Combination Gene Therapy for the Treatment of Metastatic Cancer

Abstract

The present disclosure relates to methods and compositions for localized expression of IL-12, preferably in combination with a RIG-I agonist, to activate memory T cell responses against cancer antigens. In embodiments, the methods are effective in treating metastatic disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 150,846, filed February 18, 2021, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] Technical Field The present disclosure relates to methods and compositions for treating metastatic tumors at distant sites by localized delivery and expression of IL-12, preferably in combination with a type I IFN (IFN-1) activator / inducer. [Background technology]

[0003] Cancerous diseases and tumors are one of the leading causes of death and serious illness in humans. Metastatic tumors, in particular, contribute significantly to the deaths of cancer patients, mainly because current treatments become ineffective once metastases start to form.

[0004] Treating metastatic cancer is an enormous challenge, especially when it has spread to several different sites in the body. Typically, patients with metastatic cancer are treated exclusively with systemic therapies aimed at killing cancer cells everywhere in the body. Unfortunately, the effectiveness of this approach is far from ideal. Thus, the terms "cure" and "metastatic cancer" are rarely used together. People with metastatic tumors often do not respond to existing treatments, and achieving long-term remission in these patients is much less likely than in patients with localized cancer. Rather, the goal of treatment for metastatic disease is usually to slow the growth of the cancer or alleviate symptoms caused by the cancer.

[0005] Although it is not exactly understood why metastatic cancer is difficult to treat, it is clear that metastatic tumor cells can quickly adapt and become resistant to treatment. In some cases, each metastatic tumor may grow in different organs. This makes treatment difficult, because each tumor may have its own tumor microenvironment and may respond differently to treatment. Therefore, the prognosis for people with metastatic cancer is generally poor, and metastatic cancer accounts for most cancer deaths.

[0006] Thus, there remains a need in the art for methods of inhibiting tumor cell proliferation at a second tumor site distinct from the primary cancer in an individual with a carcinoma, and for methods of treating or suppressing metastatic tumors at a site distinct from the primary cancer. Fortunately, the present disclosure addresses these and other needs. Summary of the Invention

[0007] The present disclosure solves an unmet need in the art to inhibit metastatic tumors at distant sites by locally delivering and expressing IL-12 at the primary tumor site together with a type I interferon (IFN-1) activator / inducer, such as a RIG-I agonist, a STING agonist, and / or a TLR7 / 9 agonist. As demonstrated for the first time herein, the subject therapeutic methods stimulate a robust immune response against the primary cancer, including cytotoxic CD8+ T cells and CD4+ memory T cells, with the latter cell population in particular supporting the systemic effect of the subject therapeutic methods against distant metastases. In some embodiments, the primary tumor site is a mucosal tissue. In some embodiments, the primary tumor site is other than a mucosal tissue. In preferred embodiments, the subject methods and compositions include co-expression of IL-12 and at least one RIG-I agonist.

[0008] In one aspect, the disclosure provides a method of activating a memory T cell response against a cancer antigen, comprising contacting a primary cancer with a therapeutically effective amount of a composition comprising a nucleic acid polyplex comprising a cationic polymer and / or a lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct comprising a nucleic acid encoding at least one RIG-I agonist, wherein the therapeutic nucleic acid constructs encoding IL-12 and RIG-I are the same or different nucleic acid constructs.

[0009] In some embodiments, the method is effective to treat or inhibit primary cancer. In embodiments, the primary cancer is selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, ovarian cancer, kidney cancer, brain cancer, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, kidney cancer, liver cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer. In embodiments, the primary cancer is other than mucosal cancer.

[0010] In some embodiments, the method is effective for treating or suppressing metastatic disease at a site different from the primary cancer.In some embodiments, the primary cancer is selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, ovarian cancer, kidney cancer, brain tumor, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, kidney cancer, liver cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer.In some embodiments, the site different from the primary cancer is one or more of liver, lung, bone, brain, lymph node, peritoneum, skin, prostate, breast, colon, rectum, and cervix.In some embodiments, the metastatic disease is at two or more sites different from the primary cancer.

[0011] In some embodiments, the RIG-I agonist is selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, and more preferably, is selected from the group consisting of eRNA41H and eRNA11a.

[0012] In some embodiments, the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof. In some embodiments, the cationic polymer comprises a derivatized chitosan, preferably an amino-functionalized chitosan. In some embodiments, the amino-functionalized chitosan comprises arginine and further comprises or is functionalized with a hydrophilic polyol. In some embodiments, the hydrophilic polyol is selected from gluconic acid and glucose.

[0013] In some embodiments, the nucleic acid polyplexes further comprise 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, preferably the polyanion-containing block copolymers are linear diblock and / or triblock copolymers.

[0014] In some embodiments, the therapeutic nucleic acid construct encoding IL-12 comprises SEQ ID NO:8.

[0015] In another aspect, the disclosure provides a method of treating or inhibiting a metastatic tumor at a site different from the primary cancer in an individual having a primary cancer, e.g., bladder cancer, comprising contacting the primary cancer with a therapeutically effective amount of a composition comprising a nucleic acid polyplex comprising a cationic polymer and / or a lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct comprising a nucleic acid encoding at least one RIG-I agonist, wherein the therapeutic nucleic acid constructs encoding IL-12 and RIG-I are the same or different nucleic acid constructs.

[0016] In embodiments, the primary cancer is a cancer selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, ovarian cancer, kidney cancer, brain cancer, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, kidney cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer. In one embodiment, the primary cancer is a mucosal cancer selected from the group consisting of gastrointestinal cancer, nasal cancer, or lung cancer, and genitourinary cancer. In some embodiments, the primary mucosal cancer is a gastrointestinal cancer selected from the group consisting of oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, and rectal cancer. In some embodiments, the primary mucosal cancer is a nasal cancer or lung cancer selected from the group consisting of paranasal sinus cancer, oropharynx cancer, trachea cancer, and lung cancer. In some embodiments, the primary mucosal cancer is a genitourinary cancer selected from the group consisting of bladder cancer, urothelial cancer, urethral cancer, testicular cancer, renal cancer, prostate cancer, penile cancer, adrenal cancer, uterine cancer, cervical cancer, and ovarian cancer, hi some embodiments, the genitourinary cancer is bladder cancer.

[0017] In some embodiments, the metastatic site of the tumor is in one or more of the liver, lung, bone, brain, lymph node, peritoneum, skin, prostate, breast, colon, rectum, and cervix, hi some embodiments, the metastatic tumor is in two or more different sites.

[0018] In some embodiments, the RIG-I agonist is selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20, and more preferably, is selected from the group consisting of eRNA41H and eRNA11a.

[0019] In other embodiments, the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof, hi another embodiment, the cationic polymer comprises a derivatized chitosan, preferably an amino-functionalized chitosan.

[0020] In some embodiments, the cationic polymer is an amino-functionalized chitosan that comprises arginine and further comprises or is functionalized with a hydrophilic polyol, in some embodiments, the hydrophilic polyol is selected from gluconic acid and glucose.

[0021] In some embodiments, the nucleic acid polyplexes further comprise 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, preferably the polyanion-containing block copolymers are linear diblock and / or triblock copolymers.

[0022] In some embodiments, the therapeutic nucleic acid construct encoding IL-12 comprises SEQ ID NO:8.

[0023] In some embodiments, the contacting comprises intravesical instillation. In other embodiments, the administration is oral or intrarectal / intracolonic to the gastrointestinal tract (GIT). In some embodiments, the administration is intrarectal / intracolonic to the gastrointestinal tract (GIT). In yet other embodiments, the contacting is by intratumoral injection. In yet other embodiments, the contacting is intranasal or intratracheal administration to the lungs.

[0024] Other features, objects, and advantages will become apparent from the following disclosure.

[0025] The present disclosure is disclosed with reference to the accompanying drawings. [Brief description of the drawings]

[0026] [Figure 1A]A timeline of experimental treatment of female C57BL / 6J mice with mEG-70 constructs in an orthotopic model of bladder cancer is shown. On day 1, MB49-luciferase cells (MB49-Luc; 1×105 cells) were injected into the bladder of mice. Engraftment was confirmed by in vivo imaging of luciferase signal 9 days after injection. Mice were evenly distributed into treatment groups (n=22) based on bioluminescence levels and received intravesical instillation (IVI) of mEG-70 (1 mg DNA / mL; equivalent to 80 μg DNA) on days 10 (Tx1) and 17 (Tx2), while control animals received instillation of 1% mannitol (sham treatment). A cohort of tumor-bearing animals was untreated. Survival was monitored for 85 days. [Figure 1B] mEG-70 treated animals showed prolonged survival compared to control mice, of which approximately 70% died from the disease. The survival curves for mEG-70 were significantly different from the survival rates of sham-treated (1% mannitol) or untreated mice (*p<0.05 and **p<0.01, respectively). [Figure 1C] Mice treated with mEG-70 that showed complete disease regression and no recurrence during the 76-day observation period (referred to as "mEG-70-cured") were rechallenged with MB49-Luc cells to assess protection from recurrent disease. In contrast to age-matched untreated controls, which showed robust tumor engraftment in 15 of 17 mice, all mEG-70-cured mice were resistant to tumor recurrence for up to 3 weeks after rechallenge (n=17). [Figure 2A]A timeline of experimental treatment of female C57BL / 6J mice with mEG-70 constructs in an orthotopic model of bladder cancer is shown. MB49-luciferase cells (MB49-Luc; 1 × 105 cells) were injected into the bladder of mice on day 1. Engraftment was confirmed by in vivo imaging of luciferase signal 9 days after injection and was evenly distributed into treatment groups (n = 22) based on the level of bioluminescence. Mice received intravesical instillation (IVI) of mEG-70 (1 mg DNA / mL; equivalent to 80 μg DNA) on days 10 (Tx1) and 17 (Tx2), while control animals received instillation of 1% mannitol (sham treatment). A cohort of tumor-bearing animals was untreated. Survival was monitored until all mice died of bladder cancer or were deemed tumor-free (negative bioluminescence signal and no clinical symptoms). Surviving tumor-free mEG-70-treated mice and age-matched controls were rechallenged by IVI with MB49-Luc cells (1×105 cells) on day 85. All mEG-70-treated mice remained tumor-free and were rechallenged subcutaneously in the flank on day 153 with either MB49-Luc (1×105 cells) or B16-F10 cells (1×105 cells). [Figure 2B] mEG-70-treated animals were protected from distant tumor rechallenge with MB49-Luc cells. Only one of nine animals showed tumor growth, which progressed significantly more slowly. In contrast, eight of nine mice in the untreated control cohort grew tumors. [Figure 2C] Mice were rechallenged with B16-F10 cells to assess the specificity of the response. All mice in the rechallenge and untreated control groups showed robust B16-F10 tumor engraftment (n=8 / group). [Figure 3A]A timeline of experimental treatment of female C57BL / 6J mice with mEG-70 constructs in an orthotopic model of bladder cancer is shown. MB49-luciferase cells (MB49-Luc; 1 × 105 cells) were injected into the bladder of female C57BL / 6J mice (12–16 weeks) and engraftment was confirmed by in vivo imaging of the luciferase signal 9 days after injection (using a Lumina LT IVIS imaging system). Mice were equally distributed into treatment groups (n=20) based on their levels of bioluminescence (luciferase-negative mice were excluded from the study) and received intravesical instillations (IVI) of mEG-70 (1 mg DNA / mL, equivalent to 80 μg DNA) on days 10 (Tx1) and 17 (Tx2), while control animals received instillations of 1% mannitol (sham treatment). Survival was monitored until all mice died of bladder cancer or were deemed tumor-free (negative bioluminescence signal and no clinical symptoms; data not shown). On day 167, surviving tumor-free mEG-70-treated mice and age-matched untreated controls were injected intraperitoneally with either isotype control (non-depleted), anti-CD4 antibody, or anti-CD8 antibody for 4 consecutive days to establish depletion, and then twice weekly for maintenance. After the third depleting antibody injection, mice were rechallenged with MB49-Luc cells (1×105 cells) subcutaneously in the flank (day 170; n=6). Tumors were monitored by measuring with a caliper; tumor volume was calculated using the formula (length×width2 / 2). [Figure 3B] While untreated mice receiving isotype control antibody (non-depleting) had growing subcutaneous tumors, all mEG-70-treated animals were protected from distant tumor rechallenge with MB49-Luc cells. [Figure 3C] Mice administered anti-CD4 antibodies (CD4+ T cell depleted) have growing MB49-Luc subcutaneous tumors whether untreated or previously treated with mEG-70 treatment. [Figure 3D] All untreated mice receiving anti-CD8 antibody (CD8+ T cell depletion) had growing subcutaneous tumors, whereas only 1 of 6 mEG-70-treated animals had an actively growing tumor. [Figure 4A]A timeline of experimental treatment of female C57BL / 6J mice with mEG-70 constructs is shown. MB49-luciferase cells (MB49-Luc; 2.5 × 105 cells in 100 μL) were implanted subcutaneously into the right flank of C57BL / 6J mice (12–16 weeks) under anesthesia to induce disease. When tumors reached approximately 50–200 mm3, mice were randomly assigned to treatment groups (n=10). Mice were administered mEG-70 (0.5 mg DNA / mL in 50 μL, equivalent to 25 μg DNA) directly intratumorally (IT) on days 1, 4, 8, 11, 15, and 18, while control animals received 1% mannitol (sham treatment). A cohort of tumor-bearing animals was untreated. Tumor size was monitored three times a week by measurement with a caliper; tumor volume was calculated using the formula (length x width 2 / 2). To confirm the absence of tumor recurrence in tumor-free mEG-70-cured individuals (mEG-70 "cured"; n=9), bioluminescence imaging of luciferase signal was performed on day 70 using a Lumina LT IVIS imaging system. On day 73, mEG-70-cured, age-matched controls were implanted subcutaneously in the left flank with MB49-Luc cells (2.5 x 105 cells in 100 μL). Tumors were monitored three times a week by measurement with a caliper; tumor volume was calculated using the formula (length x width 2 / 2). [Figure 4B] Intratumoral (IT) administration of mEG-70 inhibited tumor growth compared with sham-treated mice. [Figure 4C] mEG-70 "cured" mice were protected from tumor cell rechallenge in the contralateral flank. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present disclosure contemplates localized expression of IL-12 and RIG-I agonists at primary tumor sites for the treatment of metastatic disease at distant sites. For example, localized gene therapy at mucosal tissues, such as intravesical administration to the bladder, aerosolized administration to the lungs, intratumoral injection, and / or oral dosage forms to the gastrointestinal tract (GIT), presents an attractive approach to promote localized expression of immunomodulatory proteins while minimizing undesirable systemic side effects. Moreover, as demonstrated for the first time herein, it has been surprisingly found that delivery of therapeutic nucleic acids, including IL-12 and RIG-I agonists, using the non-viral vector platform disclosed herein elicits potent systemic anti-tumor activity, including both cytotoxic CD8+ T cells and CD4+ memory T cells, that can be used to treat and prevent metastatic tumors at sites distant from the primary tumor.

[0028] Without being bound by theory, activation of the IL-12 pathway at primary cancer sites according to the subject disclosure acts on effector CD4+ and CD8+ cells resulting in potent anti-tumor and anti-angiogenic functions, including induction of memory T cells, whereas 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 are combined to generate a surprising and remarkably potent inflammatory response that drives a robust and long-lasting anti-tumor immune response, coupling stimulation of the innate immune system by RIG-I agonists to IL-12-mediated stimulation of the adaptive immune response.

[0029] definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted 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.

[0030] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] The term "about" refers to and encompasses the indicated value and the 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.

[0032] The term "combinations thereof" includes all possible combinations of the elements to which the term refers.

[0033] As used herein, the term "memory T cell response" or "induction of memory T cells" refers to the "activation" of naive T cells through coordinated interactions between molecules on T cells, antigen presenting cells (APCs), and inflammatory cytokine mediators, which differentiates stimulated T cells into effectors suitable for dealing with immunological insults, e.g., cancer antigens. Memory T cell responses are known in the art. See, e.g., Pennock et al, (2013), Adv. Physiol. Educ. 37(4):273-283; Sprent et al. (2011), Nat. Immunol. 12:478-84; MacLeod et al. (2010), Immunology, 130(1):10-15.

[0034] Thus, "activating a memory T cell response" refers to the activation and programming of T cells from a naive / resting state to generate T cells capable of mediating immune defense.

[0035] The term "cancer antigen" or "tumor antigen" as used herein refers to a protein produced in a tumor cell that can act as a tumor antigen. "Cancer antigen" or "tumor antigen" is known in the art. For example, the Cancer Epitope Database and Analysis Resource (CEDAR) provides a comprehensive collection of cancer epitopes curated from the literature as well as cancer epitope prediction and analysis tools. See, for example, Kosaloglu-Yalqn1 et al. (2021), Front. Immunol. 12:1-14. Exemplary cancer antigens are also disclosed in the Cancer Antigenic Peptide Database, available on the World Wide Web, for example, at caped.icp.ucl.ac.be / Peptide / list.

[0036] As used herein, the term "primary tumor" or "primary cancer" refers to a tumor present at an anatomical site where tumor progression begins and progresses to produce a cancerous mass. Exemplary primary cancers include, but are not limited to, primary tumors of the bladder, colon, lung, vagina, ovary, cervix, kidney, stomach, gastrointestinal tract, prostate, brain, breast, pancreas, lung, thyroid, endometrium, esophagus, larynx, nasal cavity cancer, oral cancer, melanoma, pharyngeal cancer, retinoblastoma, testicular cancer, etc.

[0037] The methods disclosed herein are useful for activating strong memory T cell responses against antigens, such as cancer antigens, so that cancerous lesions or tumors can be suppressed or cured.Furthermore, the methods disclosed herein for activating strong memory T cell responses against cancer antigens provide long-lasting systemic immunity such that the methods are effective in treating or suppressing metastatic disease at sites different from the primary cancer.

[0038] The term "metastatic" as used herein refers to a tumor that develops at a site distant from the site of the primary tumor.

[0039] The term "metastatic disease" as used herein refers to a condition or symptom that may result in the spread of a tumor to another organ or tissue (or part thereof), even to a non-adjacent organ or tissue (or part thereof). In one embodiment, metastatic disease refers to cancer metastatic disease, e.g., the establishment of metastases. Some cancer cells may acquire the ability to penetrate the walls of lymphatic and / or blood vessels, and can then circulate to other sites and tissues in the body via the bloodstream (circulating tumor cells). This process is usually known as lymphatic or hematogenous spread (respectively). After tumor cells become quiescent at another site, they may re-penetrate the blood vessel or wall and continue to grow, eventually forming another clinically detectable tumor. This new tumor is known as a metastatic (or secondary or tertiary) tumor. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, a "metastasis" or "metastatic disease," and its cells resemble those of the original primary tumor. This means, for example, that if bladder cancer metastasizes to the uterus, the secondary tumor will be composed of abnormal bladder cells, not abnormal uterine cells. In this case, the tumor in the uterus is called metastatic bladder cancer rather than uterine cancer.

[0040] "Metastatic disease" includes, but is not limited to, metastatic spread of cancer from cancerous tumors, such as mucosal cancer. "Metastatic disease" also includes metastatic spread from benign tumors. Thus, in exemplary embodiments, metastatic disease includes metastatic spread from cancerous and benign tumors such as breast, colon, prostate, pancreas, skin, lung, ovary, kidney, brain, bladder, vagina, cervix, stomach, digestive tract, liver, thyroid, esophagus, nasal cancer, larynx, oral cancer, pharyngeal cancer, retinoblastoma, endometrium, and testis. In some embodiments, the metastatic disease is metastatic bladder cancer.

[0041] The methods disclosed herein are useful for preventing or treating metastatic disease in individuals with carcinoma by treating or inhibiting metastatic tumors at sites different from the primary cancer. Thus, as used herein, the phrase "preventing or treating metastatic disease" refers to the ability of 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 comprising a nucleic acid encoding at least one RIG-I agonist to limit or reduce the occurrence of metastatic disease, limit the metastatic potential of a cancer, and / or limit the number and dissemination of metastases compared to a control, or cure the disease. In some embodiments, the methods described herein are useful for preventing symptoms associated with metastatic disease or limiting the severity of symptoms associated with metastatic disease.

[0042] The methods described herein may also be useful for limiting the progression of metastatic disease. As used herein, the phrase "limiting the progression of metastatic disease" refers to the ability of 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 comprising a nucleic acid encoding at least one RIG-I agonist to delay or inhibit the appearance of metastases, limit the number of metastases, limit the size of metastases, and / or limit the number of organs or tissues containing metastases. In one embodiment, the methods described herein may also be useful for preventing symptoms associated with metastatic disease progression or limiting the severity of symptoms associated with metastatic disease progression.

[0043] Thus, "treating" or "treatment" of any disease or disorder, in certain embodiments, refers to improving the disease or disorder present in a subject. "Treating" or "treatment" includes improving at least one physical parameter that may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" includes modulating 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 an exemplary embodiment, the phrase "treating cancer" refers to inhibiting the proliferation of cancer cells, inhibiting the spread (metastasis) of cancer, inhibiting tumor growth, reducing the number of cancer cells or tumor growth, reducing the aggressiveness of cancer (e.g., increasing differentiation), or improving cancer-related symptoms. Furthermore, "treatment" as used herein includes preventing or delaying recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dosage of one or more other drugs required to treat the disease, slowing the progression of the disease, enhancing or improving quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by "treatment" is the alleviation of the pathological consequences of cancer.

[0044] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a subject composition that is effective to treat a disease or disorder when administered to a subject. For example, in exemplary embodiments, the phrase "effective amount" is used interchangeably with "therapeutically effective amount" or "therapeutically effective dose" and the like, and refers to an amount of a therapeutic agent that is effective to treat cancer. The effective amount of the compositions provided herein may vary depending on factors such as the disease state, age, sex, and weight of the animal.

[0045] As used herein, the term "subject" or "individual" refers to a mammalian subject. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, chickens, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a 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 a cancer, an autoimmune disease or condition, and / or an infectious disease.

[0046] "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 this disclosure.

[0047] Chitosan may be derivatized by functionalizing the free amino groups at the deacetylation sites. The derivatized chitosans described herein have a number of properties that are advantageous for nucleic acid delivery vehicles, including effectively binding and complexing to negatively charged nucleic acids, being able to be formed into nanoparticles of controllable size, being able to be taken up by cells, and being able to release nucleic acids at the appropriate time inside the cells. Chitosan with any degree of functionalization between 1% and 50%. (Percent functionalization is determined relative to the number of free amino moieties on the chitosan polymer before or without functionalization.) The degree of deacetylation and the degree of functionalization impart a specific charge density to the functionalized chitosan derivative.

[0048] Polyols according to the present disclosure 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 that have been functionalized with cationic moieties (e.g., molecules containing amino groups such as lysine, ornithine, guanidinium groups, molecules containing arginine, or combinations thereof).

[0049] As used herein, the term "C2-C6 alkylene" refers to a linear or branched divalent hydrocarbon radical, optionally containing one or more carbon-carbon multiple bonds. For the avoidance of doubt, as used herein, the term "C2-C6 alkylene" encompasses divalent radicals of alkanes, alkenes, and alkynes.

[0050] As used herein, unless otherwise indicated, the terms "peptide" and "polypeptide" are used interchangeably.

[0051] 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, and the like, or by substituting or modifying side chains or functional groups.

[0052] A peptidomimetic 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.

[0053] Polypeptides of the present disclosure may be produced by recognized methods such as 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).

[0054] 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.

[0055] As used herein, the "final degree of functionalization" of a cation or polyol refers to the proportion of cationic (e.g., amino) groups on the chitosan backbone that are functionalized with a cation (e.g., amino) 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: the final degree of functionalization of polyol), etc. may be used interchangeably with the terms "molar ratio" or "number ratio".

[0056] A dispersion consists of a particulate material, known as the dispersed phase, distributed throughout a continuous medium. A chitosan-nucleic acid polyplex "dispersion" is a composition that includes hydrated chitosan-nucleic acid polyplexes, where the polyplexes are distributed throughout the medium.

[0057] As used herein, a "pre-concentrated" dispersion is one that has not been subjected to a concentration process to form a concentrated dispersion.

[0058] As used herein, "substantially free" of polyplex precipitates means that the composition is essentially free of particles observable upon visual inspection.

[0059] As used herein, physiological pH refers to a pH between 6 and 8.

[0060] "Chitosan-nucleic acid polyplex" or grammatical equivalents thereof means a complex comprising a plurality of chitosan molecules and a plurality of nucleic acid molecules. In a preferred embodiment, a (e.g., doubly) derivatized chitosan is complexed with the nucleic acid.

[0061] The term "polyethylene glycol" ("PEG") as used herein is intended to mean a polymer of ethylene oxide having repeating units of -(CH2CH2-O)- and the general formula HO-(CH2CH2-O)nH.

[0062] The term "monomethoxypolyethylene glycol" ("mPEG") as used herein 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.

[0063] I. Metastatic disease Cancer metastasis refers to the spread of cancer cells from one part of the body to nearby tissues, organs, or even distant parts of the body. Usually, when cancer spreads from the primary organ to distant organs, it is considered a systemic disease and is difficult to control. Treatment options for subjects who develop metastatic disease are limited, and the prognosis is usually poor.

[0064] In the presence of metastatic disease, local therapy was considered futile. However, it is now understood that in some malignancies (e.g., kidney, breast, and prostate), treatment of the primary tumor may reduce mortality despite established metastatic spread (see, e.g., Morgan SC, et al. Nat. Rev. Clin. Oncol. 2011, Jun, 7; 8(8): 504-6; Sami-Ramzi Leyh-Bannurah et al. (2017) European Urology, 72: 118-124). Nevertheless, while treatment of the primary tumor may slow the progression of existing metastases, it does not usually result in a cure.

[0065] Fortunately, as described in detail below, it has surprisingly been discovered that the compositions disclosed herein, delivered locally to the site of a primary tumor, provide long-lasting, systemic, and specific anti-tumor immunity.

[0066] II. Composition Provided herein are chitosan compositions comprising chitosan derivative nucleic acid nanoparticles (polyplexes) complexed with a coating of polyanion-containing block copolymers, such as diblock and / or triblock copolymers, where each polymer molecule comprises 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 present disclosure is a "PEG-PA" polymer molecule comprising a polyethylene glycol (PEG) portion and a polyanion (PA) portion.

[0067] A. Chitosan Chitosan derivatives The chitosan component of the nucleic acid nanoparticles can be functionalized with cationic functional groups and / or hydrophilic moieties. Chitosan functionalized with two different functional groups is called double 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 US2007 / 0281904 and US2016 / 0235863, each of which is incorporated herein by reference.

[0068] In one embodiment, the doubly derivatized chitosan described herein comprises a 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 a chitosan having a degree of deacetylation of at least 98%.

[0069] 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 this disclosure, molecular weights ranging from 3 to 110 kDa. The embodiments described herein feature even lower average molecular weights of derivatized chitosan (less than 25 kDa, e.g., from about 5 kDa to about 25 kDa), which can have desirable delivery and transfection properties, small size, and good solubility. Since low average molecular weight derivatized chitosans are generally more soluble than those with higher molecular weights, the former produce nucleic acid / chitosan complexes that more readily release nucleic acid and increase cellular transfection. Much literature has described the optimization of all of these parameters for chitosan-based delivery systems.

[0070] Those skilled 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 between 50% and 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 have 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 a chitosan molecule 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 have a different chain length, many of which have an average molecular weight substantially equivalent or equal to a chitosan molecule having a chain length of n. For example, a 24-mer chitosan may include multiple chitosan molecules, each having 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 having a chain length of 24.

[0071] The doubly derivatized chitosan of the present disclosure may also be functionalized with a polyol or a 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 help increase the hydrophilicity of chitosan (including arginine-chitosan) and / or provide a hydroxyl group. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticle is or includes gluconic acid. See, for example, WO2013 / 138930. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticle is or includes glucose. Additionally or alternatively, the hydrophilic functional group may include a polyol. See, for example, US2016 / 0235863. Exemplary polyols for functionalization of chitosan are further described below.

[0072] The functionalized chitosan derivatives described herein include doubly derivatized chitosan compounds, such as cationic chitosan-polyol compounds. Generally, cationic chitosan-polyol compounds are functionalized with amino-containing moieties, such as arginine, lysine, ornithine, or guanidinium-containing molecules, or combinations thereof. In certain embodiments, cationic chitosan-polyol compounds have the structure of Formula I: [ka] In the formula, n is an integer of 1 to 650, α is the final degree of functionalization of cationic moieties (e.g., molecules containing amino groups such as lysine, ornithine, molecules containing guanidinium groups, arginine, or combinations thereof); β is the final degree of functionalization of the polyol; Each R 1 is independently selected from hydrogen, acetyl, a cation (e.g., arginine), and a polyol.

[0073] Preferably, the doubly derivatized chitosan of the present disclosure may be functionalized with the cationic amino acid, arginine.

[0074] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid at a final functionalization degree 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 at a final functionalization degree 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) at a final functionalization degree of about 1% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cationic moiety (e.g., arginine) at a final functionalization degree of about 10% to about 40%.

[0075] 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%.

[0076] 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%.

[0077] 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%.

[0078] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid at 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 at 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 at a final functionalization degree of about 10%.

[0079] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a hydrophilic polyol with 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 with 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 with a final degree of functionalization of about 10%.

[0080] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with glucose 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 glucose 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 glucose to a final degree of functionalization of about 10%.

[0081] In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 2% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionality of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 5% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionality of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with 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) with 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) with a final degree of functionalization of about 10% to about 40% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionalization of about 7.5% to about 15%, or about 10%.

[0082] In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 2% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionality of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 5% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionality of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with 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) with 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) with a final degree of functionalization of about 10% to about 35% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionalization of about 7.5% to about 15%, or about 10%.

[0083] In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 10% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionality of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 12% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionality of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 14% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with 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) with a final degree of functionalization of about 15% to about 30% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionalization of about 7.5% to about 15%, or about 10%.

[0084] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 25% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with 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) with a final degree of functionalization of about 28% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with 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) with a final degree of functionalization of about 25% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with 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) with a final degree of functionalization of about 28% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionalization of about 5% to about 20%.

[0085] In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) with a final functionalization degree of about 14% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 10%. In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) with a final functionalization degree of about 15% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 12%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine with a final functionalization degree of about 14% and chitosan conjugated with glucose with a final functionalization degree of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine with a final functionalization degree of about 15% and chitosan conjugated with glucose with a final functionalization degree of about 12%.

[0086] In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with a cation (e.g., arginine) with a final degree of functionalization of about 28% and chitosan conjugated with a hydrophilic polyol (e.g., glucose or gluconic acid) with a final degree of functionalization of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine with a final degree of functionalization of about 28% and chitosan conjugated with glucose with a final degree of functionalization of about 10%.

[0087] In some embodiments, DD-chitosan optionally includes DD-chitosan derivatives, such as DD-chitosan incorporating additional functionalization, such as DD-chitosan with attached ligands. "Derivatives" will be understood to include a broad class of chitosan-based polymers that contain covalently modified N-acetyl-D-glucosamine and / or D-glucosamine units, as well as chitosan-based polymers that incorporate other units or are attached 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, e.g., 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.

[0088] A.1. Chitosan-Nucleic Acid Polyplexes Chitosan derivative nanoparticle compositions generally contain at least one nucleic acid molecule, preferably a plurality of such nucleic acid molecules. Typical nucleic acid molecules contain phosphorus as a component of the nucleic acid backbone, for example in the form of a plurality of phosphodiesters or derivatives thereof (e.g., phosphorothioates). The ratio of cation-functionalized chitosan derivatives to nucleic acids can be characterized by the molar ratio of cations (+) to phosphorus (P), where (+) refers to the cation of the cation-functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, the molar ratio of (+):(P) is selected such that the chitosan-derivative-nucleic acid complex has a positive charge in the absence of the polyanion-containing block copolymer reversible coating. Thus, the molar ratio of (+):(P) is generally greater than 1. In preferred embodiments, the molar ratio of (+):(P) is greater than 1.5, at least 2, or greater than 2. In certain preferred embodiments, the molar ratio of (+):(P) is greater than 2.

[0089] 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.

[0090] 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 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 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 about 10:1 or less, about 3 to about 8:1 or less, or about 3 to about 7:1 or less.

[0091] 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 100:1. In some cases, the (+):(P) molar ratio can be greater than 2 and less than 100:1. In some cases, the (+):(P) molar ratio can be greater than 3 and less than 100:1. In some cases, the (+):(P) molar ratio can be greater than 5 and less than 100:1. In some cases, the (+):(P) molar ratio can be greater than 7 and less than 100:1. In some cases, the (+):(P) molar ratio can be greater than 2 and less than 50:1. In some cases, the (+):(P) molar ratio can be greater than 3 and less than 50:1. In some cases, the (+):(P) molar ratio can be greater than 5 and less than 50:1. In some cases, the (+):(P) molar ratio can be 7 or more and 50:1 or less. In some cases, the (+):(P) molar ratio can be greater than 2 and 25:1 or less. In some cases, the (+):(P) molar ratio can be 3 or more and 25:1 or less. In some cases, the (+):(P) molar ratio can be 5 or more and 25:1 or less. In some cases, the (+):(P) molar ratio can be 7 or more and 25:1 or less.

[0092] In some embodiments, the cationic functional group of the chitosan derivative nanoparticle 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 molecules containing guanidinium groups, lysine, ornithine, arginine, or combinations 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 molar ratio of amino (N) to phosphorus (P), where (N) refers to the nitrogen atom of the amino group of the amino-functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, the molar ratio of N:P is selected such that the chitosan derivative nucleic acid complex has a positive charge at a physiologically relevant pH in the absence of PEG-PA polymer molecules. Thus, the molar ratio of N:P is generally greater than 1. In preferred embodiments, the molar ratio of N:P is greater than 1.5, is at least 2, or is greater than 2. In certain preferred embodiments, the molar ratio of N:P is greater than 2.

[0093] 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.

[0094] 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 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 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 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.

[0095] 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 100:1. In some cases, the N:P molar ratio can be greater than 2 and less than 100:1. In some cases, the N:P molar ratio can be greater than 3 and less than 100:1. In some cases, the N:P molar ratio can be greater than 5 and less than 100:1. In some cases, the N:P molar ratio can be greater than 7 and less than 100:1. In some cases, the N:P molar ratio can be greater than 2 and less than 50:1. In some cases, the N:P molar ratio can be greater than 3 and less than 50:1. In some cases, the N:P molar ratio can be greater than 5 and less than 50:1. In some cases, the N:P molar ratio can be greater than 7 and less than 50:1. In some cases, the N:P molar ratio can be greater than 2 and less than 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.

[0096] In preferred embodiments, the subject 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., lower molecular weight (e.g., doubly) derivatized chitosans require higher N / P ratios, and vice versa.

[0097] The nucleic acids of the present disclosure will generally contain phosphodiester bonds, but may 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 made. Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids include, but are not limited to, DNA, RNA, and hybrids, and may contain any combination of deoxyribonucleotides and ribonucleotides, and 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.

[0098] In preferred embodiments, the polyplexes of the composition comprise chitosan molecules having an average molecular weight prior to 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 prior to functionalization of less than 15 kDa, less than 10 kDa, less than 7 kDa, or less than 5 kDa.

[0099] In a preferred embodiment, the polyplex comprises chitosan molecules having, on average, less than 680 glucosamine monomer units, more preferably less than 400 glucosamine monomer units, even more preferably less than 310 glucosamine monomer units, even more preferably less than 250 glucosamine monomer units, and most preferably less than 190 glucosamine monomer units. In some embodiments, the polyplex comprises chitosan molecules having, on average, less than 95 glucosamine monomer units, less than 65 glucosamine monomer units, less than 45 glucosamine monomer units, or less than 35 glucosamine monomer units.

[0100] Chitosan, including but not limited to those described herein, and (eg, doubly) derivatized chitosan nucleic acid polyplexes may be prepared by any method known in the art.

[0101] A.2. Nucleic acids As mentioned above, chitosan polyplexes can contain multiple nucleic acids. In one embodiment, the nucleic acid component comprises a therapeutic nucleic acid. The subject (e.g., double) 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 that affect cell apoptosis, factors that affect inflammation, factors that affect immune response (e.g., immune stimulators), and the like.

[0102] Therapeutic nucleic acids may be used to achieve gene therapy by serving as replacements or enhancements for defective genes, or to compensate for the lack of a particular gene product by encoding a therapeutic product. Therapeutic nucleic acids may also inhibit the expression of endogenous genes. Therapeutic nucleic acids may code for all or part of a translation product and function by recombining with DNA already present in the cell, thereby replacing the defective part of the gene. They may also code for part of a protein and exert their effect by co-suppression of the gene product.

[0103] In some embodiments, the nucleic acid component comprises a therapeutic nucleic acid construct. A therapeutic nucleic acid construct is a nucleic acid construct that can exert a therapeutic effect. A therapeutic nucleic acid construct may comprise a nucleic acid that encodes a therapeutic protein and a nucleic acid that produces a transcript that is a therapeutic RNA.

[0104] In a preferred embodiment described and exemplified herein, the therapeutic nucleic acid construct comprises a nucleic acid encoding IL-12, alone or in combination with additional immune stimulatory molecule(s). IL-12 is a heterodimeric type 1 cytokine with a four α-helical bundle structure. The active heterodimer, also known as IL-12p70, is composed of 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 of 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 of 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 of mouse IL-12B are, for example, NM_001303244 and NP_001290173.1.

[0105] In some embodiments, a single chain IL-12 protein 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 is secreted as a result of the inclusion of a signal peptide from the subunit 5' of the linker, while 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 (VPGVGVPGVG) derived from bovine elastin and composed of valine (V), proline (P) and glycine (G) residues. In some embodiments, the linker sequence may contain G residues such as (GGGGS)n and / or serine (S) residues. In other embodiments, the linker sequence may contain additional amino acids including, but not limited to, G, S, and P, arginine (R), lysine (K), threonine (T) and glutamic acid (E). In exemplary embodiments, the linker is selected from the group consisting of GSGSSRGSGSGGSGGGGSK (SEQ ID NO: 1), GSTSG(A / S)GKSSEGKG (SEQ ID NO: 2), GSTGSSGKPGSGEGSTKG (SEQ ID NO: 3), GGGGGGS (SEQ ID NO: 4) or GGGGSGGGSGGGGS (SEQ ID NO: 5).

[0106] In an exemplary embodiment, the nucleic acid sequence encoding hIL-12p40p35 comprises:

[0107] In an exemplary embodiment, the hIL-12p40p35 amino acid sequence comprises: M C H Q Q L V I S W F S L V F L A S P L V A I W E L K K D V Y V V E L D W Y P D A P G E M V V L T C D T P E E D G I T W T L D Q S S E V L G S G K T L T I Q V K E F G D A G Q Y T C H K G G E V L S H S L L L L H K K E D G I W S T D I L K D Q K E P K N K T F L R C E A K N Y S G R F T C W W L T T I S T D L T F S V K S S R G S S D P Q G V T C G A A T L S A E R V R G D N K E Y E Y S V E C Q E D S A C P A A E E S L P I E V M V D A V H K L K Y E N Y T S S F F I R D I I K P D P P K N L Q L K P L K N S R Q V E V S W E Y P D T W S T P H S Y F S L T F C V Q V Q G K S K R E K K D R V F T D K T S A T V I C R K N A S I S V R A Q D R Y Y S S S W S E W A S V P C S V P G V G V P G V G A R N L P V A T P D P G M F P C L H H S Q N L L R A V S N M L Q K A R Q T L E F Y P C T S E E I D H E D I T K D K T S T V E A C L P L E L T K N E S C L N S R E T S F I T N G S C L A S R K T S F M M A L C L S S I Y E D L K M Y Q V E F K T M N A K L L M D P K R Q I F L D Q N M L A V I D E L M Q A L N F N S E T V P Q K S S LEEPDFYKTKIKLCILLHAFRI RAVTIDRVMSYLNAS Stop (SEQ ID NO: 7)

[0108] In another exemplary embodiment, the therapeutic nucleic acid comprises a 4156 bp plasmid DNA (pDNA) comprised of a codon-optimized human interleukin-12 gene designated opt-hIL-12 encoding a polypeptide having the sequence of SEQ ID NO:7 (SEQ ID NO:8) linked to a constitutively active cytomegalovirus (CMV) promoter on an NTC9385R backbone with a sucrose-based antibiotic-free selection marker (RNA-OUT). The 4156 bp plasmid (SEQ ID NO:8) is shown in Table 1. [Table 1-1] [Table 1-2]

[0109] The R6K origin of replication restricts replication of the plasmid to certain 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) to monomerize p40 to p35 by adding a short repeated elastin linker sequence. The plasmid also consists of 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, which further recruits more immune cells to the local tissue. In a further embodiment, the therapeutic nucleic acid is packaged in a dual-derivatized chitosan polymer that is functionalized with arginine and glucose and coated with a removable PEG-b-PLE excipient to form the pharmaceutical composition EG-70. The composition is formulated as a nanoparticle aqueous dispersion in 1 w / w% mannitol solution, sterilized by filtration, lyophilized to a dry powder, and stored at 4° C. The average particle size of the nanoparticle dispersion is in the range of 75 to 175 nanometers.

[0110] Therapeutic nucleic acids also include therapeutic DNA in the form of circular double-stranded DNA plasmids, minicircular DNA (Science Report 6:2315, 2016) or closed-ended linear double-stranded DNA (Li et al, PLoS One, 8(8):e69879, 2013).

[0111] Therapeutic nucleic acids also include therapeutic RNA, which is an RNA molecule that can exert a therapeutic effect in mammalian cells.Therapeutic RNA includes, but is 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 RNA 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).

[0112] A.3. Expression control region In a preferred embodiment, the polyplexes of the present disclosure comprise a therapeutic nucleic acid that is a therapeutic construct that includes an expression control region operably linked to a coding region. The therapeutic construct produces a therapeutic nucleic acid, which may be therapeutic in its own right or may encode a therapeutic protein.

[0113] 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. By "dynamic" expression is meant expression that varies over time. Dynamic expression may include several such 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.

[0114] 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.

[0115] 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 the complete or non-mutant function (e.g., retain some degree of nutritional regulation or cell / tissue specific expression). As used herein, the term "functional" and grammatical variations thereof, when used in reference to a nucleic acid sequence, subsequence, or fragment, means that the sequence has 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 substitutions, deletions, or additions of a sequence, or other modifications (e.g., chemical derivatives, such as modified forms that are resistant to nucleases).

[0116] As used herein, the term "operably linked" refers to the physical juxtaposition of the described components that allows them to function in the 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. Usually, 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 usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.

[0117] 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. Usually, 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.

[0118] Numerous regulatable promoters are known in the art. Preferred inducible expression control regions include those that contain inducible promoters stimulated by small molecule chemical compounds. In one embodiment, the expression control region responds to chemicals that are orally deliverable but are 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.

[0119] Promoter / enhancer sequences of particular interest include: [Table 2]

[0120] In some embodiments of the present disclosure, the therapeutic construct is contained within a plasmid that includes an origin, 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 U.S. Pat. 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, 9,487,789, 9,573,091 ... See US Pat. Nos. 9,506,082, 9,550,998, 9,725,725, 9,737,620, 9,950,081, 10,047,365, 10,144,935, and 10,167,478. In some embodiments, the plasmid is "engineered" to remove antibiotic selection agents and / or to increase expression levels.

[0121] For further teachings, see WO2008 / 020318, which is expressly incorporated herein by reference in its entirety. In one embodiment, the nucleic acids of the (eg, doubly) derivatized chitosan-nucleic acid polyplexes are artificial nucleic acids.

[0122] 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.

[0123] In one embodiment, the therapeutic nucleic acid is DNA.

[0124] In one embodiment, the therapeutic nucleic acid comprises a nucleic acid sequence encoding a therapeutic protein, hi one embodiment, the therapeutic protein is IL-12.

[0125] B. Polyol Chitosan derivative nanoparticles can be functionalized with polyol. In general, polyols useful in the present disclosure 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 cationic moieties such as amino groups and polyols are called "doubly derivatized chitosan nanoparticles".

[0126] In some embodiments, the chitosan derivative nanoparticles comprise a polyol of Formula II: [ka] During the ceremony, 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.

[0127] In some embodiments, the chitosan derivative nanoparticles are functionalized with a polyol of formula II, where 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.

[0128] In some embodiments, the chitosan derivative nanoparticles comprise a polyol of formula III: [ka] During the ceremony, -----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;

[0129] [ka] represents the bond between the polyol and the derivatized chitosan.

[0130] In one embodiment, polyols according to the present disclosure having 3-7 carbons may have one or more carbon-carbon multiple bonds. In a preferred embodiment, polyols according to the present disclosure include carboxyl groups. In a more preferred embodiment, polyols according to the present disclosure include aldehyde groups. When polyols according to the present disclosure include aldehyde groups, one of skill in the art will recognize that such polyols include both open chain structures (aldehydes) and cyclic structures (hemiacetals).

[0131] Non-limiting examples of polyols include gluconic acid, threonic acid, glucose, and threose. Examples of other such 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. Patent 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.

[0132] 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.

[0133] 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.

[0134] In some embodiments, the polyol may be a compound of Formula IV or Formula V. [ka]

[0135] In a preferred embodiment, the polyol is a compound of formula IV: Optionally, the polyol of formula IV is attached to chitosan by reductive amination.

[0136] A hydrophilic polyol having 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 groups of the hydrophilic polyol may be attacked by unbound amines on the chitosan backbone according to a nucleophilic substitution reaction mechanism.

[0137] Those skilled in the art will recognize that when such hydrophilic polyols are attached to Arg-chitosan, the nucleophilic substitution reaction is likely to occur primarily at the amine groups of the chitosan backbone, although it is also possible that small amounts of the hydrophilic polyol may form covalent bonds with the amine groups of Arg via the same mechanism.

[0138] 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.

[0139] C. Polymer: Polyplex Composition Chitosan polyplexes can be mixed with multiple polymers, which include hydrophilic uncharged portions and negatively charged (anionic) portions. 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 polymer of the polymer component is unbranched. In some embodiments, the polymer is branched. In some cases, the polymer component comprises a mixture of branched and unbranched polymers.

[0140] In some embodiments, the polymer component is released from the chitosan polyplex after administration, after entering the cell, and / or after endocytosis. Without wishing to be bound by theory, it is hypothesized that by complexing a polyplex and an anionic moiety-containing polymer, 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 reduced mucoadhesive properties, for example, as compared to the same polyplex without the separate polymer component.

[0141] In preferred embodiments, the polyplex:polymer compositions have a low net positive, neutral, or 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 non-specific binding to ubiquitous anionic components in vivo. Such properties may enhance the movement of such compositions (e.g., enhanced diffusion through mucus) in contact with cells resulting in enhanced intracellular release of nucleic acids.

[0142] In preferred embodiments, the polyplex:polymer particle composition has 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 composition has 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 composition has 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 composition has 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 composition has 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.

[0143] 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%.

[0144] In one embodiment, the polyplex:polymer composition has an average zeta potential of +10 mV to -10 mV at physiological pH, and most preferably, +5 mV to -5 mV at physiological pH.

[0145] 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, even more preferably less than 0.3, even more preferably less than 0.25, and most preferably less than 0.2.

[0146] 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.

[0147] 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 lyophilization and rehydration. 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 spray drying and rehydration. 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 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, 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 when concentrated to a nucleic acid concentration of 125 μg / mL to about 1,000 μg / mL. In some cases, the dispersion of the polyplex:polymer composition exhibits 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, the dispersion of the polyplex:polymer composition exhibits 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, the dispersion of the polyplex:polymer composition exhibits 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, a dispersion of the polyplex:polymer composition exhibits 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.

[0148] In general, 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 fluids or simulated physiological fluids. For example, in some embodiments, the polyplex:polymer compositions described herein are stable when stored in simulated intestinal fluid, in mammalian urine, and / or in (e.g., in contact with) urine, and / or in a mammalian bladder.

[0149] As described above, the polyplex:polymer compositions described herein are preferably substantially size stable in composition. In a preferred embodiment, the compositions of the present disclosure comprise polyplex:polymer particles that increase in average diameter by less than 100%, more preferably less than 50%, and most preferably less than 25% at room temperature for 6 hours, more preferably less than 12 hours, more preferably less than 24 hours, and most preferably less than 48 hours. In a particularly preferred embodiment, the compositions of the present disclosure comprise polyplex:polymer particles that increase in average diameter by less than 25% at room temperature for at least 24 hours or at least 48 hours.

[0150] The polyplex:polymer particles of the subject compositions are preferably substantially size stable under cooling conditions, in a preferred embodiment, the compositions of the present disclosure comprise polyplex:polymer particles that increase in average diameter 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.

[0151] The polyplex:polymer particles of the subject compositions are preferably substantially size stable under freeze-thaw conditions. In a preferred embodiment, the compositions of the present disclosure comprise polyplexes that increase in average diameter by less than 100%, more preferably less than 50%, and most preferably less than 25% at room temperature for 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours after thawing from freezing at -20 to -80°C.

[0152] In a preferred embodiment, the composition has a nucleic acid concentration of 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 of 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.

[0153] 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 formulations and therapeutic applications.

[0154] In certain embodiments, the polyplex:polymer particle composition is not coated by lowering the pH, and can release all or part of the polymer coat, such as PEG. In certain embodiments, the polymer coat is released by incubating the particle under pH conditions below the pKa of the polyanionic anchor region of the polymer. For example, if the polymer coat is a polyglutamate, the polymer coat can be released by incubating the particle at a pH below the pKa of the polyglutamate, for example, at a pH below about 4.25. In certain embodiments, the polymer coat can be released by incubating the particle under pH conditions that are at least 0.25 pH units or at least 0.5 pH units below the pKa of the polyanionic anchor region of the polymer coat.

[0155] In certain embodiments, polyplex:polymer particle compositions can be uncoated by exposing the particles to high ionic strength, releasing all or a portion of the polymer coat, such as, for example, PEG.

[0156] Without wishing to be bound by theory, it is hypothesized that certain physiological conditions can 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, low pH conditions in certain intracellular compartments (e.g., endosomes, early endosomes, late endosomes, or lysosomes) can promote the release of the polymer coat. As another example, certain extracellular conditions can 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, the high ionic strength and / or acidic pH conditions commonly encountered at certain locations in the gastrointestinal tract can 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.

[0157] 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 polyplexes remain PEGylated, thereby facilitating transfection of the target cell. In some embodiments, 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%) 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 the cell, tissue, or body compartment (e.g., the intestine, small intestine, large intestine, colon, lung, or bladder).

[0158] 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 intended conditions.Similarly, it will be understood that the pH, volume, and ionic strength, as well as other conditions of the formulation, can be adjusted to promote or inhibit release under intended conditions.For example, for delivery to the intestine via the low pH stomach environment, the PEGylated polyplex formulation can be enteric coated and / or delivered in a buffer to increase 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.

[0159] Compositions comprising chitosan polyplexes complexed with anionic moiety-containing polymers can be characterized by the ratio of cationic functional groups (+) of the (e.g., doubly) derivatized chitosan polyplexes to anionic moieties (-) of the polymer, referred to as the "(+):(-) molar ratio." This (+):(-) molar ratio can vary from greater than about 1:100 to less than about 10:1.

[0160] In certain embodiments, the (+):(-) molar ratio can be greater than about 1:75 to less than about 8:1. In some cases, the (+):(-) molar ratio can be greater than 1:10 to less than 10:1. In some cases, the (+):(-) molar ratio can be from 1:10 or about 1:10 to 1:10 or about 1:10. In some cases, the (+):(-) molar ratio can be from 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 to less than about 10:1. In some cases, the (+):(-) molar ratio can be greater than 1:25 to less than about 10:1. In some cases, the (+):(-) molar ratio can be greater than 1:10 to less than about 7:1. In some cases, the (+):(-) molar ratio can be greater than 1:8 to less than about 7:1. In some cases, the (+):(-) molar ratio can be greater than 1:8 to less than about 6:1.

[0161] In certain embodiments, when the cationic functional groups of a (e.g., doubly) derivatized chitosan polyplex are amino moieties, a composition comprising a chitosan polyplex complexed with an anionic moiety-containing polymer can be characterized by the ratio of the amino groups (N) of the (e.g., doubly) derivatized chitosan polyplex to the 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.

[0162] In certain embodiments, the N:A molar ratio can be greater than about 1:75 to less than about 8:1. In some cases, the N:A molar ratio can be greater than 1:10 to less than 10:1. In some cases, the N:A molar ratio can be from 1:10 or about 1:10 to 10:1 or about 10:1. In some cases, the N:A molar ratio can be from 1:8 or about 1:8 to 8:1 or about 8:1. In certain embodiments, the N:A molar ratio can be greater than 1:50 to less than about 10:1. In some cases, the N:A molar ratio can be greater than 1:25 to less than about 10:1. In some cases, the N:A molar ratio can be greater than 1:10 to less than about 7:1. In some cases, the N:A molar ratio can be greater than 1:8 to less than about 7:1. In some cases, the N:A molar ratio can be greater than 1:8 to less than about 6:1.

[0163] 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 and anionic moieties (-) of the polymer (referred to as the "(+):P:(-) molar ratio").

[0164] In certain embodiments, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:40 to about 40:1. In certain embodiments, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:40 to about 1:10. In some embodiments, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:25 to about 25:1. In some embodiments, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:25 to about 1:10. In some cases, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:20 to about 20:1. In some cases, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:20 to about 1:10. In some cases, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:10 to about 10:1. In some cases, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:25 to about 2:1. In some cases, when (+):P is at least 2:1 to 20:1 or less, the molar ratio of (+):(-) can vary from at least 1:20 to about 1:1.

[0165] In certain preferred embodiments, (+):P:(-) is 3:1:3.5 to 3:1:17.5. In certain preferred embodiments, (+):P:(-) is 5:1:3.5 to 5:1:17.5. In certain preferred embodiments, (+):P:(-) is 7:1:3.5 to 7:1:17.5. In certain preferred embodiments, (+):P:(-) 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, (+):P:(-) 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.

[0166] One of skill 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 the amino functional groups (N) of the (e.g., doubly) derivatized chitosan polyplex to the phosphorus atoms (P) of the nucleic acid and the anionic moieties (A) of the polymer (referred to as the "N:P:A molar ratio"). In certain embodiments, the molar ratio of P:A can vary from at least 1:40 to about 40:1, with N:P being at least 2:1 to no more than 20:1.

[0167] In certain embodiments, when N:P is at least 2:1 to 20:1 or less, the molar ratio of P:A can vary from at least 1:40 to about 1:10. In certain embodiments, when N:P is at least 2:1 to 20:1 or less, the molar ratio of P:A can vary from at least 1:25 to about 25:1. In certain embodiments, when N:P is at least 2:1 to 20:1 or less, the molar ratio of P:A can vary from at least 1:25 to about 1:10. In some cases, when N:P is at least 2:1 to 20:1 or less, the molar ratio of P:A can vary from at least 1:20 to about 20:1. In some cases, when N:P is at least 2:1 to 20:1 or less, the molar ratio of P:A can vary from at least 1:20 to about 1:10. In some cases, when N:P is at least 2:1 and up to 20:1, the molar ratio of P:A can vary from at least 1:10 to about 10:1. In some cases, when N:P is at least 2:1 and up to 20:1, the molar ratio of P:A can vary from at least 1:25 to about 2:1. In some cases, when N:P is at least 2:1 and up to 20:1, the molar ratio of P:A can vary from at least 1:20 to about 1:1.

[0168] In certain preferred embodiments, N:P:A is 3:1:3.5 to 3:1:17.5. In certain preferred embodiments, N:P:A is 5:1:3.5 to 5:1:17.5. In certain preferred embodiments, N:P:A is 7:1:3.5 to 7:1:17.5. In certain preferred embodiments, N:P:A is 10:1:10 to 10:1:40. In certain preferred embodiments, N:P:A 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, N:P:A 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.

[0169] C.1. Hydrophilic uncharged moiety The hydrophilic uncharged portion of the polymer can be or include a polyalkylene polyol or polyalkyleneoxy polyol moiety, or a combination thereof. The hydrophilic uncharged portion of the polymer can be or include a polyalkylene glycol or polyalkyleneoxy glycol 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 a polyethylene glycol. The hydrophilic uncharged portion of the polymer can be or include other biologically compatible polymer(s), such as polylactic acid.

[0170] 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 portions of polymers 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).

[0171] The hydrophilic portion can 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.

[0172] C.2. Anionic Polymer Moiety The anionic polymer portion of the polymer can include 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, provided that such anionic polymers can be provided as components of a polymer having a hydrophilic, uncharged polymer portion and can form (e.g., reversible) charge:charge complexes with positively charged (e.g., doubly) derivatized chitosan-nucleic acid nanoparticles.

[0173] 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 that have 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.

[0174] In some cases, the anionic polymer can 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, keratin sulfate, heparin, heparin sulfate, hyaluronic acid, or combinations thereof.In certain embodiments, the anionic polymer portion of the polymer is or includes hyaluronic acid.

[0175] Additional or alternative anionic carbohydrate polymers may include polymers containing dextran sulfate.

[0176] In some cases, the polyanion portion is or includes a polyanion selected from the group consisting of polymethacrylic acid and its salts, polyacrylic acid and its salts, copolymers of methacrylic acid and its salts, and copolymers of acrylic acid and / or methacrylic acid and its salts, e.g., polyalkylene oxides, polyacrylic acid copolymers.

[0177] In some cases, the polyanionic portion 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, phosphate or sulfate groups, predominantly negatively charged polyamino acids, and biocompatible polyphenolic materials.

[0178] 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 moiety has a weight average molecular weight of about 1,000 Da to about 2,000 Da. In some cases, the anionic moiety has a weight average molecular weight of 1,500 Da or about 1,500 Da.

[0179] As used herein, "block copolymer," "block co-polymer," and the like, refer to a copolymer that contains distinct homopolymer regions. A diblock copolymer contains two distinct homopolymer regions. A triblock copolymer contains three distinct homopolymer regions. The three distinct regions can be different (e.g., AAAA-BBBB-CCCC), or the two regions can be the same (e.g., AAAA-BBBB-AAAA) or similar (e.g., AAAA-BBBB-AAA), where "A," "B," and "C" represent the inclusion of 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 this disclosure include those listed in the following non-exhaustive list: [Table 3-1] [Table 3-2]

[0180] In one embodiment, the block copolymer is or includes a PEG-polyglutamic acid polymer having the following structure: [ka]

[0181] In one embodiment, the block copolymer is or includes a PEG-polyaspartic acid polymer having the following structure: [ka]

[0182] In one embodiment, the block copolymer is or includes a PEG-hyaluronic acid polymer having the following structure: [ka]

[0183] D. Alternative Cationic Polymers and Lipids The nucleic acid polyplexes of the present disclosure function to condense and protect nucleotides from enzymatic degradation. In addition to chitosan and its derivatives, alternative materials that may also be advantageously used for this purpose include other positively charged (i.e., cationic) polymers and / or lipids.

[0184] Examples of cationic polymers that can be used to form polyplexes with the therapeutic nucleic acid constructs of the present disclosure include polyamines; polyorganoamines (e.g., polyethyleneimine (PEI), polyethyleneimine cellulose and its derivatives); poly(amidoamines) (PAMAM and its derivatives); polyamino acids (e.g., polylysine (PLL), polyarginine and its derivatives); polysaccharides (e.g., cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyltrialkylammonium), poly(4(-vinyl-N-alkyl-pyridium), 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).

[0185] Examples of positively charged lipids include esters of phosphatidic acid with amino alcohols, such as esters of dipalmitoylphosphatidic acid or distearoylphosphatidic acid with hydroxyethylenediamine. Further 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-dimethylhydroxyethylammonium 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 cationic lipids as described, for example, in Martin et al., Current Pharmaceutical Design, 2005, 11, 375-394.

[0186] Blends of lipids and polymers at any concentration and in any ratio can also be used. Mixing different polymer types in different ratios using various grades can result in properties borrowed from each of the contributing polymers. Various end group chemistries can also be employed.

[0187] III. How to make As noted above, one of skill in the art will appreciate that the polyplex:polymer particles of the present disclosure may be produced in a variety of ways. For example, polyplex particles may be produced and then contacted with a polymer. In an exemplary, non-limiting embodiment, the polyplex particles are prepared by providing and combining functionalized chitosan and nucleotide feedstocks. Feedstock concentrations may be adjusted to accommodate various amino to phosphate (N / P) ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly small batches, e.g., batches less than 2 mL, the functionalized chitosan and nucleotide feedstock may be mixed by gradually dropping the nucleotide feedstock into the functionalized chitosan feedstock while vortexing the vessel. In other embodiments, the functionalized chitosan and nucleotide feedstock 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.

[0188] Similarly, a polyplex particle source (e.g., an aqueous solution containing a polyplex composition) can be provided (e.g., isolated from a reaction mixture described above) and combined with a polymer source (e.g., an aqueous solution containing a polymer). The source concentrations can be adjusted to accommodate various amino-to-anion ratios (N / A), amino-to-phosphorus (N:P), N:P:A ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly in small batches, e.g., batches less than 2 mL, the sources can be mixed by slowly dropping a first source (e.g., polyplex) into 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).

[0189] 1. Powder formulation The polyplex:polymer compositions of the present disclosure include powders. In a preferred embodiment, the present disclosure provides dry powder polyplex:polymer compositions. In a preferred embodiment, the dry powder polyplex:polymer compositions are produced via dehydration (e.g., spray drying or freeze drying) of the chitosan-nucleic acid polyplex dispersions of the present disclosure.

[0190] 2. Pharmaceutical preparations The present disclosure also provides "pharmacologically acceptable" or "physiologically acceptable" formulations comprising the polyplex:polymer compositions of the present disclosure. Such formulations can be administered in vivo to a subject to practice the disclosed methods of treatment.

[0191] As used herein, the terms "pharmacologically acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients, etc. that can be administered to a subject, preferably without causing 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.

[0192] Pharmaceutical preparations 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 preparations can be contained in tablets (coated or uncoated), capsules (hard or soft), microbeads, emulsions, powders, granules, crystals, suspensions, syrups, or elixirs. Among other additives, auxiliary active compounds and preservatives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, can also be present.

[0193] The excipients may include salts, isotonicity agents, serum proteins, buffers or other pH adjusting agents, antioxidants, thickening agents, uncharged polymers, preservatives, or cryoprotectants. The excipients used in the compositions of the present disclosure may further include isotonicity agents and buffers or other pH adjusting agents. These excipients may be added to obtain a preferred range of pH (about 6.0-8.0) and osmolality (about 50-400 mmol / L). 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 any of 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 osmolality that is the same 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 particular isotonicity 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 specifically 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 composition of the present disclosure may further contain a preservative. Examples of preservatives are polyhexamethylene-biguanidine, benzalkonium chloride, stable oxychloro complexes (known as Purite®), phenylmercuric acetate, 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 composition of the present disclosure may also contain a cryopreservative. Preferred cryopreservation agents are glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran with a preferred molecular weight of less than 100,000 g / mol, glycerol, and polyethylene glycol with a molecular weight of less than 100,000 g / mol, or mixtures thereof. Most preferred are glucose, trehalose, and polyethylene glycol.Typically, such cryopreservatives are present at a concentration of about 0.01-10%.

[0194] 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, troches, capsules, such as gelatin capsules, or coatings, such as enteric coatings (Eudragit® or Sureteric®). Pharmaceutically compatible binding agents, and / or auxiliary substances can be included in the oral formulations. 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.

[0195] The formulation may also include a carrier to protect 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.

[0196] 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.

[0197] Additional pharmaceutical formulations suitable for administration are known in the art and are applicable to the methods and compositions of the present disclosure (see, 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)).

[0198] IV. Administration In one embodiment, the use of polyplex:polymer compositions provides long-term stability of the polyplexes at physiological pH, which provides for effective administration.

[0199] Any of a number of administration routes for contacting cells or tissues are possible, and the choice of a particular route will depend, in part, on the target cells or tissues. Syringes, endoscopes, cannulas, intubation tubes, catheters, nebulizers, inhalers, and other items may be used for administration.

[0200] In some embodiments, the cancer is bladder cancer. Intravesical administration of chemotherapy is the standard treatment for some bladder cancers. Briefly, intravesical therapy involves injecting therapeutic agents directly into the bladder by inserting a urinary catheter. In some embodiments, the subject compositions provide enhanced stability in urine, thereby improving localized expression.

[0201] Although preventing or inhibiting the progression or worsening of a disorder or condition, or symptoms, is a good outcome, a dose or "effective amount" for treating a subject is preferably sufficient to improve one, some, or all of the symptoms of the condition to a measurable or detectable extent. Thus, for a condition or disorder 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 disclosed method 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, sex, age, etc.). An effective amount can be ascertained by measuring the relevant physiological effect.

[0202] Veterinary applications are also contemplated by the present disclosure. Thus, in one embodiment, the present disclosure provides a method of treating a non-human mammal comprising administering a polyplex:polymer composition of the present disclosure to a non-human mammal in need of treatment. The compositions of the present disclosure may also be administered to a mucosa. For example, the compositions can be administered to mucosal cells or tissues of the digestive 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, airway epithelium, lung, vagina, and bladder. Other target cells or tissues include, but are not limited to, cells of the breast, colon, prostate, pancreas, skin, lung, ovary, kidney, brain, bladder, vagina, cervix, stomach, digestive tract, kidney, liver, thyroid, esophagus, nasal cancer, larynx, oral cancer, pharyngeal cancer, retinoblastoma, endometrium, and testis.

[0203] Typical formulations for this purpose include solutions, gels, hydrogels, solutions, creams, foams, films, implants, sponges, fibers, powders, and microemulsions.

[0204] The compounds of the present disclosure can typically be administered intranasally or to mucous membranes by inhalation 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), or as an aerosol spray from a pressurized container, pump, spray, atomizer, or nebulizer, with or without the use of a suitable propellant.

[0205] Capsules, blisters, and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the present disclosure, a suitable powder base such as lactose or starch, and a performance modifier such as I-leucine, mannitol, or magnesium stearate.

[0206] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release.

[0207] The compounds of the present disclosure may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

[0208] Formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release.

[0209] The compounds of the present disclosure may also be administered directly to the eye or ear, usually in the form of liquid 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.

[0210] Formulations for ocular / aural administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed release.

[0211] 1. Mucosal Administration In some embodiments, the composition of the present disclosure is administered to mucosa.For example, the composition can be administered to mucosa cells or mucosa tissues of the digestive tract, including but not limited to mucosa cells or mucosa tissues of the bladder, and mucosa cells or mucosa tissues of the small intestine and / or large intestine and / or colon.Other target mucosa cells or mucosa tissues include but are not limited to eye, airway epithelium, lung, vagina, and bladder cells or tissues.

[0212] Typical formulations for this purpose include solutions, gels, hydrogels, solutions, creams, foams, films, implants, sponges, fibers, powders, and microemulsions.

[0213] In an exemplary embodiment relating to the bladder mucosa, the compounds described herein can be administered using intravesical therapy. Intravesical therapy involves injecting a therapeutic agent directly into the bladder by insertion of a urethral catheter. The agent is placed in the bladder for 0.5 to 6 hours. It is the standard administration route for bladder cancer chemotherapy. It takes advantage of the external anatomical access available to deliver the agent directly to the diseased site in the bladder, thereby avoiding unnecessary exposure of the injected agent to healthy tissue elsewhere in the body.

[0214] Formulations for bladder administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted, or programmed release.

[0215] The compounds of the disclosure 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), or as an aerosol spray from a pressurized container, pump, spray, atomizer, or nebulizer, with or without the use of a suitable propellant.

[0216] Capsules, blisters, and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the present disclosure, a suitable powder base such as lactose or starch, and a performance modifier such as I-leucine, mannitol, or magnesium stearate.

[0217] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release.

[0218] The compounds of the present disclosure may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

[0219] Formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release.

[0220] The compounds of the present disclosure may also be administered directly to the eye or ear, usually in the form of liquid 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.

[0221] Formulations for ocular / aural administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed release.

[0222] 2. Intratumoral administration In some embodiments, the composition of the present disclosure is administered directly to a tumor (cancer). For example, 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 comprising a nucleic acid encoding at least one RIG-I agonist can be directly contacted with the cancer and administered locally, and the composition can be administered to the cancer in tissues such as breast, prostate, skin, lung, brain, bladder, stomach, kidney, etc., in which the therapeutic nucleic acid constructs encoding IL-12 and RIG-I are the same or different nucleic acid constructs.

[0223] Intratumoral injections are known in the art (see, e.g., Melero et al., (2021), Nature Reviews Clinical Oncology, 18:558-576).

[0224] V. Therapeutic Applications The methods disclosed herein activate strong memory T cell responses against cancer antigens. Thus, the therapeutic proteins contemplated for use in the present disclosure have a wide variety of activities and are used in the treatment of a wide variety of disorders. Thus, the following description of therapeutic protein activity and the description of indications treatable with the therapeutic nucleic acids and proteins of the present disclosure are exemplary and are not intended to be exhaustive. The term "subject" refers to animals, with mammals being preferred, and humans being particularly preferred. Specific non-limiting examples of therapeutic embodiments are described below.

[0225] In some therapeutic embodiments, the therapeutic polyplex:polymer composition is applied directly to the tumor, for example, by intratumoral injection. When the therapeutic effect is applied to metastatic disease, the cells or tissues contacted by the polyplex:polymer compositions described herein are neoplastic, but the therapeutic effect is distal to the primary tumor or primary target tissue.

[0226] In some cases, therapeutic embodiments are applied to mucosal tissues but are intended to affect non-mucosal target tissues, cells, or organs. In such embodiments, where the therapeutic effect is non-mucosal, it is understood that the cells or tissues contacted by the polyplex:polymer compositions described herein are mucosal. In some embodiments, 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 immune stimulating molecule. However, in other embodiments where the therapeutic effect is non-mucosal, such as metastatic disease, the cells or tissues contacted by the polyplex:polymer compositions described herein are mucosal, but the therapeutic effect is distal to the primary tumor or primary mucosal target tissue.

[0227] In one embodiment, the polyplex:polymer compositions of the present disclosure may be used for therapeutic or prophylactic treatment. Such compositions may be referred to herein as therapeutic compositions. As noted above, the subject compositions and methods primarily employ therapeutic nucleic acids encoding IL-12, alone or in conjunction with additional innate and / or adaptive immune stimulatory molecules, e.g., RIG-I agonists. In some embodiments, the therapeutic nucleic acid further encodes an IFN-1 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.

[0228] Suitable IFN-1 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., CDN, i.e., cyclic dinucleotides), PRRago (e.g., CpG, Imiquimod or poly I:C), and TLR agonists including TRL7 and TLR9 (e.g., CPG-1826, GS-9620, AED-1419, CYT-003-QbG10, AVE-0675 or PF-7909), 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.

[0229] In a preferred embodiment, the IFN-1 activator / inducer is a RIG-I agonist. RIG-I (retinoic acid-inducible gene I, encoded by Ddx58) is a cytoplasmic antiviral helicase that acts as an RNA sensor, detecting viral RNA in the cytoplasm and being activated upon its recognition. A pattern recognition receptor, RIG-I contains an RNA helicase domain and two N-terminal caspase recruitment domains (CARDs), which relay the signal to the downstream signaling adaptor MAVS (mitochondrial antiviral-signaling protein). RIG-I signaling through MAVS leads to various responses, including induction of type I IFN responses, including IFNα and IFNβ, and activation of caspase 8-dependent apoptosis, via TBK1 and IRF7 / 8. They are found in most tissues, including cancer cells (Kato et al., Immunol. Rev. 243(1):91-98(2011)).

[0230] The response to RIG-I induction 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 ligands of RIG-I are viral short blunt ends of double-stranded RNA containing 5' triphosphate or 5' 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). The potent antitumor activity of RIG-I ligands is in part due to their ability to bind CD8 + and 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 models of viral infection such as influenza (Weber-Gerlach & Weber, 2016).

[0231] 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 specifically used as agonists in this disclosure. These include, but are not limited to, (i) eRNA11a, an immunostimulatory dsRNA expressed by convergent transcription, combined with (ii) eRNA41H, an 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, alternative polyphosphorylated RNAs with a stable tetraloop at one end (Jiang et al., J. Exp. Med. 216:2854-68(2019)).

[0232] Additional RIG-I agonists that may be advantageously used in the compositions and methods described herein include SB-9200, a broad spectrum antiviral natural 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)), MK 4621 (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 DVG (Xu et al. al., mBio, 65:e01265-15 (2015)), 5'pppRNA with a uridine-rich sequence with a 99-nucleotide hairpin (M8) (Chiang et al. J. Virol. 89:8011-25 (2015)), and 3pRNA.

[0233] In accordance with the foregoing embodiments, suitable RIG-I agonists for co-expression with IL-12 in the subject compositions and methods include RIG-I DNA vaccines, plasmid-encoded RNA polymerase III-expressed RNA-based RIG-I agonists, such as eRNA11a, adenovirus VA RNA1, eRNA41H (Nature Technology Corp), GFP2, Lamin A / C and Lamin VSV, tri-GFPs, SAD ΔPLp, tri-G-AC-U, Flu vRNA, RNase L 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 Ellermeier et al., Cancer Research, (2013), 73 (6) disclosed siRNA.Similarly, STING agonists suitable for co-expression with IL-12 include, but are not limited to, DExD / H helicase, including DDX41, and TLR agonists include, but are not limited to, CpG dinucleotides, such as, for example, CpG-1826 (ODN1826, Invivogen).

[0234] In accordance with the foregoing embodiments, modulators of immune checkpoint molecules suitable for co-expression with IL-12 in the subject compositions and methods include, for example, single domain antibodies (sdAbs) directed to 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) (see also 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 (e.g., competitive antagonists) with high affinity for PD-L1 (Maute, PNAS (2015)); and CD80 mutant(s) with increased binding to CD28 (e.g., WO2017 / 181152).

[0235] In some cases, the IFN-1 agonist and / or the immune checkpoint inhibitor is encoded by: - said therapeutic nucleic acid construct in said derivatized chitosan-nucleic acid polyplex; - different therapeutic nucleic acid constructs in said derivatized chitosan-nucleic acid polyplexes; - therapeutic nucleic acid constructs within different derivatized chitosan nucleic acid polyplexes (e.g., not including a construct encoding IL-12); - Therapeutic nucleic acid constructs (e.g., formulated in alternative nucleic acid delivery formulations such as PEI or cationic lipid formulations).

[0236] The therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding the IFN-1 agonist and / or the immune checkpoint inhibitor can be administered simultaneously or sequentially. In some cases, the therapeutic nucleic acid construct encoding the IFN-1 agonist and / or the immune checkpoint inhibitor are co-administered in a single formulation or in 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-1 agonist and / or the immune checkpoint inhibitor are administered sequentially.

[0237] The immunostimulatory molecules of the present disclosure may encode shRNA (short hairpin RNA) molecules designed to inhibit a protein(s) involved in the growth or maintenance of tumor cells or other hyperproliferative cells. The plasmid DNA may simultaneously encode a therapeutic protein and one or more shRNAs. Furthermore, the nucleic acid of the composition may be a mixture of plasmid DNA and synthetic RNA, including sense RNA, antisense RNA, or ribozymes.

[0238] VI. Treatment Methods Hyperproliferative disorders The subject compositions and methods are advantageously used in the treatment of hyperproliferative disorders. Exemplary hyperproliferative disorders include those of the breast, colon, prostate, pancreas, skin, lung, ovary, kidney, brain, bladder, vagina, cervix, stomach, digestive tract, kidney, liver, thyroid, esophagus, nasal cavity, larynx, oral cavity, throat, retina, endometrium, testes, etc. Of particular interest are compositions and methods for treating hyperproliferative disorders that have metastasized from a primary cancer / tumor to a site different from the primary cancer.

[0239] In some embodiments, the hyperproliferative disorder is a hyperproliferative disorder in or near mucosal tissue.The methods and compositions of the present disclosure may be used to treat gastrointestinal cancer, including but not limited to oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, and rectal cancer.Nasal and lung cancers that can be treated by the methods and compositions of the present disclosure include but are not limited to paranasal sinus cancer, oropharynx cancer, tracheal cancer, and lung cancer.Genitourinary cancers that can be treated by the methods and compositions of the present disclosure 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.

[0240] In some embodiments of any one of the methods provided above, the method further comprises administering (systemically or locally to the site of the tumor) a non-nucleic acid based immune stimulatory molecule.

[0241] In some embodiments, the immune stimulatory 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 PD-L1 or an inhibitor of PD-L1. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab. In some embodiments, the immunomodulatory agent is an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, e.g., ipilimumab or tremelimumab. In some embodiments, the inhibitor of PD-L1 is an anti-PD-L1 antibody, e.g., atezolizumab.

[0242] In some embodiments, the immunomodulatory agent is an IFN-1 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, US993 US20200055871A1, US20200016253A1, US20190076463A1, US20180195063A1, US20160287623A1.

[0243] 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-dimethylxanthenone-4-acetic acid (DMXAA), MK-1454 (Merck), ML RR-S2 CDG, ML RR-S2CDA (ADU-S100), SB11285 (Springbank Pharmaceuticals), MAVU (AbbVie), DiABZI, disodium dithio-(Rp1Rp)-[cyclic[A(2'5')pA(3'5')p]][Rp,Rp]-cyclic 9 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 US10,176,292, US9,724,408, US10,011,630, US10,435,469, US10,414,747, US10,413,612, US10,131,686, US10,106,574, US10,047,115, US10,045,961, US10,011,630, US9,994,607, US9,937,247, US9,840,533, US9,770,467, US9,724,408, US9,718,848, and US9,642,830.

[0244] Suitable TLR7 and TLR9 agonists for co-administration with IL-12 include imidazoquinolines and their analogs, including resiquimod and imiquimod (Aldara), hydroxychloroquine, chloroquine (chloroquire), bropirimine, loxoribine, isatoribine, CpG oligonucleotides, stabilized immunomodulatory RNA (SIMRA), AST-008 (Exicure), MEDI9197, and US434,064, US10,413,612, US10,407,431, US10 ,370,342, US10,364,266, US10,208,037, US10,202,386, US9,944,649, US9,902,730, US9,868,955, US9,359,360, US9,295,732, US9,243,050, US9,228,184, US9,216,192, US9,2206,430, US8,735,421, US8,728,486, US8,399,423 and US8,242,106.

[0245] In some embodiments, the non-nucleic acid based immunomodulatory agent and the subject compositions are administered simultaneously, such as in the same composition, hi some embodiments, the non-nucleic acid based immunomodulatory agent and the subject compositions are administered sequentially.

[0246] In some embodiments, the methods for treating cancer provided herein further comprise administering to the subject at least one additional therapeutic agent. In further embodiments, the additional therapeutic agent is a chemotherapeutic agent or a radiotherapeutic agent. In some embodiments, chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, paclitaxel, docetaxel, 5-fluorouracil 1, bleomycin, methotrexate, ifosamide, oxaliplatin, cyclophosphamide, dacarbazine, temozolomide, gemcitabine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, pentostatin, thioguanadine, 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, rubrutinib, imatinib, ipilimumab, lapatinib, nilotinib, and the like. The additional therapeutic agent may include, but is not limited to, nib, obinutuzumab, ofatumumab, panitumumab, pazopanib, pertuzumab, ponatinib, regorafenib, rituximab, romidepsin, ruxolitinib, sipuleucel-T, sorafenib, temsirolimus, tocilizumab, tofacitinib, tositumomab, trametinib, trastuzumab, vandetanib, vemurafenib, vismodegib, vorinostat, Ziv-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.

[0247] In some embodiments, the cancer treated using the methods disclosed herein is bladder cancer. The conventional bladder cancer treatment currently approved in the United States is the intraurethral Bacillus Calmette-Guerin vaccine. This antigen vaccine is believed to stimulate bladder cells to express interferon, which then recruits 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, this vaccine is ineffective. Similarly, intravesical instillation of exogenously produced interferon polypeptides has been investigated but has not shown efficacy. The subject compositions and methods can also be advantageously employed in conjunction with these conventional approaches to enhance and improve immune responses.

[0248] The examples presented herein illustrate some embodiments of the present disclosure, but should not be construed as limiting the scope of the disclosure in any way. EXAMPLES

[0249] Example 1: Evaluation of long-lasting anti-tumor immunity in an orthotopic model of bladder cancer To evaluate the long-lasting antitumor immunity of mEG-70 nanoparticles, an orthotopic model of mouse bladder cancer was used. Briefly, the codon-optimized mouse IL-12 (opto-mouse IL-12p40p35) gene encodes two subunits (p40 and p35) of the cytokine protein IL-12. To ensure 1:1 stoichiometry of the subunits, the mEG-70 plasmid was designed to contain a single open reading frame (ORF) that monomerizes p40 to p35 by adding a short repeated elastin linker sequence. The codon-optimized sequence was cloned into the NTC9385R or NTC9385R-eRNA41H vector backbone and expression was confirmed as disclosed in WO2020 / 183239, which is incorporated herein by reference in its entirety. The plasmid contains genes for eRNA11a (immunostimulatory double-stranded ribonucleic acid [dsRNA]) and adenovirus VA RNA1. The RNA products of two of these genes stimulate the RIG-I pathway, thereby recruiting more immune cells to local tissues.

[0250] This therapeutic nucleic acid is packaged into a dual-derivatized chitosan polymer that is functionalized with arginine and glucose and coated with a removable PEG-b-PLE excipient to form the pharmaceutical composition mEG-70 as disclosed in WO2020 / 183239.

[0251] Disease was established by pretreating mouse bladders with poly-L-lysine to promote the detachment of the urothelial surface layer and facilitate the implantation of cancer cells. Urothelial cancer cells stably overexpressing the luciferase gene (MB49-Luc) were then injected into the mouse bladders (100,000 cells per mouse) and luciferase expression was confirmed using an in vivo imaging system (IVIS) 9 days after injection. The intensity of the bioluminescent signal was used to assign animals to treatment groups (n=22) based on the level of bioluminescence. Animals without a positive bioluminescent signal were excluded from the study.

[0252] Under isoflurane anesthesia, mice received intravesical instillations (IVI) of mEG-70 (1 mg DNA / mL; equivalent to 80 μg DNA) on days 10 (Tx1) and 17 (Tx2), while control animals received instillations of 1% mannitol (sham treatment). This dosing regimen was selected based on evaluation of protein expression kinetics determined as disclosed in WO2020 / 183239. A cohort of tumor-bearing animals was untreated.

[0253] Following injection of MB49-Luc cells, survival was monitored for 85 days. Statistical significance was analyzed by log-rank (Mantel-Cox) test (*p<0.05 and **p<0.01 for mEG-70 compared to sham and untreated, respectively). (C) Surviving tumor-free mEG-70-treated mice (negative bioluminescence signal and no clinical symptoms) and age-matched controls were injected with MB49-Luc cells (1×10 5 Tumor engraftment was monitored by in vivo bioluminescence imaging 7, 14, and 21 days after reloading (study days 92, 99, and 106, respectively).

[0254] As shown in Figure 1B, mEG-70 treated animals showed prolonged survival compared to control mice, of which approximately 70% died from the disease. The survival curves for mEG-70 were significantly different from the survival rates of sham-treated (1% mannitol) or untreated mice (*p<0.05 and **p<0.01, respectively).

[0255] Treated mice that showed complete disease regression and no recurrence during the 76-day observation period (referred to as "mEG-70 cured") were rechallenged with MB49-Luc cells to assess protection from recurrent disease. In contrast to age-matched untreated controls, which showed robust tumor engraftment in 15 of 17 mice, all mEG-70 cured mice were resistant to tumor recurrence for up to 3 weeks after rechallenge (n=17). See Figure 1C.

[0256] Taken together, these data suggest that long-lasting systemic antitumor immunity is established in response to mEG-70 treatment.

[0257] Example 2: Distant Tumor Rechallenge As described in Example 1, MB49-luciferase cells (MB49-Luc; 1 × 10 5 Cells) were injected into the bladder of female C57BL / 6J mice (12-16 weeks old) and engraftment was confirmed by in vivo imaging of luciferase signal 9 days after injection (using a Lumina LT IVIS imaging system). Mice were evenly distributed into treatment groups (n=22) based on their levels of bioluminescence (luciferase-negative mice were excluded from the study) and received intravesical instillation (IVI) of mEG-70 nanoparticles (1 mg DNA / mL, equivalent to 80 μg DNA, see Example 1) on days 10 (Tx1) and 17 (Tx2), while control animals received instillation of 1% mannitol (sham treatment). A cohort of tumor-bearing animals was untreated.

[0258] Survival was monitored until all mice died of bladder cancer or were deemed tumor-free (negative bioluminescence signal and no clinical symptoms). On day 85, surviving tumor-free mEG-70-treated mice and age-matched controls were inoculated with MB49-Luc cells (1 × 10 5 All mEG-70-treated mice remained tumor-free and were rechallenged by IVI with MB49-Luc (1 × 10 5 cells; Fig. 2B) or B16-F10 cells (1 × 10 5 The tumors were then reloaded subcutaneously in the flank with either 0.1% T4+ cells (Figure 2C). A cohort of age-matched animals was also included as a control for subcutaneous cell implantation. Tumors were monitored by measuring with a caliper; tumor volume was calculated according to the formula (length × width). 2 / 2).

[0259] As shown in Figure 2B, mEG-70-treated animals were protected from distant tumor rechallenge with MB49-Luc cells. Only one of nine animals showed tumor growth, which progressed significantly more slowly. In contrast, tumors grew in eight of nine mice in the untreated control cohort.

[0260] Mice were rechallenged with B16-F10 cells to assess the specificity of the response. All mice in the rechallenge and untreated control groups showed robust B16-F10 tumor engraftment (n=8 / group). See Figure 2C.

[0261] Taken together, these data suggest that long-lasting systemic and specific antitumor immunity is established in response to mEG-70 treatment.

[0262] Example 3: T cell depletion during distant tumor rechallenge The following examples demonstrate that the long-lasting systemic anti-tumor immunity achieved with the subject therapeutic methods is T cell dependent.

[0263] As described in Example 1, MB49-luciferase cells (MB49-Luc; 1 × 10 5 Cells) were injected into the bladder of female C57BL / 6J mice (12-16 weeks old) and engraftment was confirmed by in vivo imaging of luciferase signal 9 days after injection (using a Lumina LT IVIS imaging system). Mice were equally distributed into treatment groups (n=20) based on the level of bioluminescence (luciferase-negative mice were excluded from the study). Figure 3A provides a schematic of the experimental timeline.

[0264] Mice received intravesical instillations (IVI) of mEG-70 nanoparticles (1 mg DNA / mL; equivalent to 80 μg DNA, see Example 1) on days 10 (Tx1) and 17 (Tx2), while control animals received instillations of 1% mannitol (sham treatment). Survival was monitored until all mice died of bladder cancer or were deemed tumor-free (negative bioluminescence signal and no clinical symptoms).

[0265] On day 167, surviving tumor-free mEG-70-treated mice and age-matched untreated controls were injected intraperitoneally with isotype control (non-depleted), anti-CD4 antibody, or anti-CD8 antibody for 4 consecutive days to establish depletion, and then twice weekly for maintenance. After the third depleting antibody injection, mice were injected with MB49-Luc cells (1 × 10 5 The tumors were reloaded (day 170; n=6). Tumors were monitored by caliper measurement. Tumor volume was calculated according to the formula [length × width 2 The results are summarized below and shown in Figure 3 (B–D).

[0266] All untreated mice that received an isotype control antibody (non-depleting) had growing subcutaneous tumors. In contrast, all mice treated with mEG-70 were protected from rechallenge of distant tumors with MB49-Luc cells (Figure 3B).

[0267] All mice that received anti-CD4 antibodies (CD4 + T cell depleted) had growing MB49-Luc subcutaneous tumors, whether untreated or previously treated with mEG-70 treatment ( Fig. 3C ).

[0268] All untreated mice that received anti-CD8 antibodies (CD8+ T cell depletion) had growing subcutaneous tumors. In contrast, only one of six animals treated with mEG-70 had an actively growing tumor (Figure 3D). Notably, two of six mEG-70-treated mice that were depleted of CD8+ T cells had small tumor masses that were quiescent and had delayed growth.

[0269] Thus, protection from rechallenge in mEG-70-treated mice is compromised in the absence of CD4+ T cells, thus providing long-lasting and systemic antitumor immunity that is primarily mediated by CD4+ T cells.

[0270] Example 4: Reloading on the Contralateral Flank The following examples demonstrate that mEG-70 administered by direct intratumoral injection in subcutaneous tumors produces antitumor responses that are both long-lasting and systemic.

[0271] MB49-luciferase cells (MB49-Luc; 2.5 × 10 in 100 μL) 5 The cells were subcutaneously transplanted into the right flank of anesthetized C57BL / 6J mice (12-16 weeks old). 3 Once this was reached, mice were randomly assigned to treatment groups (n=10).

[0272] Mice were administered mEG-70 nanoparticles (0.5 mg DNA / mL in 50 μL, equivalent to 25 μg DNA) directly intratumorally (IT) on days 1, 4, 8, 11, 15, and 18, whereas control animals received 1% mannitol (sham treatment). A cohort of tumor-bearing animals was untreated. Tumor size was monitored three times a week by measurement with a vernier caliper and tumor volume was calculated according to the formula [length × width 2 / 2] was used to calculate the concentration (Figure 4A).

[0273] To confirm that tumors had not recurred, bioluminescence imaging of luciferase signal was performed using a Lumina LT IVIS imaging system on day 70 in tumor-free mEG-70-treated mice (mEG-70 "cured"; n=9). On day 73, mEG-70-cured and age-matched controls received MB49-Luc cells (2.5×10 in 100 μL) in the left flank. 5 Tumors were monitored three times a week by measuring with a caliper; tumor volume was calculated by the formula [length × width 2 / 2] was used to calculate the coefficient of variation.

[0274] As shown in Figure 4B, intratumoral (IT) administration of mEG-70 inhibited tumor growth compared to sham-treated mice. Furthermore, mEG-70 "cured" mice were protected from tumor cell rechallenge in the contralateral flank (Figure 4C).

[0275] Thus, mEG-70 administered by direct intratumoral injection in subcutaneous tumors produced a long-lasting and systemic antitumor response.

[0276] Example 5: Human Clinical Trial in Metastatic Bladder Cancer 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 by 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).

[0277] After confirming the presence of bladder cancer from pathology, physicians develop an ongoing treatment plan that often involves BCG therapy. Despite significant adverse effects and a failure rate of 30%-40%, intravesical immunotherapy with BCG is the primary treatment used to prevent recurrence and / or progression in patients with advanced (Ta or higher) NMIBC. Although NMIBC patients who do not respond to BCG are highly unlikely to benefit from further treatment with BCG and therefore represent a unique population for the study of novel therapies, BCG is often administered in a second maintenance course to achieve a disease-free state (Jarow et al., 2015).

[0278] In the absence of pharmacological intervention or cystectomy, BCG-refractory NMIBC will persist and progress, with or without resected disease. Currently, there is no effective treatment available for patients who fail BCG, as gemcitabine and mitomycin, often administered after TURBT, are not effective salvage agents. Thus, the treatment for BCG-refractory disease is radical cystectomy, which surgically removes all tumor (regardless of BCG refractory or recurrent) to ensure disease-free survival. The scarcity of treatment options available for NMIBC and the fact that patients continue to undergo radical organ resections for early stage disease explain a truly large unmet medical need. More effective treatments that are effective in refractory patients are urgently needed in NMIBC.

[0279] In an exemplary embodiment, the therapeutic nucleic acid comprises a 4156 bp plasmid DNA (pDNA) composed of a codon-optimized human interleukin-12 gene, designated opt-hIL-12, linked to a constitutively active cytomegalovirus (CMV) promoter on an NTC9385R backbone with a sucrose-based antibiotic-free selection marker (RNA-OUT), as set forth in SEQ ID NO:8.

[0280] The R6K origin of replication restricts replication of the plasmid to certain 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) that monomerizes p40 through p35 by adding a short repeated elastin linker sequence. The plasmid also consists of 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 the local tissue. In a further embodiment, the therapeutic nucleic acid is packaged into a dual-derivatized chitosan polymer functionalized with arginine and glucose and coated with a removable PEG-b-PLE excipient to form the pharmaceutical composition EG-70. The composition is formulated as a nanoparticle aqueous dispersion in 1 w / w% mannitol solution, sterilized by filtration, lyophilized to a dry powder, and stored at 4° C. The average particle size of the nanoparticle dispersion is in the range of 75 to 175 nanometers.

[0281] This study will evaluate the safety of intravesical administration of EG-70 at a distal site and its effect on bladder tumors in patients who have failed BCG therapy and are awaiting radical cystectomy. The study will be a classical dose escalation study with three patients treated in each cohort. The initial dose of EG-70 will be based on non-clinical toxicology and non-clinical efficacy data and will be at least 1 / 5 of the minimally toxic dose seen in GLP-toxicology studies. Projected Phase I dose escalation will be performed in up to 1 / 2-log increments with successive cohorts treated without dose-limiting toxicity (DLT).

[0282] Patients are monitored to assess whether delivery of nanoparticles to the bladder is sufficient to stimulate the immune system to prevent / eradicate secondary tumor growth. * * * *

[0283] 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 disclosure above may encompass multiple separate disclosures having independent utility. While each of these disclosures is disclosed in a preferred form(s), the specific embodiments thereof disclosed and exemplified herein should not be considered in a limiting sense, as numerous variations are possible. The subject matter of this disclosure includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations that are deemed to be novel and non-obvious. Disclosures implemented with other combinations and subcombinations 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 directed to a different disclosure or to the same disclosure, and whether broader, narrower, equal or different in scope than the original claims, are deemed to be within the scope of the subject matter of this disclosure.

Claims

1. 1. A composition for use in activating a memory T cell response against a primary cancer in a patient in need thereof, said composition comprising a nucleic acid polyplex comprising a cationic polymer and / or a lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct comprising a nucleic acid encoding at least one RIG-I agonist, wherein said therapeutic nucleic acid constructs encoding IL-12 and at least one RIG-I agonist are the same or different nucleic acid constructs.

2. The composition of claim 1 , wherein the composition is effective to treat or inhibit a primary cancer other than a mucosal tumor in the patient.

3. The composition of claim 1 , wherein the composition is effective to treat or inhibit metastatic disease in the patient at a site different from the primary cancer.

4. 2. The composition of claim 1, wherein the primary cancer is selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, pulmonary cancer, ovarian cancer, kidney cancer, brain cancer, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, liver cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer.

5. the site distinct from the primary cancer is one or more of the liver, lung, bone, brain, lymph node, peritoneum, skin, prostate, breast, colon, rectum, and cervix; 4. The composition of claim 3, optionally wherein the metastatic disease is at two or more sites different from the primary cancer.

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

7. the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof; Optionally, the cationic polymer comprises a derivatized chitosan.

8. the derivatized chitosan is an amino-functionalized chitosan; 8. The composition of claim 7, wherein the amino-functionalized chitosan optionally comprises arginine and further comprises or is functionalized with a hydrophilic polyol.

9. The composition of claim 8, wherein the hydrophilic polyol is selected from gluconic acid and glucose.

10. 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; Optionally, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer.

11. the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is contained within a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, and NTC9385R; 2. The composition of claim 1, wherein optionally, the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is comprised within a NTC9385R plasmid.

12. 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 promoters; Optionally, 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.

13. 1. A composition for use in treating or inhibiting a metastatic tumor at a site different from the site of the primary cancer in a patient in need thereof, comprising: The composition comprises a nucleic acid polyplex comprising a cationic polymer and / or a lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct comprising a nucleic acid encoding at least one RIG-I agonist, wherein the therapeutic nucleic acid constructs encoding IL-12 and the at least one RIG-I agonist are the same or different nucleic acid constructs.

14. 14. The composition of claim 13, wherein the primary cancer is selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, pulmonary cancer, ovarian cancer, kidney cancer, brain cancer, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, liver cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer.

15. The primary cancer is gastrointestinal cancer selected from the group consisting of oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, and rectal cancer; Nasal or lung cancer selected from the group consisting of paranasal sinus cancer, oropharyngeal cancer, tracheal cancer, and lung cancer; or 15. The composition of claim 14, wherein the cancer is a genitourinary cancer selected from the group consisting of bladder cancer, urothelial cancer, urethral cancer, testicular cancer, kidney cancer, prostate cancer, penile cancer, adrenal cancer, uterine cancer, cervical cancer, and ovarian cancer.

16. The composition of claim 15, wherein the genitourinary cancer is bladder cancer.

17. the metastatic site of the tumor is in one or more of the liver, lung, bone, brain, lymph node, peritoneum, skin, prostate, breast, colon, rectum and cervix; Optionally, the metastatic tumors are at two or more different sites.

18. the at least one RIG-I agonist is selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20; Optionally, the at least one RIG-I agonist is eRNA41H and / or eRNA11a.

19. the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof; Optionally, the cationic polymer comprises a derivatized chitosan.

20. the derivatized chitosan is an amino-functionalized chitosan; 20. The composition of claim 19, wherein the amino-functionalized chitosan optionally comprises arginine and further comprises or is functionalized with a hydrophilic polyol.

21. 21. The composition of claim 20, wherein the hydrophilic polyol is selected from gluconic acid and glucose.

22. 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; Optionally, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer.

23. the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is contained within a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, and NTC9385R; Optionally, the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is comprised within a NTC9385R plasmid.

24. 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 promoters; Optionally, 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.

25. The composition of claim 13, wherein the composition is in a dosage form for intravesical instillation.

26. 14. The composition of claim 13, wherein the composition is in a dosage form for oral administration to the gastrointestinal tract (GIT) or for rectal / colonic administration.

27. The composition of claim 13 , wherein the composition is in a form for intratumoral injection.

28. 14. The composition of claim 13, wherein the composition is in a formulation for intranasal or intratracheal administration to the lungs.

29. 23. The composition of claim 10 or 22, wherein the cationic polymer is a derivatized chitosan functionalized with arginine and glucose; the polyanion-containing block copolymer is PEG-b-PLE; and the therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding at least one RIG-I agonist are identical and comprise SEQ ID NO:

8.

30. 1. Use of a composition in the manufacture of a medicament for activating a memory T cell response against a primary cancer in a patient in need thereof, the composition comprising a nucleic acid polyplex comprising a cationic polymer and / or a lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct comprising a nucleic acid encoding at least one RIG-I agonist, wherein the therapeutic nucleic acid constructs encoding IL-12 and the at least one RIG-I agonist are the same or different nucleic acid constructs.

31. 31. The use of claim 30, wherein the composition is effective to treat or inhibit a primary cancer other than a mucosal tumor in the patient.

32. 31. The use of claim 30, wherein the composition is effective to treat or inhibit metastatic disease in the patient at a site different from the primary cancer.

33. 31. The use of claim 30, wherein the primary cancer is selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, pulmonary cancer, ovarian cancer, kidney cancer, brain cancer, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, liver cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer.

34. the site distinct from the primary cancer is one or more of the liver, lung, bone, brain, lymph node, peritoneum, skin, prostate, breast, colon, rectum, and cervix; 34. The use of claim 33, optionally wherein the metastatic disease is at two or more sites different from the primary cancer.

35. the at least one RIG-I agonist is selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20; 31. The use of claim 30, optionally wherein the at least one RIG-I agonist is eRNA41H and / or eRNA11a.

36. the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof; 31. The use of claim 30, optionally wherein the cationic polymer comprises a derivatized chitosan.

37. the derivatized chitosan is an amino-functionalized chitosan; 37. The use according to claim 36, wherein optionally the amino-functionalized chitosan comprises arginine and further comprises or is functionalized with a hydrophilic polyol.

38. 38. The use according to claim 37, wherein the hydrophilic polyol is selected from gluconic acid and glucose.

39. 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; Optionally, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer.

40. the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is contained within a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, and NTC9385R; Optionally, the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is comprised within a NTC9385R plasmid.

41. 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 promoters; Optionally, 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.

42. 1. Use of a composition in the manufacture of a medicament for treating or inhibiting a metastatic tumor at a site different from the site of the primary cancer in a patient in need thereof, comprising: The use, wherein the composition comprises a nucleic acid polyplex comprising a cationic polymer and / or a lipid, a therapeutic nucleic acid construct encoding interleukin-12 (IL-12), and a therapeutic nucleic acid construct comprising a nucleic acid encoding at least one RIG-I agonist, and the therapeutic nucleic acid constructs encoding IL-12 and the at least one RIG-I agonist are the same or different nucleic acid constructs.

43. 43. The use of claim 42, wherein the primary cancer is selected from breast cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lung cancer, pulmonary cancer, ovarian cancer, kidney cancer, brain cancer, sarcoma, bladder cancer, vaginal cancer, cervical cancer, gastric cancer, gastrointestinal cancer, liver cancer, thyroid cancer, esophageal cancer, nasal cancer, laryngeal cancer, oral cancer, pharyngeal cancer, retinoblastoma, endometrial cancer, and testicular cancer.

44. The primary cancer is gastrointestinal cancer selected from the group consisting of oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, colon cancer, and rectal cancer; Nasal or lung cancer selected from the group consisting of paranasal sinus cancer, oropharyngeal cancer, tracheal cancer, and lung cancer; or 44. The use of claim 43, wherein the cancer is a genitourinary cancer selected from the group consisting of bladder cancer, urothelial cancer, urethral cancer, testicular cancer, kidney cancer, prostate cancer, penile cancer, adrenal cancer, uterine cancer, cervical cancer, and ovarian cancer.

45. 45. The use of claim 44, wherein the genitourinary cancer is bladder cancer.

46. the metastatic site of the tumor is in one or more of the liver, lung, bone, brain, lymph node, peritoneum, skin, prostate, breast, colon, rectum and cervix; 43. The use of claim 42, optionally wherein the metastatic tumors are at two or more different sites.

47. the at least one RIG-I agonist is selected from the group consisting of eRNA11a, VA RNA1, eRNA41H, MK4621, SLR10, SLR14, and SLR20; Optionally, the at least one RIG-I agonist is eRNA41H and / or eRNA11a.

48. the cationic polymer is selected from the group consisting of polyethyleneimine (PEI), PAMAM, polylysine (PLL), polyarginine, chitosan, and derivatives thereof; 43. The use of claim 42, optionally wherein the cationic polymer comprises a derivatized chitosan.

49. the derivatized chitosan is an amino-functionalized chitosan; 49. The use of claim 48, wherein optionally the amino-functionalized chitosan comprises arginine and further comprises or is functionalized with a hydrophilic polyol.

50. 50. The use according to claim 49, wherein the hydrophilic polyol is selected from gluconic acid and glucose.

51. 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; Optionally, the polyanion-containing block copolymer is a linear diblock and / or triblock copolymer.

52. the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is contained within a plasmid selected from the group consisting of gWIZ, pVAX, NTC8685, and NTC9385R; Optionally, the therapeutic nucleic acid construct encoding IL-12 and / or the therapeutic nucleic acid construct encoding at least one RIG-I agonist is comprised within a NTC9385R plasmid.

53. 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 promoters; Optionally, 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.

54. 43. The use according to claim 42, wherein the composition is in a dosage form for intravesical instillation.

55. 43. The use according to claim 42, wherein the composition is in a dosage form for oral administration to the gastrointestinal tract (GIT) or for rectal / colonic administration.

56. 43. The use according to claim 42, wherein the composition is in a form for intratumoral injection.

57. 43. The use according to claim 42, wherein the composition is in a dosage form for intranasal or intratracheal administration to the lungs.

58. 52. The use of claim 39 or 51, wherein the cationic polymer is a derivatized chitosan functionalized with arginine and glucose; the polyanion-containing block copolymer is PEG-b-PLE; and the therapeutic nucleic acid construct encoding IL-12 and the therapeutic nucleic acid construct encoding at least one RIG-I agonist are identical and comprise SEQ ID NO:8.

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