders (100,000 cells per mouse). Luciferase expression was confirmed 12 days after instillation using an in vivo imaging system (IVIS). Animals were randomized into treatment groups based on the intensity of the bioluminescent signal. Furthermore, animals without a positive bioluminescent signal were excluded from the study. Mice received two weekly intravesical administrations of nanoparticles on days 13 and 20 after instillation. This administration regimen was selected based on evaluation of protein expression kinetics. An additional group of animals underwent a sham procedure with the administration of the nanoparticle vehicle, trehalose (5%). The experiment was terminated on day 29 to measure bladder weight as a function of intravesical tumor burden.

[0301] As shown in Figures 27A and 27B, sham-treated tumor-bearing animals exhibited an average bladder weight of approximately 75 mg, while animals treated with a 20 μg dose of the mEG-70 prototype of plasmid DNA had bladder weights (approximately 20 mg) that were nearly indistinguishable from naive animals.

[0302] Example 22 Dynamics of mouse IL-12 mRNA expression in the mouse bladder To evaluate the kinetics of codon-optimized mouse Il12p40p35 gene expression in the mouse bladder, healthy female C57Bl / 6J mice (12–16 weeks) under anesthesia received a single intravesical instillation (IVI) of IL-12 / eRNA11a / VA1-containing RXG nanoparticles or polyplexes containing empty vector control DNA via intravesical instillation (exposure time = 60 min). Animals received mEG-70 prototype or control nanoparticles (RXG-PEG-N9) at 0.25 mg / mL, c250. At the indicated times post-instillation, bladder tissue was harvested and lysed by homogenization in the presence of RNA extraction buffer. RT-qPCR was performed using 1 μg of input RNA and TaqMan primers / probes recognizing the codon-optimized mIL-12p40p35. Absolute quantification was performed using a standard curve of mouse Il12p40p35 RNA generated by in vitro transcription (Figure 23).

[0303] As shown in Figure 23, mouse Il12p40p35 mRNA levels in bladder tissue were detectable as early as 4 hours after administration, and expression persisted up to 96 hours after administration in mice receiving mEG-70 prototype nanoparticles. As expected, mice receiving nanoparticles containing a plasmid without an IL-12 transgene (N9, negative control) did not have any detectable Il12p40p35 mRNA (less than two copies).

[0304] Example 23 Dynamics of mouse IL-12p70 protein expression in the mouse urinary bladder To assess the kinetics of mouse IL12p70 protein expression in the mouse bladder, healthy female C57Bl / 6J mice (12–16 weeks old) received a single intravenous infusion of 20 μg of mEG-70 prototype nanoparticles. Bladder tissue was harvested at the indicated times, and tissue lysates were prepared for immunoassay of mouse IL12p70 on the Mesoscale Discovery (MSD) platform.

[0305] As shown in Figure 24, mice administered a single IVI of mEG-70 prototype nanoparticles exhibited detectable levels of IL-12p70 protein as early as 24 hours after administration. Protein expression peaked at 48 hours and was detected as late as 96 hours after administration. There was no detectable IL-12p70 in the bladder tissue of mice receiving nanoparticles containing N9 plasmid DNA (negative control).

[0306] Example 24 Dose-response of murine IL-12p40p35 protein expression in the mouse bladder To assess whether there is a dose-dependent expression of mouse IL12p70 protein in the mouse bladder, healthy female C57Bl / 6J mice (12-16 weeks old) received a single intravenous infusion of mEG70 prototype nanoparticles at doses ranging from 0.1 to 80 µg of plasmid DNA. Negative controls were administered up to 20 µg. Bladder tissue was harvested 48 hours after administration, and tissue lysates were prepared for mouse IL12p70 immunoassays on the MSD platform.

[0307] As shown in Figure 25, mice administered a single IVI of mEG-70 prototype nanoparticles showed low levels and frequency (25% of mice) of IL-12p70 protein at the lowest dose of N9-m12-R (0.1 μg). Protein expression was detected at comparable levels for all doses ranging from 180 μg. There was no detectable IL-12p70 in the bladder tissue of mice receiving nanoparticles containing N9 plasmid DNA (negative control).

[0308] Example 25 Expression of PEGylated nanoparticles in vivo A study was conducted to evaluate any potential effects of PEGylation on murine IL-12p70 protein expression. Briefly, female C57Bl / 6J mice (12–16 weeks old) received a single dose of nanoparticles containing 20 μg of plasmid DNA. Mice received a single intravenous infusion of mEG-70 or mEG-70 prototype nanoparticles. Bladder tissue was collected 24 hours to 7 days after administration, and tissue protein lysates were obtained for quantification of IL-12p70 protein by immunoassay.

[0309] As shown in Figure 26, mice receiving a single IVI of either mEG-70 prototype or mEG-70 exhibited comparable levels and kinetics of murine IL-12p70 protein expression.

[0310] Example 26 Human clinical studies in NMIBC Bladder cancer is the fourth and tenth most common malignancy in men and women in the United States (US), respectively (American Cancer Society 2019). Non-muscle-invasive bladder cancer (NIMBC) is typically managed with surgical resection (TURBT), often followed by a single dose of intravesical chemotherapy (gemcitabine or mitomycin) within 24 hours, to reduce recurrence rates by 35% (Sylvester et al., 2016).

[0311] After confirming the presence of bladder cancer through pathology, physicians often develop an ongoing treatment plan that includes BCG therapy. Despite significant side effects and a 30%–40% failure rate, intravesical immunotherapy with BCG is the mainstay treatment used to prevent recurrence and / or progression in patients with high-grade (Ta or higher) NMIBC. BCG is often given as a second maintenance course to achieve a disease-free state, but NMIBC patients who do not respond to BCG are highly unlikely to benefit from further treatment, including BCG, and therefore represent a unique population for research into novel therapies (Jarow et al., 2015).

[0312] Without pharmacological intervention or cystectomy, BCG-unresponsive NMIBC will persist and progress, regardless of whether the disease is resected. To date, no effective treatment is available for patients who fail BCG, as gemcitabine and mitomycin, often administered after TURBT, are not effective rescue medications. Therefore, the treatment for BCG-unresponsive disease (regardless of whether it is BCG-refractory or recurrent) is radical cystectomy, which surgically removes all tumor and ensures disease-free survival. The fact that few treatment options are available for NMIBC and patients continue to undergo radical organ removal for early-stage disease represents a truly significant unmet medical need. More effective treatments that are effective for refractory patients are desperately needed for NMIBC.

[0313] In an exemplary embodiment of the invention, the therapeutic nucleic acid comprises a 4156 bp plasmid DNA (pDNA) consisting of a codon-optimized human interleukin-12 gene, designated opt-hIL-12 (SEQ ID NO: 7), linked to a constitutively active cytomegalovirus (CMV) promoter on an NTC9385R backbone with a sucrose-based antibiotic-free selectable marker (RNA-OUT), as set forth in the table below. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0314] The R6K origin of replication restricts replication of the plasmid to specific strains of Escherichia coli (E. coli). The opt-hIL12 gene encodes two subunits (p40 and p35) of the cytokine protein IL-12. To ensure 1:1 stoichiometry of the subunits, the EG-70 plasmid was designed to contain a single open reading frame (ORF), with the addition of a short, repeating elastin linker sequence to monomerize p40 to p35. The plasmid also contains genes for eRNA11a (immunostimulatory double-stranded ribonucleic acid [dsRNA]) and adenovirus VA RNA1. The two RNA products of these genes stimulate the RIG-I pathway, thereby recruiting more immune cells to local tissues. In a further embodiment, the therapeutic nucleic acid is packaged in a dual-derivatized chitosan polymer functionalized with arginine and glucose and coated with a separable PEG-b-PLE excipient to form the pharmaceutical composition EG-70. The composition is formulated as an aqueous nanoparticle dispersion in a 1% w / w mannitol solution, filter sterilized, lyophilized to a dry powder, and stored at 4° C. The mean particle size of the nanoparticle dispersion ranges from 75 to 175 nanometers.

[0315] This study will evaluate the safety of intravesical administration of EG-70 and its effect on bladder tumors in patients with NMIBC who have failed BCG therapy and are undergoing radical cystectomy. The study will be a representative dose-escalation trial, with three patients in each cohort treated. The initial dose of EG-70 will be based on nonclinical toxicity data, as well as nonclinical efficacy data, and will be at least 1 / 5 the minimally toxic dose seen in GLP-toxicity studies. The anticipated Phase 1 dose escalation will be up to 1 / 2 log increments, with consecutive cohorts treated without dose-limiting toxicities (DLTs).

[0316] equivalent All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The above disclosure may encompass multiple separate inventions having independent utility. While each of these inventions is disclosed in a preferred form(s), the specific embodiments thereof disclosed and exemplified herein are susceptible to numerous variations and should not be considered limiting. The subject matter of the inventions includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or properties disclosed herein. The following claims particularly point out certain combinations and sub-combinations that are deemed to be novel and non-obvious. Inventions embodying other combinations and sub-combinations of features, functions, elements, and / or properties may be claimed in this application, in applications claiming priority to this application, or in related applications. Such claims, whether broader, narrower, equal, or different in scope compared to the original claims, whether directed to different inventions or the same invention, are also deemed to be included within the inventive subject matter of this disclosure.

Claims

1. A composition comprising a nucleic acid polyplex, comprising a cationic polymer and / or lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct encoding at least one RIG-I agonist, wherein the therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding at least one RIG-I agonist are the same or different nucleic acid constructs.

2. The composition according to claim 1, wherein the at least one RIG-I agonist is selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20.

3. The composition according to claim 1, wherein the at least one RIG-I agonist is eRNA41H or eRNA11a.

4. The composition according to claim 1, wherein the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof.

5. The composition according to claim 1, wherein the cationic polymer is a derivatized chitosan.

6. The composition according to claim 5, wherein the derivatized chitosan is an amino-functionalized chitosan.

7. The composition according to claim 6, wherein the amino-functionalized chitosan comprises arginine.

8. The composition according to claim 6, wherein the amino-functionalized chitosan further comprises a hydrophilic polyol or is functionalized with a hydrophilic polyol.

9. The composition according to claim 8, wherein the hydrophilic polyol is glucose or gluconic acid.

10. The composition according to claim 1, wherein the nucleic acid polyplex further comprises a reversible coating comprising one or more polyanion-containing block copolymers having at least one polyanionic anchor region and at least one hydrophilic tail region.

11. The composition according to claim 10, wherein the polyanion-containing block copolymer is a linear binary block or a ternary block copolymer.

12. The composition according to claim 1, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is contained in a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, or NTC9385R.

13. The composition according to claim 12, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist are contained within the NTC9385R plasmid.

14. The composition according to claim 1, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises an expression control element selected from the group consisting of CMV, EF1a, CMV / EF1a, CAG, and CMV / EF1a / HTLV promoter.

15. The composition according to claim 14, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises a CMV promoter.

16. The composition according to claim 1, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises a synthetic β-globin-based intron.

17. The composition according to claim 1, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises HTLV-IR.

18. The composition according to claim 1, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises a kanamycin-selective or sucrose-based selective element.

19. The composition according to claim 18, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises a sucrose-based selection element.

20. The composition according to claim 1, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist further comprises a pUC or an R6K replication origin.

21. The composition according to claim 20, wherein the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist comprises an R6K replication origin.

22. The composition according to claim 11, wherein the cationic polymer is a derivatized chitosan functionalized with arginine and glucose, the polyanion-containing block copolymer is PEG-b-PLE, the therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding at least one RIG-I agonist are the same, and the composition includes Sequence ID No.

7.

23. Use of the composition according to any one of claims 1 to 22 in the manufacture of a pharmaceutical product for the local expression of IL-12 in the mucosal tissue of a patient.

24. Use of the composition according to any one of claims 1 to 22 in the manufacture of a pharmaceutical product for inhibiting the growth of mucosal cancer cells in a patient.

25. Use of the composition according to any one of claims 1 to 22 in the manufacture of a pharmaceutical product for treating bladder cancer in a patient.

26. The composition according to any one of claims 1 to 22 for use in local expression of IL-12 in the mucosal tissue of a patient.

27. ​​The composition according to any one of claims 1 to 22 for use in suppressing the growth of mucosal cancer cells in a patient.

28. The composition according to any one of claims 1 to 22 for use in the treatment of bladder cancer in a patient.

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