Reversible coating of chitosan-nucleic acid nanoparticles and method of use thereof

Abstract

Described herein are polyplex compositions containing a reversibly attached polymer coat. In some cases, the compositions exhibit surprisingly improved stability and / or mucosal diffusion compared to uncoated particles. In some cases, the reversibly attached polymer coat does not interfere with or enhances transfection of target cells or tissues compared to uncoated particles.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Provisional Applications No. 62 / 818,425 filed on 14 March 2019, No. 62 / 923,403 filed on 18 October 2019, and No. 62 / 924,131 filed on 21 October 2019, which are incorporated herein by reference and for all purposes. [Background technology]

[0002] Chitosan is a deacetylated form of chitin. It is a non-toxic cationic polymer of N-acetyl-D-glucosamine and D-glucosamine. Chitosan can form complexes with nucleic acids and, as a biocompatible, non-toxic polysaccharide, is used as a DNA delivery medium for transfecting cells. Due to the complexity and potential toxicity of viral vectors, much interest has focused on the use of chitosan in non-viral delivery of nucleic acids.

[0003] Numerous chitosan / DNA complexes, including modified chitosan and nucleic acid complexes, have been investigated to identify compositions well-suited for gene transfection. See, for example, WO2010 / 088565;WO2008 / 082282. These complexes have been found to differ in important physicochemical and biological properties, along with other characteristics such as solubility, aggregation tendency, complex stability, particle size, DNA release ability, and transfection efficiency.

[0004] Chitosan-nucleic acid polyplexes with improved transfection efficiency have been developed by functionalizing the chitosan skeleton with arginine and hydrophilic polyols. See, for example, U.S. Patent Nos. 10,046,066 and 9,623,112. In particular, these polyplexes exhibit remarkable stability in the intestinal microenvironment, which presents unique challenges compared to systemic circulation due to significant pH changes, enzymatic activity, and complex mucosal interactions.

[0005] Due to the dense network of mucin fibers that efficiently trap foreign particles via adhesion and steric interactions, followed by rapid clearance, the mucosal barrier presents a particular challenge for nanoparticle delivery systems. Indeed, especially in the case of oral drug delivery, the intestinal mucosa can turn over in as little as 50 minutes, which results in very efficient clearance of administered particles. See, for example, Lehr et al., Int'l J.Pharm. 70:235-40 (1991). Mucin fibers contain highly glycosylated segments with high affinity for positively charged particles, and periodic hydrophobic domains that can bind hydrophobic materials with high bonding strength, including many commonly used drug delivery materials such as poly-(lactic acid-coglycolic acid) (PLGA). These complex charge interactions, combined with steric hindrance, have proven difficult for nanoparticle drug delivery forms to overcome without some form of shielding. Conversely, however, conventional shielding approaches often result in nanoparticles with significantly reduced transfection capabilities.

[0006] Polyethylene glycol (PEG) has emerged as a key component of a wide range of shielding strategies, based on its demonstrated ability to increase mucosal adhesion, at least initially, via interpenetrating network effects, thereby extending drug delivery. See, for example, Huckaby and Lai, Advanced Drug Delivery Reviews 124:125-39 (2018). Unfortunately, however, these mucosal adhesion strategies do not work for therapies requiring intracellular delivery, such as nucleic acids. This is because enhanced mucosal adhesion prevents particles from reaching the underlying epithelium or other relevant target tissues. Paradoxically, perhaps PEG coatings have also found use in mucosal-inactive approaches that seek to enhance particle diffusion. However, recent findings and conclusions, particularly regarding these types of systems, highlight the importance of high PEG graft density rather than minimizing mucin affinity and enhancing mucus penetration. See, Id., Wang and Lai, Angew Chem 47:9726-9729 (2008). These exceptionally high-density, covalently PEGylated strategies present problems for efficient gene transfection.

[0007] Therefore, there remains a need for new compositions and methods for in vivo gene transfer with improved mucosal penetration properties. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Compositions and methods for reducing mucosal adhesion and enhancing mucosal penetration of chitosan-nucleic acid nanoparticles are provided herein using a reversible (i.e., non-covalently bonded) polymer coating comprising a plurality of linear block copolymers having a load-charged anchor region and at least one (e.g., hydrophilic) non-charged tail region. In preferred embodiments, the linear block copolymer is a binary block or ternary block copolymer comprising at least one polyanion anchor region and at least one PEG tail region. In some embodiments, the compositions described herein surprisingly provide reduced mucosal adhesion and enhanced mucosal penetration at a lower surface coating density than previously intended. In some embodiments, the compositions described herein surprisingly provide consistent physical and / or chemical stability despite the presence of polyanions. In some embodiments, the compositions described herein surprisingly provide consistent and / or equivalent transfection efficiency despite containing a reversible polymer coating. In some embodiments, the compositions described herein surprisingly remain stable during long-term storage (e.g., several months) in bulk form and / or coated or encapsulated form. In some embodiments, the compositions described herein are surprisingly stable when in contact with mucosal barriers and / or mucus-related fluids (e.g., urine, or gastric or intestinal fluid). [Means for solving the problem]

[0009] In one embodiment, the present invention provides a chitosan composition comprising chitosan derivative nucleic acid nanoparticles (polyplexes) complexed with a plurality of polyanion-containing block copolymers, e.g., linear binary block and / or ternary block copolymers, each copolymer comprising at least one polyanion anchor region and at least one hydrophilic (e.g., uncharged) tail region. Typically, the polyplex forms a core, and the polyanion hydrophilic polymer forms, for example, at least a partial outer membrane. The complex between the polyplex and the polyanion-containing block copolymer is typically formed by a reversible and non-covalent electrostatic interaction between the polyanion anchor region of the polymer and the net positive charge of the uncoated polyplex. In some cases, the complex is reversible in that all or part of the polyanion hydrophilic polymer can be released from the complex by increasing the ionic strength to reduce the intensity of the electrostatic interaction between the polyplex and the polyanion anchor regions of the polymer, and / or decreasing the pH to protonate the anionic portion of the polyanion region of the polymer.

[0010] An exemplary binary block copolymer molecule useful in the methods and compositions of the present invention is a "PEG-PA" copolymer molecule comprising a polyethylene glycol (PEG) tail region and a polyanion (PA) anchor region. An exemplary ternary block copolymer molecule useful in the methods and compositions of the present invention is a "PEG-PA" copolymer molecule comprising a central PA anchor region [PEG-PA-PEG] adjacent to two PEG tail regions, or two PA anchor regions [PA-PEG-PA] located laterally to a central PEG tail region.

[0011] In some embodiments, the present invention provides a complex comprising an amino-functionalized chitosan and chitosan derivative nanoparticles comprising at least one nucleic acid molecule, wherein the at least one nucleic acid molecule is non-covalently bonded to the chitosan derivative nanoparticle in an amino-to-phosphorus (N:P) molar ratio greater than 3:1, thereby forming a positively charged derivatized chitosan nucleic acid complex; and a plurality of linear block copolymers are non-covalently bonded to the chitosan derivative nanoparticles, wherein the block copolymers have at least one polyanion (PA) anchor region and at least one polyethylene glycol (PEG) tail region, and the composition comprises an amino-to-anion (N:A) molar ratio greater than about 1:100 and less than about 10:1.

[0012] In some embodiments, the linear block copolymer is a binary block copolymer comprising PA anchor regions and PEG tail regions. In some embodiments, the linear block copolymer is a ternary block copolymer comprising a central PA anchor region adjacent to two PEG tail regions, or alternatively, a central PEG tail region adjacent to two PA anchor regions.

[0013] In some embodiments, the PA anchor region comprises a polypeptide, and the polypeptide is the load charge. In some embodiments, the PA anchor region of the PEG-PA molecule comprises a carbohydrate, and the carbohydrate is the load charge. In some embodiments, the carbohydrate comprises multiple phosphate and / or sulfate moieties. In some embodiments, the carbohydrate comprises multiple carboxylate moieties. In some embodiments, the carbohydrate comprises multiple carboxylate moieties and multiple phosphate and / or sulfate moieties. In some embodiments, the carbohydrate comprises a higher proportion or number of carboxylate moieties than phosphate and / or sulfate moieties. In exemplary embodiments, the carbohydrate is a glycosaminoglycan.

[0014] In certain embodiments, the PEG-PA molecule includes PEG-polyglutamic acid (PEG-PGA) molecules; PEG-polyaspartic acid (PEG-PAA) molecules; or PEG-hyaluronic acid (PEG-HA) molecules, or a combination thereof.

[0015] In some embodiments, the PEG tail region of the PEG-PA molecule contains a weight-average molecular weight (Mw) of about 500 Da to about 50,000 Da, preferably about 1,000 Da to about 10,000 Da, more preferably about 1,500 Da to about 7,500 Da, even more preferably about 3,000 Da to about 5,000 Da, and most preferably about 5,000 Da. In some embodiments, the PA tail region of the PEG-PA molecule contains a weight-average molecular weight (Mw) of about 500 Da to about 3,000 Da, more preferably about 1,000 Da to about 2,500 Da, and more preferably about 1,500 Da.

[0016] In some embodiments, the N:P molar ratio is greater than about 3:1 and less than about 100:1, more preferably greater than about 5:1 and less than about 50:1, even more preferably greater than about 5:1 and less than about 30:1, even more preferably greater than about 5:1 and less than about 20:1, and even more preferably about 5:1 and less than about 10:1. In some cases, the N:P molar ratio is about 3:1 to about 30:1. In some cases, the N:P molar ratio is about 3:1 to about 20:1. In some cases, the N:P molar ratio is about 3:1 to about 10:1. In some cases, the N:P molar ratio is about 7:1.

[0017] In some embodiments, the N:A molar ratio is greater than about 1:75 and less than about 8:1, more preferably greater than about 1:50 and less than about 6:1, even more preferably greater than about 1:25 and less than about 6:1, even more preferably greater than about 1:10 and less than about 6:1, and even more preferably greater than about 1:5 and less than about 6:1.

[0018] In some embodiments, the N:P molar ratio is about 1:8 to about 8:1, the P:A molar ratio is about 0.02 to about 0.2, more preferably the N:A molar ratio is about 0.1 to about 5, more preferably about 0.2 to about 2, more preferably about 0.3 to about 1.5, more preferably about 0.4 to about 1, and even more preferably the N:P:A ratio is about 7:1:7, about 7:1:12, or about 7:1:17.

[0019] In some embodiments, the N:P molar ratio is about 1:8 to about 30:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is about 1:5 to about 20:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is about 1:2 to about 10:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1.

[0020] In some embodiments, the N:P molar ratio is about 1:1 to about 30:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is about 1:1 to about 20:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is about 1:1 to about 15:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is about 1:1 to about 10:1, the P:A molar ratio is about 1:50 to about 1:5, more preferably the N:A molar ratio is about 1:10 to about 5, more preferably about 1:5 to about 2, more preferably about 1:3 to about 1.5, and even more preferably about 1:2.5 to about 1.

[0021] In some embodiments, the N:P molar ratio is from about 2:1 to about 30:1, the P:A molar ratio is from about 1:50 to about 1:5, more preferably, the N:A molar ratio is from about 1:10 to about 5, more preferably, from about 1:5 to about 2, more preferably, from about 1:3 to about 1.5, even more preferably, from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 2:1 to about 20:1, the P:A molar ratio is from about 1:50 to about 1:5, more preferably, the N:A molar ratio is from about 1:10 to about 5, more preferably, from about 1:5 to about 2, more preferably, from about 1:3 to about 1.5, even more preferably, from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 2:1 to about 15:1, the P:A molar ratio is from about 1:50 to about 1:5, more preferably, the N:A molar ratio is from about 1:10 to about 5, more preferably, from about 1:5 to about 2, more preferably, from about 1:3 to about 1.5, even more preferably, from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 2:1 to about 10:1, the P:A molar ratio is from about 1:50 to about 1:5, more preferably, the N:A molar ratio is from about 1:10 to about 5, more preferably, from about 1:5 to about 2, more preferably, from about 1:3 to about 1.5, even more preferably, from about 1:2.5 to about 1.

[0022] In some embodiments, the amino-functionalized chitosan is functionalized with arginine, lysine, or ornithine functional groups, preferably functionalized with arginine functional groups. In some embodiments, the amino-functionalized chitosan derivative nanoparticles further comprise a polyol. In some embodiments, the amino-functionalized chitosan further comprises a polyol. In some embodiments, the amino-functionalized chitosan derivative nanoparticles are also functionalized with a polyol. In some embodiments, the amino-functionalized chitosan is also functionalized with a polyol.

[0023] In some embodiments, the composition is stable in artificial intestinal fluid on an empty stomach for 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months, or for at least 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months. In some embodiments, the composition is stable in an aqueous dispersion such as a dispersion of the composition in purified water at 4°C for 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months, or for at least 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months. In some embodiments, the composition is stable in purified water at 4°C for 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months, or for at least 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months. In some embodiments, the composition is stable in urine at 4°C for 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months, or for at least 1 hour, 24 hours, 48 hours, 1 week, or 1 or 2 months. In some embodiments, the composition is stable in that there is substantially no precipitate agglomerate (less than 10%) in artificial intestinal fluid and / or aqueous dispersion and / or urine and / or purified water after a predetermined time, for example, after 24 hours, 48 hours, 1 week, or 1 or 2 months.

[0024] In some embodiments, the composition is stable in an aqueous dispersion after freezing / thawing and / or lyophilization / rehydration. In some embodiments, the composition exhibits a polydispersity index of less than 0.2 after being in an aqueous solution at 4°C for 48 hours, 1 week, or 1 or 2 months, or for at least 48 hours, 1 week, or 1 or 2 months. In some embodiments, the composition is dried (e.g., by lyophilization, spray drying, evaporation, supercritical drying, spray freeze drying, etc.) and then rehydrated and is stable in an aqueous dispersion.

[0025] In some embodiments, the composition is stable in mammalian urine for at least 1 hour. In some embodiments, the composition is stable in mammalian urine for at least 1 hour at room temperature or 37°C. In some embodiments, the composition exhibits a polydispersity index of less than 0.2 after at least 1 hour in mammalian urine (e.g., at 37°C).

[0026] In some embodiments, the composition transfects cells with therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is transcribed into a therapeutic protein. In some embodiments, the therapeutic nucleic acid suppresses the expression of a gene encoding an endogenous protein. In some embodiments, the therapeutic nucleic acid suppresses the expression of an endogenous gene.

[0027] In some embodiments, the composition further comprises a surfactant, an excipient, and / or a storage stabilizer. In some embodiments, the storage stabilizer is a monosaccharide, disaccharide, polysaccharide, or a reduced alcohol thereof, and more preferably, the storage stabilizer is selected from trehalose and mannitol. In some embodiments, the surfactant comprises poloxamer, and more preferably, the poloxamer is poloxamer 407.

[0028] In some embodiments, at least one nucleic acid comprises RNA. In some embodiments, at least one nucleic acid comprises DNA. [Brief explanation of the drawing]

[0029] [Figure 1] Polyplex: A polymer composition and a method for preparing the composition are shown.

[0030] [Figure 2] This specification shows the inverse relationship between the zeta potential and degree of PEGylation for the specific polyplex:polymer compositions described herein. As the molar ratio of polyanion-PEG(A) to amino-functionalized chitosan(N) increases, the zeta potential decreases at higher PEG densities, reaching a value from nearly neutral to slightly negative.

[0031] [Figure 3] Polyplex after freeze-thaw cycles: This demonstrates the stability of the polymer composition. Polyplex PEGylated in the tested ratio of [N+] to [A-] remained stable after freeze-thaw cycles.

[0032] [Figure 4] This shows the stability of polyplex:polymer compositions in artificial intestinal fluid at different volume:volume ratios. FaSSIF-V2 = Fasting artificial intestinal fluid V2.

[0033] [Figure 5] Polyplex: Indicates the ability of a polymer composition to retain nucleic acids complexed in artificial intestinal fluid. FaSSIF-V1 = Fasting artificial intestinal fluid V1. FaSSIF-V2 = Fasting artificial intestinal fluid V2. PP = Polyplex.

[0034] [Figure 6] This paper demonstrates in vitro transfection using PEGylated DD-chitosan-nucleic acid polyplex.

[0035] [Figure 7] Using a fluorescence microscope, the method and results of a mucoagulation assay for the polyplex:polymer compositions described herein are shown.

[0036] [Figure 8] This specification describes a method for performing a mucous penetration assay on the polyplex:polymer compositions described herein using the Transwell diffusion assay.

[0037] [Figure 9] The results of the Transwell diffusion assay for the polyplex:polymer compositions described herein are shown.

[0038] [Figure 10]The results of the Transwell diffusion assay of the polyplex:polymer compositions described herein in the presence of poloxamer 407 are shown.

[0039] [Figure 11] This specification provides a scalable method for preparing the polyplex:polymer compositions described herein.

[0040] [Figure 12] This demonstrates the stability of the polyplex:polymer compositions described herein under freeze-drying and rehydration conditions, as well as under high-concentration conditions.

[0041] [Figure 13] This shows the stability of freeze-dried PEGylated polyplexes for up to 4 weeks at room temperature and 4°C.

[0042] [Figure 14] The stability of the polyplex:polymer compositions described herein, after freeze-drying in the absence of excipients and storage at 4°C for up to 4 weeks, is demonstrated.

[0043] [Figure 15] The stability of the polyplex:polymer composition described herein, as administered to the bladder of mice and recovered in the collected urine, is demonstrated.

[0044] [Figure 16] (Measured by mRNA expression of the transgene) This shows significantly improved in vivo gene delivery of the polyplex:polymer compositions described herein when delivered by intracolonic infusion (ICI). The administered polyplex formulation contained 150 μg / mL of nucleic acid and was administered as 3 x 150 μL intracolonic infusions; colon sections were collected 24 hours after administration, and human PD-L1-Fc mRNA was quantified using cell lysates.

[0045] [Figure 17](Measured by protein expression of the transgene) This demonstrates significantly improved in vivo gene delivery of the polyplex:polymer compositions described herein when delivered by intracolonic infusion (ICI). The administered polyplex formulation contained 150 μg / mL of nucleic acid and was administered as 3 x 150 μL intracolonic infusion; colon sections were collected 24 hours after administration, and human PD-L1-Fc protein was quantified using a custom-designed Meso Scale Discovery immunoassay.

[0046] [Figure 18] (Measured by transgene protein expression) This demonstrates significantly improved in vivo gene delivery of the polyplex:polymer compositions described herein when delivered by intracolonic infusion (ICI). The administered polyplex formulation contained 1000 μg / mL of nucleic acid and was administered as 3 x 150 μL intracolonic infusion; colon sections were collected 24 hours after administration, and human PD-L1-Fc protein was quantified using a custom-designed Meso Scale Discovery immunoassay.

[0047] [Figure 19] This shows an improvement in the % supercoiled DNA content of PEGylated DDX polyplexes compared to non-PEGylated DDX polyplexes.

[0048] [Figure 20] This demonstrates the storage stability of PEGylated DDX polyplex.

[0049] [Figure 21] This study demonstrates the rehydration properties of PEGylated and unPEGylated DDX polyplex formulations. PEGylated DDX polyplex was able to rehydrate stably at a higher final concentration (10 mg / mL) compared to unPEGylated DDX polyplex (2 mg / mL).

[0050] [Figure 22]This shows the stability of the DDX polyplex formulation when incubated in the bladder of mammals.

[0051] [Figure 23] This shows the filterability of the DDX polyplex formulation under the indicated conditions when tested on a small scale.

[0052] [Figure 24] This shows the filterability of the DDX polyplex formulation under the indicated conditions when tested on a medium scale.

[0053] [Figure 25] This indicates the stability of a specified DDX polyplex formulation after freeze-thaw (FT) and freeze-drying / rehydration (FD), expressed as nanoparticle size (nm), zeta potential (mV), and % supercoil content (%SC).

[0054] [Figure 26] This demonstrates in vivo transfection of canine small intestine by direct delivery of PEGylated double-derivatized (DDX) chitosan DNA polyplex to the small intestine.

[0055] [Figure 27] The study shows a significant increase in zeta potential (mv) when an aqueous polyplex formulation is mixed with a low pH acetate buffer in a ratio sufficient to lower the pH to below the pKa (approximately 4.25) of the PEG-polyglutamate polymer (indicating decoating of the PEGylated polyplex at pH less than 4).

[0056] [Figure 28] The particle sizes of reversibly PEGylated polyplexes formed with various N:P:A ratios are shown. Different polyglutamic acid (PLE) polyanion anchor regions were tested: PLE5 (5 glutamic acid polyglutamic acid regions), PLE10 (10 glutamic acid polyglutamic acid regions), and PLE25 (25 glutamic acid polyglutamic acid regions).

[0057] [Figure 29] This indicates the pH of the aqueous dispersion of the reversibly PEGylated polyplex.

[0058] [Figure 30] The results of zeta potential measurements of the reversibly PEGylated polyplex are shown.

[0059] [Figure 31] The results of polyaspartate (PAA) induction of reversibly PEGylated polyplexes are shown. Nucleic acid release was monitored as a function of PAA concentration. Different polyglutamate (PLE) polyanion anchor regions of PEG-PLE were tested: PLE5 (5 glutamate polyglutamate regions), PLE10 (10 glutamate polyglutamate regions), and PLE25 (25 glutamate polyglutamate regions) with different NPA ratios.

[0060] [Figure 32] This shows the level of PAA required for DNA release from PEGylated polyplexes. Nucleic acids from PEGylated polyplexes created using PEG-PLE25 were looser bound than those from polyplexes created using PEG-PLE5 or PEG-PLE10.

[0061] [Figure 33] The effects of fasting artificial intestinal fluid (FaSSIF v2) on the particle size and polydispersity index (PDI) of the PEGylated polyplexes are shown.

[0062] [Figure 34] The effects of fasting artificial intestinal fluid (FaSSIF v2) on the zeta potential and pH of the particles in the resulting aqueous dispersion of the PEGylated polyplex are shown.

[0063] [Figure 35]This shows the stability (measured by particle size) of PEGylated polyplexes (PLE10, PLD10, and PLD50) after freeze-thaw cycles. PEG-PLD50 polyplex with a 7:1:30 N:P:A ratio showed an increase in particle size after freeze-thaw cycles.

[0064] [Figure 36] This shows the stability of PEGylated polyplexes (PLE10, PLD10, and PLD50) after freeze-thaw cycles (measured by particle size (top) and zeta potential (center)). PEG-PLD50 polyplexes with a 7:1:30 N:P:A ratio showed an increase in particle size and a decrease in zeta potential after freeze-thaw cycles. The polyplexes, as aqueous dispersions, showed a gradually increasing pH as the P:A ratio decreased (A increased) and the number of anionic subunits increased (bottom).

[0065] [Figure 37] The results of analyzing polyplexes that were reversibly PEGylated by agarose gel electrophoresis to detect nucleic acids that did not form complexes are shown.

[0066] [Figure 38] The diagrams show the expected behavior of polyplexes that are reversibly PEGylated (top panel) and covalently PEGylated (bottom panel) at different pH levels.

[0067] [Figure 39] The solution behavior of polyplexes reversibly PEGylated at two pH levels, 2 and 6 (top panel), and two different covalently PEGylated polyplexes (middle and bottom panels), in response to polyaspartic acid (PAA) induction, is shown.

[0068] [Figure 40] This shows the transfection efficiency of reversibly PEGylated polyplexes compared to non-PEGylated polyplexes.

[0069] [Figure 41]This shows the transfection efficiency of reversibly PEGylated polyplexes compared to covalently PEGylated polyplexes.

[0070] [Figure 42] This demonstrates the transfection efficacy of reversibly PEGylated polyplexes prepared by a one-step or two-step method after intracolonic delivery to mice.

[0071] [Figure 43] This demonstrates the transfection efficacy of reversibly PEGylated polyplexes prepared by a one-step or two-step method after intravesicular administration to mice. [Modes for carrying out the invention]

[0072] 1.Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings generally understood by those skilled in the art to which the present invention relates. In some cases, terms having generally understood meanings are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference beyond what is generally understood in the art. The methods and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methodologies, e.g., the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Procedures, including the use of commercially available kits and reagents where necessary, are generally carried out according to the manufacturer's defined protocols and / or parameters unless otherwise specified.

[0073] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise specified by the context.

[0074] The term "approximately" indicates and encompasses the given value and its upper and lower range. In certain embodiments, the term "approximately" indicates a given value ± 10%, ± 5%, or ± 1%. In certain embodiments, where indicated, the term "approximately" indicates a given value ± 1 standard deviation of that value.

[0075] The term "those combinations" includes all possible combinations of the elements referred to by that term.

[0076] In certain embodiments, “treating” or “treating” any disease or disorder means improving the disease or disorder present in the subject. In other embodiments, “treating” or “treating” includes improving at least one physical parameter that may not be identifiable by the subject. In yet another embodiment, “treating” or “treating” includes alleviating the disease or disorder either physically (e.g., stabilizing identifiable symptoms) or physiologically (e.g., stabilizing a physical parameter) or both. In yet another embodiment, “treating” or “treating” includes delaying or preventing the onset of the disease or disorder.

[0077] As used herein, the terms “therapeutic effective dose” or “effective dose” refer to the amount of antibody or composition that, when administered to a subject, is effective in treating a disease or disorder.

[0078] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has cancer, an autoimmune disease or condition, and / or an infection that can be treated with antibodies provided herein. In some embodiments, the subject is a human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0079] Chitosan is a partially or completely deacetylated form of chitin, which is a polymer of N-acetylglucosamine. Chitosan with a degree of deacetylation exceeding 50% is used in this invention.

[0080] Chitosan may be derivatized by functionalizing the free amino groups at the deacetylation site. The derivatized chitosans described herein have numerous properties advantageous to nucleic acid delivery vehicles, including: effectively binding and complexing with load-bearing nucleic acids; being able to form nanoparticles of controllable size; being able to be taken up by cells; and being able to release nucleic acids into cells in an appropriate time. Chitosan having any final degree of functionalization from 1% to 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 final degree of functionalization give the functionalized chitosan derivative a specific charge density.

[0081] The polyols according to the present invention may have a carbon skeleton of 3, 4, 5, 6, or 7 carbon atoms and may have at least 2 hydroxyl groups. Such polyols, or combinations thereof, may be useful for conjugating to a chitosan skeleton, such as chitosan, which is functionalized with a cationic moiety (e.g., a molecule containing an amino group such as lysine ornithine, a molecule containing a guanidinium group, an arginine group, or a combination thereof).

[0082] As used herein, the term "C2-C6 alkylene" refers to a linear or branched divalent hydrocarbon radical that optionally contains one or more carbon-carbon multiple bonds. To avoid misunderstanding, as used herein, the term "C2-C6 alkylene" encompasses divalent radicals of alkanes, alkenes, and alkynes.

[0083] As used herein, unless otherwise indicated, the terms "peptide" and "polypeptide" are interchangeable.

[0084] The term "polypeptide" is used in its 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 peptide mimics that retain desired functional activity. Peptide equivalents may differ from conventional peptides by replacing one or more amino acids with related organic acids, amino acids, etc., or by substituting or modifying side chains or functional groups.

[0085] The term "acidic amino acid" refers to naturally occurring or unnatural amino acids that have negatively charged side chains in an aqueous buffer solution at pH 7. Non-limiting examples of acidic amino acids include aspartic acid and glutamic acid.

[0086] The peptide mimeograph may have one or more peptide bonds replaced by alternative bonds, as is known in the art. As is known in the art, some or all of the peptide backbone may also be replaced with conformationally constrained cyclic alkyl or aryl substituents to restrict the mobility of the functional amino acid side chains.

[0087] The polypeptides of the present invention may be produced by recognized methods, such as recombinant and synthetic methods, which are well known in the art. Peptide synthesis methods 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).

[0088] As used herein, “linear polypeptide” refers to a polypeptide that lacks branching groups covalently bonded to its constituent amino acid side chains. As used herein, “branched polypeptide” refers to a polypeptide that contains branching groups covalently bonded to its constituent amino acid side chains.

[0089] As used herein, the “final degree of functionalization” of a cation or polyol refers to the percentage of cation (e.g., amino) groups on a chitosan skeleton functionalized with a cation (e.g., amino) group or polyol, respectively. Therefore, terms such as “α:β ratio” and “final degree of functionalization ratio” (e.g., ratio of final degree of functionalization of Arg to final degree of functionalization of polyol) can be used interchangeably with the terms “molar ratio” or “numerical ratio.”

[0090] A dispersion system consists of particulate matter known as a dispersed phase, distributed throughout a continuous medium. A "dispersion" of chitosan nucleic acid polyplex is a composition containing hydrated chitosan nucleic acid polyplex, in which the polyplex is distributed throughout the medium.

[0091] As used herein, a “pre-concentrated” dispersion is one that has not undergone a concentration process to form a concentrated dispersion.

[0092] As used herein, "substantially free" of polyplex precipitates means that the composition is essentially free of particles that can be observed by visual inspection.

[0093] As used herein, physiological pH refers to a pH of 6 to 8.

[0094] "Chitosan nucleic acid polyplex" or its grammatical equivalent means a complex comprising multiple chitosan molecules and multiple nucleic acid molecules. In preferred embodiments, (e.g., double) derivatized chitosan is complexed with the nucleic acid.

[0095] As used herein, the term "polyethylene glycol" ("PEG") is intended to mean a polymer of ethylene oxide having a repeating unit of -(CH2CH2-O)- and the general formula HO-(CH2CH2-O)nH.

[0096] As used herein, the term “monomethoxypolyethylene glycol” (“mPEG”) is intended to mean a polymer of ethylene oxide having repeating units of -(CH2CH2-O)- and the general formula CH3O-(CH2CH2-O)nH, such as PEG, which is capped at one end with a methoxy group.

[0097] 2. Composition Chitosan compositions comprising chitosan derivative nucleic acid nanoparticles (polyplexes) complexed with binary block and / or ternary block copolymer coatings are provided herein, each polymer molecule comprising a charged anchor region and one or more uncharged hydrophilic tail regions. An example of a charged anchor region is a polyanion anchor region comprising repeating units, each of which comprises one or more charged subunits, e.g., one or more acidic amino acids. An example of a hydrophilic tail region is, but is not limited to, PEG tail regions and their derivatives, polyvinyl alcohol tail regions and their derivatives, polyoxazoline tail regions and their derivatives, polysarcosine tail regions and their derivatives, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) tail regions and their derivatives, and combinations thereof.

[0098] A useful example polymer molecule in the methods and compositions of the present invention is a "PEG-PA" polymer molecule containing a polyethylene glycol (PEG) moiety and a polyanion (PA) moiety.

[0099] 2.1. Chitosan The chitosan components of chitosan derivative nucleic acid nanoparticles can be functionalized with cationic functional groups and / or hydrophilic moieties. Chitosan functionalized with two different functional groups is called double-derivatized chitosan (DD-chitosan). Exemplary DD-chitosans are functionalized with both hydrophilic moieties (e.g., polyols) and cationic functional groups (e.g., amino groups). Exemplary chitosan derivatives are also described, for example, in U.S. Patent No. 2007 / 0281904 and U.S. Patent No. 2012 / 0235863 (both incorporated herein by reference).

[0100] In one embodiment, the bi-derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 50%. In one embodiment, the degree of deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. In a preferred embodiment, the bi-derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 98%.

[0101] The chitosan derivatives described herein have a broad average molecular weight that is soluble at neutral and physiological pH, and for the purposes of the present invention, include molecular weights in the range of 3 to 110 kDa. Embodiments described herein feature a lower average molecular weight of the derivatized chitosan (less than 25 kDa, e.g., about 5 kDa to about 25 kDa), which may have desirable delivery and transfection properties, a smaller size, and good solubility. Derivatized chitosan with a low average molecular weight is generally more soluble than that with a high polymer content, and the former produces nucleic acid / chitosan complexes that more readily release nucleic acids and increase cellular transfection. Much literature has been dedicated to optimizing all of these parameters for chitosan-based delivery systems.

[0102] Those skilled in the art will understand that chitosan refers to multiple molecules having the structure of formula I (wherein n is any integer, and each R1 is independently selected from acetyl or hydrogen, with the degree of selection of R1 from hydrogen being 50% to 100%). Furthermore, chitosan referred to as having an average molecular weight of 3 kD to 110 kD generally refers to multiple chitosan molecules, each having a weight-average molecular weight of, for example, 3 kD to 110 kD, and each of these chitosan molecules may have a different chain length (n+2). It is also well recognized that chitosan referred to as "n-mer chitosan" does not necessarily contain chitosan molecules of formula I (wherein each chitosan molecule has a chain length of n+2). Rather, as used herein, "n-mer chitosan" refers to multiple chitosan molecules, each of which may have a different chain length, and many have an average molecular weight substantially equivalent to or equal to that of a chitosan molecule with a chain length of n. For example, 24-mer chitosan may contain multiple chitosan molecules, each having a different chain length ranging, for example, from 7 to 50, which has a weight-average molecular weight substantially equivalent to or equal to that of a chitosan molecule with a chain length of 24.

[0103] The double-derivatized chitosan of the present invention may also be functionalized with a polyol, or a hydrophilic functional group such as a polyol. While not wishing to be bound by theory, it is assumed that functionalization with a hydrophilic group such as a polyol may help increase the hydrophilicity of chitosan (including Arg-chitosan) and / or donate a hydroxyl group. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticles is or contains gluconic acid. See, for example, WO2013 / 138930. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticles is or contains glucose. Additionally or alternatively, the hydrophilic functional group may include a polyol. See, for example, US2016 / 0235863. Exemplary polyols for the functionalization of chitosan are further described below.

[0104] The functionalized chitosan derivatives described herein include biderivatized chitosan compounds, such as cationic chitosan polyol compounds. Generally, cationic chitosan polyol compounds are functionalized with an amino-containing moiety, such as a molecule containing arginine, lysine, ornithine, or guanidinium, or a combination thereof. In certain embodiments, the cationic chitosan polyol compound has the following structure of formula I: [ka] In the formula, n is an integer between 1 and 650. α is the final degree of functionalization of the cationic moiety (e.g., molecules containing amino groups such as lysine, ornithine, guanidinium groups, arginine, or combinations thereof). β is the final degree of functionalization of the polyol. Each R 1 This is independently selected from hydrogen, acetyl, cations (e.g., arginine), and polyols.

[0105] Preferably, the bi-derivative chitosan of the present invention may be functionalized with arginine, which is a cationic amino acid.

[0106] In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to gluconic acid at a final functionalization degree of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30%, or higher. In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to glucose at a final functionalization degree of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30%, or higher. In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to a cationic moiety (e.g., arginine) at a final functionalization degree of about 1% to about 25%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to a cationic moiety (e.g., arginine) at a final functionalization degree of about 10% to about 40%.

[0107] In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) at a final functionalization degree of about 10% to about 35%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) at a final functionalization degree of about 20% to about 35%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) at a final functionalization degree of about 25% to about 35%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) at a final functionalization degree of about 25% to about 30%, preferably 28%.

[0108] In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 15% to about 40%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 15% to about 35%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 15% to about 30%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 15% to about 28%.

[0109] In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 10% to about 35%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 10% to about 30%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 10% to about 28%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a cationic moiety (e.g., arginine) with a final functionalization degree of about 28%.

[0110] In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to gluconic acid at a final functionalization degree of approximately 2% to approximately 30%, approximately 5% to approximately 30%, approximately 7.5% to approximately 30%, approximately 5% to approximately 25%, approximately 5% to approximately 22%, approximately 5% to approximately 20%, approximately 5% to approximately 15%, or approximately 5% to approximately 10%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to gluconic acid at a final functionalization degree of approximately 7.5% to approximately 25%, approximately 7.5% to approximately 20%, approximately 7.5% to approximately 15%, or approximately 7.5% to approximately 12%. In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to gluconic acid at a final functionalization degree of approximately 10%.

[0111] In one embodiment, the chitosan derivative nanoparticles contain chitosan bonded to a hydrophilic polyol 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 contain chitosan bonded to a hydrophilic polyol 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 contain chitosan bonded to a hydrophilic polyol at a final functionalization degree of about 10%.

[0112] In one embodiment, the chitosan derivative nanoparticles contain chitosan bound to glucose 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 contain chitosan bound to glucose 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 contain chitosan bound to glucose at a final functionalization degree of about 10%.

[0113] In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 2% to about 40% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 5% to about 40% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 7.5% to about 40% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 10% to about 40%, and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 15%, or about 10%.

[0114] In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 2% to about 35% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 5% to about 35% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 7.5% to about 35% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 10% to about 35%, and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 15%, or about 10%.

[0115] In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 10% to about 30% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 12% to about 30% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 14% to about 30% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 15% to about 30%, and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 15%, or about 10%.

[0116] In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 25% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 15%. In another embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 28% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 7.5% to about 15%. In yet another embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 25% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 5% to about 20%. In one embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 28% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 5% to about 20%.

[0117] In a preferred embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 14% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 15% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 12%. In yet another preferred embodiment, the chitosan derivative nanoparticles include chitosan bonded to arginine with a final functionalization degree of about 14% and chitosan bonded to glucose with a final functionalization degree of about 10%. In yet another preferred embodiment, the chitosan derivative nanoparticles include chitosan bonded to arginine with a final functionalization degree of about 15% and chitosan bonded to glucose with a final functionalization degree of about 12%.

[0118] In a preferred embodiment, the chitosan derivative nanoparticles include chitosan bonded to a cation (e.g., arginine) with a final functionalization degree of about 28% and chitosan bonded to a hydrophilic polyol (e.g., glucose or gluconic acid) with a final functionalization degree of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles include chitosan bonded to arginine with a final functionalization degree of about 28% and chitosan bonded to glucose with a final functionalization degree of about 10%.

[0119] In some embodiments, DD-chitosan may include, as needed, DD-chitosan derivatives, e.g., DD-chitosan incorporating additional functionalization, e.g., DD-chitosan having a bound ligand. “Derivatives” would be understood to include a broad classification of chitosan polymers containing covalently modified N-acetyl-D-glucosamine and / or D-glucosamine units, as well as chitosan polymers incorporating other units or having other parts bound to them. Derivatives are often based on modification of the hydroxyl or amine group of glucosamine, as is done in arginine-functionalized chitosan. Examples of chitosan derivatives include, but are not limited to, trimethylated chitosan, PEGylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, PEI-integrated chitosan, uronic acid-modified chitosan, glycol chitosan, and others. For further information regarding chitosan derivatives, see, for example, pp. 63-74 of “Non-viral Gene Therapy”, K. Taira, K. Kataoka, T. Niidome (editors), Springer-Verlag Tokyo, 2005, ISBN 4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007, vol. 52(23), pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55(2004) 77-93.

[0120] 2.2. Chitosan Nucleic Acid Polyplex Chitosan derivative nanoparticle compositions generally contain at least one nucleic acid molecule, preferably multiple such nucleic acid molecules. Typical nucleic acid molecules include, for example, multiple phosphodiesters or derivatives thereof (e.g., phosphorothioates) containing phosphorus as a component of the nucleic acid backbone. The ratio of cationic functionalized chitosan derivative to nucleic acid can be characterized by a cation (+) to phosphorus (P) molar ratio, where (+) refers to the cation of the cationic functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, the (+):(P) molar ratio is selected so that the chitosan-derivative-nucleic acid complex has a positive charge in the absence of PEG-PA polymer molecules. Thus, the (+):(P) molar ratio is generally greater than 1. In preferred embodiments, the (+):(P) molar ratio is greater than 1.5, at least 2, or greater than 2. In certain preferred embodiments, the (+):(P) molar ratio is greater than 2.

[0121] In some cases, the (+):(P) molar ratio is 3:1 or approximately 3:1. In some cases, the (+):(P) molar ratio is 4:1 or approximately 4:1. In some cases, the (+):(P) molar ratio is 5:1 or approximately 5:1. In some cases, the (+):(P) molar ratio is 6:1 or approximately 6:1. In some cases, the (+):(P) molar ratio is 7:1 or approximately 7:1. In some cases, the (+):(P) molar ratio is 8:1 or approximately 8:1. In some cases, the (+):(P) molar ratio is 9:1 or approximately 9:1. In some cases, the (+):(P) molar ratio is 10:1 or approximately 10:1.

[0122] 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.

[0123] In certain embodiments, the (+):(P) molar ratio is 100:1, preferably less than 100:1. For example, in certain embodiments, the (+):(P) molar ratio may be greater than 1 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 2 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 3 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 5 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 7 and less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 2 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio may be greater than 3 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio may be greater than 5 and less than or equal to 50:1. In some cases, the (+):(P) molar ratio can be between 7 and 50:1. In some cases, the (+):(P) molar ratio can be between 2 and 25:1. In some cases, the (+):(P) molar ratio can be between 3 and 25:1. In some cases, the (+):(P) molar ratio can be between 5 and 25:1. In some cases, the (+):(P) molar ratio can be between 7 and 25:1.

[0124] In some embodiments, the cationic functional group of the chitosan derivative nanoparticles is an amino group or contains an amino group. Examples of such amino-functionalized chitosan derivative nanoparticles include, but are not limited to, those containing chitosan functionalized with guanidinium or a molecule containing a guanidinium group, lysine, ornithine, arginine, or a combination thereof. In preferred embodiments, the cationic functional group is arginine. The ratio of amino-functionalized chitosan derivative to nucleic acid can be characterized by an amino(N) to phosphorus(P) molar ratio, where (N) refers to the nitrogen atom of the amino group in the amino-functionalized chitosan derivative and (P) refers to the phosphorus of the amino acid backbone. Typically, the N:P molar ratio is selected so 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 N:P molar ratio is generally greater than 1. In preferred embodiments, the N:P molar ratio is greater than 1.5, at least 2, or greater than 2. In certain preferred embodiments, the N:P molar ratio is greater than 2.

[0125] In some cases, the N:P molar ratio is 3:1 or approximately 3:1. In some cases, the N:P molar ratio is 4:1 or approximately 4:1. In some cases, the N:P molar ratio is 5:1 or approximately 5:1. In some cases, the N:P molar ratio is 6:1 or approximately 6:1. In some cases, the N:P molar ratio is 7:1 or approximately 7:1. In some cases, the N:P molar ratio is 8:1 or approximately 8:1. In some cases, the N:P molar ratio is 9:1 or approximately 9:1. In some cases, the N:P molar ratio is 10:1 or approximately 10:1.

[0126] In some cases, the N:P molar ratio is greater than 1 to approximately 20:1 or less, approximately 2 to approximately 20:1 or less, or approximately 2 to approximately 10:1 or less. In some cases, the N:P molar ratio is greater than approximately 2 to approximately 20:1 or less, or approximately 2 to approximately 10:1 or less. In some cases, the N:P molar ratio is approximately 3 to approximately 20:1 or less, approximately 3 to approximately 10:1 or less, approximately 3 to approximately 8:1 or less, or approximately 3 to approximately 7:1 or less. In some cases, the N:P molar ratio is approximately 3 to approximately 20:1 or less, approximately 3 to approximately 10:1 or less, approximately 3 to approximately 8:1 or less, or approximately 3 to approximately 7:1 or less.

[0127] 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 may be greater than 1 and less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than 2 and less than or equal to 100:1. In some cases, the N:P molar ratio may be 3 or more and less than or equal to 100:1. In some cases, the N:P molar ratio may be 5 or more and less than or equal to 100:1. In some cases, the N:P molar ratio may be 7 or more and less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than 2 and less than or equal to 50:1. In some cases, the N:P molar ratio may be 3 or more and less than or equal to 50:1. In some cases, the N:P molar ratio may be 5 or more and less than or equal to 50:1. In some cases, the N:P molar ratio may be 7 or more and less than or equal to 50:1. In some cases, the N:P molar ratio may be greater than 2 and less than or equal to 25:1. In some cases, the N:P molar ratio can be between 3 and 25:1. In some cases, the N:P molar ratio can be between 5 and 25:1. In some cases, the N:P molar ratio can be between 7 and 25:1.

[0128] In preferred embodiments, the polyplex has an amine-to-phosphate (N / P) ratio of 2 to 100, for example, 2 to 50, for example, 2 to 40, for example, 2 to 30, for example, 2 to 20, for example, 2 to 5. Preferably, the N / P ratio is inversely proportional to the molecular weight of the chitosan, i.e., (e.g., double) derivatized chitosan with a smaller molecular weight requires a higher N / P ratio, and vice versa.

[0129] The nucleic acids of the present invention will generally contain phosphodiester bonds, but in some cases, they will include nucleic acid analogs that may have alternative skeletons or other modifications or parts incorporated for any of the following purposes, e.g., stability and protection. Other analog nucleic acids intended include those having a non-ribose skeleton. In addition, naturally occurring nucleic acids, analogs, and mixtures of both can be created. Nucleic acids may be single-stranded or double-stranded, or may contain parts of both double-stranded and single-stranded sequences. Examples of nucleic acids include, but are not limited to, DNA, RNA, and hybrids, and nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, etc. Nucleic acids include any form of DNA, any form of RNA including triple-stranded, double-stranded, or single-stranded RNA, antisense, siRNA, ribozyme, deoxyribozyme, polynucleotide, oligonucleotide, chimeric, microRNA, and derivatives thereof. Nucleic acids include, but are not limited to, peptide nucleic acids (PNA), phosphorodiamidate morpholino oligos (PMO), locked nucleic acids (LNA), glycol nucleic acids (GNA), and threose nucleic acids (TNA). For phosphorus-free synthetic nucleic acids, it will be understood that equivalent measurements of the (+):P or N:P ratio can be approximated by the number of nucleotide (or nucleotide analog) bases.

[0130] In preferred embodiments, the polyplex of the composition comprises chitosan molecules having an average molecular weight 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 before functionalization. In some embodiments, the polyplex of the composition comprises chitosan having an average molecular weight of less than 15 kDa, less than 10 kDa, less than 7 kDa, or less than 5 kDa before functionalization.

[0131] In preferred embodiments, the polyplex contains chitosan molecules having an average of less than 680 glucosamine monomer units, more preferably less than 400 glucosamine monomer units, more preferably less than 310 glucosamine monomer units, more preferably less than 250 glucosamine monomer units, and most preferably less than 190 glucosamine monomer units. In some embodiments, the polyplex contains chitosan molecules having an average of 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.

[0132] Chitosan and (e.g., double) derivatized chitosan nucleic acid polyplexes may be prepared by any method known in the art, including, but not limited to, those described herein.

[0133] 2.2.1. Nucleic acids As described above, chitosan polyplexes may contain multiple nucleic acids. In one embodiment, the nucleic acid component includes therapeutic nucleic acids. This (e.g., doubly) derivatized chitosan nucleic acid polyplex is suitable for use with any therapeutic nucleic acid known in the art. Therapeutic nucleic acids include therapeutic RNA, which is an RNA molecule capable of exerting a therapeutic effect in mammalian cells. Therapeutic RNA includes, but is not limited to, messenger RNA, antisense RNA, siRNA, short hairpin RNA, microRNA, and enzyme RNA. Therapeutic nucleic acids include, but are not limited to, nucleic acids intended to form triplicates, protein-binding nucleic acids, ribozymes, deoxyribozymes, and small nucleotide molecules.

[0134] Many types of therapeutic RNA are known in this field. See, for example, Meng et al., A new developing class of gene delivery: messenger RNA-based therapeutics, Biomator. 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; and 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).

[0135] Therapeutic nucleic acids also include nucleic acids that encode therapeutic proteins, including cytotoxic proteins and prodrugs.

[0136] In preferred embodiments, the nucleic acid components include a therapeutic nucleic acid construct. The therapeutic nucleic acid construct is a nucleic acid construct capable of exerting a therapeutic effect. The therapeutic nucleic acid construct may include a nucleic acid encoding a therapeutic protein, as well as a nucleic acid that produces a transcript which is therapeutic RNA. The therapeutic nucleic acid may be used to perform gene therapy by serving as a replacement or enhancement of a defective gene, or to compensate for the absence of a specific gene product by encoding a therapeutic product. The therapeutic nucleic acid may also inhibit the expression of endogenous genes. The therapeutic nucleic acid may encode all or part of its translation product and may function by recombining with DNA already present in the cell, thereby replacing a defective portion of the gene. It may also encode part of a protein and exert its effect by co-repression of a gene product. In preferred embodiments, the therapeutic nucleic acid is selected from those disclosed in US2011 / 0171314 (expressly incorporated herein by reference).

[0137] In preferred embodiments, the therapeutic nucleic acid encodes a therapeutic protein selected from the group consisting of hormones, enzymes, cytokines, chemokines, antibodies, mitogen factors, growth factors, differentiation factors, factors affecting angiogenesis, factors affecting blood clot formation, factors affecting blood glucose levels, factors affecting glucose metabolism, factors affecting lipid metabolism, factors affecting blood cholesterol levels, factors affecting blood LDL or HDL levels, factors affecting cell apoptosis, factors affecting food intake, factors affecting energy expenditure, factors affecting appetite, factors affecting nutrient absorption, factors affecting inflammation, and factors affecting bone formation. Particularly preferred are therapeutic nucleic acids encoding insulin, leptin, glucagon antagonists, GLP-1, GLP-2, ghrelin, cholecystokinin, growth hormone, coagulation factors, PYY, erythropoietin, anti-inflammatory agents, IL-10, IL-12, IL-17 antagonists, TNFα antagonists, growth hormone-releasing hormone, or parathyroid hormone.

[0138] 2.2.1.1. Expression regulatory regions In a preferred embodiment, the polyplex of the present invention comprises a therapeutic nucleic acid, which is a therapeutic construct comprising an expression regulatory region operably linked to a coding region. The therapeutic construct generates a therapeutic nucleic acid, which may be therapeutic in itself or encode a therapeutic protein.

[0139] In some embodiments, the expression regulatory region of the therapeutic construct has constitutive activity. In many preferred embodiments, the expression regulatory region of the therapeutic construct does not have constitutive activity. This provides dynamic expression of the therapeutic nucleic acid. "Dynamic" expression means expression that changes over time. Dynamic expression may include several such periods in which expression is low or absent, separated by periods of detectable expression. In many preferred embodiments, the therapeutic nucleic acid is operably linked to a regulating promoter. This provides regulating expression of the therapeutic nucleic acid.

[0140] The expression regulatory region includes regulatory polynucleotides (sometimes referred to as elements herein), such as promoters and enhancers, which affect the expression of operably linked therapeutic nucleic acids.

[0141] The expression regulatory elements included herein may be derived from bacteria, yeast, plants, or animals (mammalian or non-mammalian). Expression regulatory regions include full-length promoter sequences, e.g., native promoters and enhancer elements, as well as partial sequences or polynucleotide variants that retain all or part of the complete or non-mutant function (e.g., retain some degree of trophic regulation or cell / tissue-specific expression). As used herein, the terms “functional” and its grammatical variations, when used with respect to nucleic acid sequences, partial sequences, or fragments, mean that the sequence possesses one or more functions of a native nucleic acid sequence (e.g., a non-mutant or unmodified sequence). As used herein, the term “mutant” means a substitution, deletion, or addition of a sequence, or other modification (e.g., a chemical derivative such as a nuclease-resistant modified form).

[0142] As used herein, the term “operable linkage” refers to the physical juxtaposition of components described in such a way that they enable them to function in the manner intended. In the example of an expression regulatory element operably linked to a nucleic acid, the relationship is such that the regulatory element modulates the expression of the nucleic acid. Typically, the expression regulatory region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., “upstream”). The expression regulatory 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). The expression regulatory element can be located at a distance from the transcribed sequence (e.g., 100–500, 500–1000, 2000–5000+ nucleotides from the nucleic acid). A concrete example of an expression regulatory element is a promoter, which is typically located at the 5' end of the transcribed sequence. Another example of an expression regulatory element is an enhancer, which can be located at the 5' or 3' end of the transcribed sequence, or within the transcribed sequence.

[0143] Some regulatory regions provide regulated expression to operably ligated therapeutic nucleic acids. A signal (sometimes called a stimulus) can increase or decrease the expression of therapeutic nucleic acids operably ligated to such regulatory regions. Such regulatory regions that increase expression in response to a signal are often called inductive. Such regulatory regions that decrease expression in response to a signal are often called repressive. Typically, the amount of increase or decrease provided by such elements is proportional to the amount of signal present; the greater the signal, the greater the increase or decrease in expression.

[0144] Numerous modulo promoters are known in the art. Preferred inducible regulatory regions include those comprising an inducible promoter stimulated by a small molecule chemical compound. In one embodiment, the regulatory region responds to a chemical that is orally deliverable but not typically found in food. Specific examples can be found, for example, in U.S. Patents 5,989,910; 5,935,934; 6,015,709; and 6,004,941.

[0145] In one embodiment, the therapeutic construct further includes an embedding sequence. In one embodiment, the therapeutic construct includes a single embedding sequence. In another embodiment, the therapeutic construct includes first and second embedding sequences that embed a therapeutic nucleic acid or a portion thereof into the genome of a target cell. In a preferred embodiment, the embedding sequence(s) are functional in combination with embedding means selected from the group consisting of Mariner, Sleeping Beauty, FLP, Cre, ФC31, R, and Lambda, as well as embedding means derived from embedding viruses such as AAV, retroviruses, and lentiviruses.

[0146] In one embodiment, the composition further comprises a non-therapeutic construct in addition to a therapeutic construct, the non-therapeutic construct comprising a nucleic acid sequence encoding an embedding means operably linked to a second expression regulatory region. This second expression regulatory region and the expression regulatory region operably linked to the therapeutic nucleic acid may be the same or different. The means encoded for embedding are preferably selected from the group consisting of Mariner, Sleeping Beauty, FLP, Cre, ФC31, R, Lambda, and embedding means derived from embedding viruses such as AAV, retroviruses, and lentiviruses.

[0147] For further teachings, see WO2008 / 020318 (the whole is expressly incorporated herein by reference). In one embodiment, the nucleic acid of the (e.g., double) derivatized chitosan nucleic acid polyplex is an artificial nucleic acid.

[0148] Preferred artificial nucleic acids include, but are not limited to, peptide nucleic acids (PNA), phosphorodiamidate morpholino oligos (PMO), locked nucleic acids (LNA), glycol nucleic acids (GNA), and threose nucleic acids (TNA).

[0149] 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 enzyme RNA.

[0150] In one embodiment, the therapeutic nucleic acid is DNA.

[0151] In one embodiment, the therapeutic nucleic acid includes a nucleic acid sequence that encodes a therapeutic protein.

[0152] 2.3. Polyols Chitosan derivative nanoparticles can be functionalized with polyols. Generally, polyols useful in this invention are typically hydrophilic. In some cases, chitosan derivative nanoparticles are functionalized with a cationic component such as an amino group and a polyol. Such chitosan derivative nanoparticles functionalized with an amino group and a cationic moiety such as a polyol are called "double-derivatized chitosan nanoparticles."

[0153] In some embodiments, the chitosan derivative nanoparticles include a polyol of formula II: [ka] (In the formula, R 2 is selected from H and hydroxyl, R 3 is selected from H and hydroxyl, X is selected from C2-C6 alkylenes optionally substituted with one or more hydroxyl substituents.

[0154] In some embodiments, the chitosan derivative nanoparticles are polyols of formula II (wherein R 2 is selected from H and hydroxyl; R 3 (where is selected from H and hydroxyl; X is functionalized with C2-C6 alkylenes optionally substituted with one or more hydroxyl substituents).

[0155] In some embodiments, the chitosan derivative nanoparticles include a polyol of formula III: [ka] (In the formula, -----Y is, =O And, R 2 is selected from H and hydroxyl, X is selected from C2-C6 alkylenes optionally substituted with one or more hydroxyl substituents. [ka] (This indicates the bond between the polyol and the derivatized chitosan.)

[0156] In one embodiment, the polyol according to the present invention having 3 to 7 carbon atoms may have one or more carbon-carbon multiple bonds. In a preferred embodiment, the polyol according to the present invention contains a carboxyl group. In a more preferred embodiment, the polyol according to the present invention contains an aldehyde group. When the polyol according to the present invention contains an aldehyde group, those skilled in the art will recognize that such a polyol encompasses both open-chain conformations (aldehydes) and cyclic conformations (hemiacetals).

[0157] Non-limiting examples of polyols include gluconic acid, threonic acid, glucose, and threose. Examples of other such polyols that may have carboxyl groups and / or aldehyde groups, or that may be sugars or acidic forms thereof, are described in further 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 specific stereochemistry.

[0158] In preferred embodiments, 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.

[0159] In preferred embodiments, 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.

[0160] In some embodiments, the polyol may be a compound of formula IV or formula V. [ka]

[0161] In preferred embodiments, the polyol is a compound of formula IV. In some cases, the polyol of formula IV is bonded to chitosan by reductive amination.

[0162] A hydrophilic polyol having a carboxyl group may be bonded to a cationic functionalized chitosan, such as chitosan or amine-functionalized chitosan (e.g., Arg-bonded chitosan (Arg-chitosan)). In some embodiments, the polyol is bonded at a reaction pH of 6.0 ± 0.3. At this pH, the carboxylic acid group of the hydrophilic polyol can be attacked by an unbonded amine on the chitosan skeleton according to a nucleophilic substitution reaction mechanism.

[0163] Hydrophilic polyols, which are natural sugars, may be bonded to chitosan, such as cationically functionalized chitosan, such as amine-functionalized chitosan (e.g., Arg-bonded chitosan (Arg-chitosan)), by reductive amination followed by reduction with NaCBH3 or NaBH.

[0164] 2.4. Polymers: Polyplex compositions Chitosan polyplexes can be mixed with multiple polymers, each polymer comprising a hydrophilic uncharged moiety and a charged (anionic) moiety. As described above, chitosan polyplexes are formulated to either not complex with anionic moiety-containing polymers or to have a positive charge before complexation. Therefore, under suitable conditions, the polymer components will form reversible charge-to-charge complexes with chitosan derivative nucleic acid polyplexes. In some embodiments, the polymers of the polymer components are unbranched. In some embodiments, the polymers are branched. In some cases, the polymer components include a mixture of branched and unbranched polymers.

[0165] In some embodiments, polymer components are released from the chitosan polyplex after administration, after entry into cells, and / or after endocytosis. While we do not wish to be bound by theory, it is hypothesized that by complexing a polyplex with an anion-partially containing polymer, the polyplex:polymer composition thus formed may provide improved in vitro, in solution, and / or in vivo stability without substantially hindering transfection efficiency. In some embodiments, the polyplex:polymer composition thus formed may provide, for example, reduced mucosal adhesion properties compared to the same polyplex without the polymer components.

[0166] In preferred embodiments, the polyplex:polymer composition has a low net positive zeta potential, a net neutral zeta potential, or a net negative zeta potential (approximately +10 mV to approximately -20 mV) at physiological pH. Such compositions may exhibit reduced aggregation under physiological conditions and reduced nonspecific binding to ubiquitous anionic components in vivo. These properties may enhance the movement of such compositions (e.g., enhanced diffusion in mucus) to bring them into contact with cells and result in enhanced intracellular release of nucleic acids.

[0167] 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 specific 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 specific 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 specific 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 specific 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.

[0168] In one embodiment, the polyplex polymer composition has a supercoil DNA content of 80%, at least 80%, preferably 90%, and more preferably at least 90%.

[0169] In one embodiment, the polyplex:polymer composition has an average zeta potential of +10mV to -10mV at physiological pH, most preferably +5mV to -5mV at physiological pH.

[0170] The polyplex:polymer composition is preferably homogeneous with respect to particle size. Therefore, in preferred embodiments, the composition has a low mean polydispersity index ("PDI"). In particularly preferred embodiments, the dispersion of the polyplex:polymer composition has a PDI of less than 0.5, more preferably less than 0.4, more preferably less than 0.3, even more preferably less than 0.25, and most preferably less than 0.2.

[0171] In some cases, the dispersion of the polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range after one or more freeze-thaw cycles. In some cases, the dispersion of the polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range after being stored in solution at 4°C for at least 48 hours. In some cases, the dispersion of the polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range after being stored in solution at 4°C for at least one or two weeks.

[0172] In some cases, the dispersion of the Polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range after freeze-drying and rehydration. In some cases, the dispersion of the Polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range after spray-drying and rehydration. In some cases, the dispersion of the Polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range when concentrated to a nucleic acid concentration of at least 250 μg / mL (e.g., by ultrafiltration such as tangential flow filtration). In some cases, the dispersion of the Polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to approximately 1,000 μg / mL. In some cases, a dispersion of the polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range when concentrated to nucleic acid concentrations of 125 μg / mL to approximately 25,000 μg / mL. In some cases, a dispersion of the polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range when concentrated to nucleic acid concentrations of 125 μg / mL to approximately 2,000 μg / mL. In some cases, a dispersion of the polyplex polymer composition exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range when concentrated to nucleic acid concentrations of 125 μg / mL to approximately 5,000 μg / mL. In some cases, the dispersion of polyplex polymer compositions exhibits one or more of the above-mentioned PDI, mean zeta potential, % supercoiled DNA, or mean particle size (nm) or size range when concentrated to nucleic acid concentrations of 125 μg / mL to approximately 10,000 μg / mL.

[0173] In general, the polyplex:polymer compositions described herein exhibit good solution behavior (e.g., stability and / or non-aggregation) (measured by PDI or average particle size) in the absence of excipients, such as cryoprotectants, antifreezes, surfactants, rehydrating or wetting agents. In some cases, the polyplex:polymer compositions described herein exhibit favorable solution behavior (e.g., stability and / or non-aggregation) (measured by PDI or average particle size) in physiological or artificial physiological fluids. For example, in some embodiments, the polyplex:polymer compositions described herein are stable in artificial intestinal fluid, intestinal fluid, artificial urine, and mammalian urine when incubated in a mammalian bladder (e.g., in contact with urine) and / or in the intestines (e.g., colon, small intestine, or large intestine, preferably the colon). In some embodiments, the polyplex:polymer compositions are stable for at least about 10 minutes, or about 10 minutes to about 1 hour, or at least about 1 hour, or 1 hour to about 2 hours under one or more of the conditions described herein (e.g., in artificial intestinal fluid).

[0174] As described above, the polyplex:polymer compositions described herein are preferably substantially size-stable in composition. In preferred embodiments, the compositions of the present invention include polyplex:polymer particles whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% over 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours at room temperature. In particularly preferred embodiments, the compositions of the present invention include polyplex:polymer particles whose average diameter increases by less than 25% over at least 24 hours or at least 48 hours at room temperature.

[0175] The polyplex:polymer particles of this composition are preferably substantially size-stable under cooling conditions. In preferred embodiments, the composition of the present invention comprises polyplex:polymer particles whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% over 6 hours, more preferably 12 hours, more preferably 24 hours, most preferably 48 hours, at 2-8°C.

[0176] The polyplex polymer particles of this composition are preferably substantially size-stable under freeze-thaw conditions. In preferred embodiments, the composition of the present invention comprises polyplex whose average diameter increases 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 at -20 to -80°C.

[0177] In preferred embodiments, the composition has a nucleic acid concentration greater than 0.5 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 0.6 mg / ml, more preferably at least 0.75 mg / ml, more preferably at least 1.0 mg / ml, more preferably at least 1.2 mg / ml, and most preferably at least 1.5 mg / ml, and is substantially free of precipitated polyplexes. In another preferred embodiment, the composition has a nucleic acid concentration greater than 2 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 2.5 mg / ml, more preferably at least 5 mg / ml, more preferably at least 10 mg / ml, more preferably at least 15 mg / ml, and most preferably at 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 preferred embodiments, the composition is substantially free of nucleic acids that do not form complexes.

[0178] In preferred embodiments, the polyplex:polymer particle composition is isotonic. Achieving isotonicity while maintaining the stability of the polyplex is highly desirable for formulating pharmaceutical compositions, and these preferred compositions are well-suited for pharmaceutical formulations and therapeutic applications.

[0179] In certain embodiments, the polyplex:polymer particle composition can be decoated to release all or part of the polymer coating by lowering the pH, for example, to release PEG. In certain embodiments, the polymer coating is released by incubating the particles under pH conditions lower than the pKa of the polyanion anchor region of the polymer. For example, if the polymer coating is polyglutamate, the polymer coating can be released by incubating the particles at a pH lower than the pKa of the polyglutamate, e.g., a pH less than about 4.25. In certain embodiments, the polymer coating can be released by incubating the particles under pH conditions that are at least 0.25 pH units or at least 0.5 pH units lower than the pKa of the polyanion anchor region of the polymer coating.

[0180] In certain embodiments, the polyplex:polymer particle composition can be decoated by subjecting the particles to high ionic intensity, for example, to release all or part of the PEG.

[0181] While we do not wish to be bound by theory, it is hypothesized that certain physiological conditions may promote partial (e.g., greater than 5%), substantial (greater than 50%), extensive (e.g., greater than 90%), or complete decoating 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) may promote polymer coat release. As another example, certain extracellular conditions may promote partial (e.g., greater than 5%), substantial (greater than 50%), extensive (e.g., greater than 90%), or complete (100%) decoating of the reversibly PEGylated chitosan DNA polyplexes described herein. In some cases, the high ionic strength and / or acidic pH conditions that typically occur at specific locations in the gastrointestinal tract may promote partial (e.g., greater than 5%), substantial (greater than 50%), extensive (e.g., greater than 90%), or complete (100%) decoating of the reversibly PEGylated chitosan DNA polyplexes described herein.

[0182] In certain embodiments, the PEGylated polyplexes described herein are formulated for delivery to cells, tissues, or body compartments (e.g., the intestine, small intestine, large intestine, colon, lung, or bladder), and as a result, the polyplexes remain PEGylated, thereby facilitating transfection to target cells. In some embodiments, the PEGylated polyplexes described herein release a polymer coating partially (e.g., more than 5%), substantially (more than 50%), extensively (e.g., more 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 cells, tissues, or body compartments (e.g., the intestines, small intestines, large intestines, colon, lungs, or bladder), and as a result, the PEGylated polyplexes described herein release the polymer coating partially (e.g., more than 5%), substantially (more than 50%), extensively (e.g., more than 90%), or completely (100%) upon delivery to cells, tissues, or body compartments (e.g., the intestines, small intestines, large intestines, colon, lungs, or bladder).

[0183] It will be understood that the anionic charge density and / or pKa of the polymer's anionic anchor region can be adjusted to promote or inhibit release under intended conditions. Similarly, it will be understood that 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, to deliver to the intestines via the low-pH environment of the stomach, pegylated polyplex formulations can be enterically coated and / or delivered in a buffer to enhance the pH of the stomach environment. Optimized reversibly PEGylated particle compositions and formulations can be identified by assaying stability and transfection efficiency using the assays described herein.

[0184] A composition comprising a chitosan polyplex complexed with an anionic moiety-containing polymer may be characterized by a ratio, referred to as the "(+):(-) molar ratio," of the cationic functional group of the (e.g., double) derivatized chitosan polyplex (+) to the anionic moiety of the polymer (-). This (+):(-) molar ratio can vary from about 1:100 to less than about 10:1.

[0185] In certain embodiments, the (+):(-) molar ratio may be greater than about 1:75 and less than about 8:1. In some cases, the (+):(-) molar ratio may be greater than 1:10 and less than 10:1. In some cases, the (+):(-) molar ratio may be 1:10 or about 1:10 to 10:1 or about 10:1. In some cases, the (+):(-) molar ratio may be 1:8 or about 1:8 to 8:1 or about 8:1. In certain embodiments, the (+):(-) molar ratio may be greater than 1:50 and less than about 10:1. In some cases, the (+):(-) molar ratio may be greater than 1:25 and less than about 10:1. In some cases, the (+):(-) molar ratio may be greater than 1:10 to less than about 7:1. In some cases, the (+):(-) molar ratio may be greater than 1:8 to less than about 7:1. In some cases, the (+):(-) molar ratio can be greater than 1:8 and less than approximately 6:1.

[0186] In certain embodiments where the cationic functional group (e.g., double)-derivativeized chitosan polyplex is an amino moiety, compositions comprising the chitosan polyplex complexed with an anionic moiety-containing polymer can be characterized by a ratio called the "N:A molar ratio," which is the ratio of the amino groups (N) of the (e.g., double)-derivativeized chitosan polyplex to the anionic (A) moiety of the polymer. This N:A molar ratio can vary from more than about 1:100 to less than about 10:1.

[0187] In certain embodiments, the N:A molar ratio may be greater than about 1:75 and less than about 8:1. In some cases, the N:A molar ratio may be greater than 1:10 and less than 10:1. In some cases, the N:A molar ratio may be 1:10 or about 1:10 to 10:1 or about 10:1. In some cases, the N:A molar ratio may be 1:8 or about 1:8 to 8:1 or about 8:1. In certain embodiments, the N:A molar ratio may be greater than 1:50 and less than about 10:1. In some cases, the N:A molar ratio may be greater than 1:25 and less than about 10:1. In some cases, the N:A molar ratio may be greater than 1:10 and less than about 7:1. In some cases, the N:A molar ratio may be greater than 1:8 to less than about 7:1. In some cases, the N:A molar ratio may be greater than 1:8 to less than about 6:1.

[0188] Additionally or alternatively, compositions comprising a chitosan polyplex complexed with an anionic moiety-containing polymer may be characterized by a three-component ratio called the "(+):P:(-) molar ratio," which is the ratio of the cationic functional group (+) of the (e.g., double) derivatized chitosan polyplex to the phosphorus atom (P) of the nucleic acid to the anionic moiety (-) of the polymer.

[0189] In certain embodiments where (+):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 where (+):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 where (+):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 where (+):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 where (+):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 where (+):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 where (+):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 where (+):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 where (+):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.

[0190] 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 approximately 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 approximately 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 approximately 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 approximately 10:1:10, 10:1:15, 10:1:20, 10:1:25, 10:1:30, or 10:1:40.

[0191] Those skilled in the art will understand that amino-functionalized chitosan polyplex particles complexed with an anionic moiety-containing polymer can be characterized by a three-component ratio, referred to as the "N:P:A molar ratio," of amino functional groups of the (e.g., double) derivatized chitosan polyplex (N) to phosphorus atoms of the nucleic acid (P) to the anionic moiety of the polymer (A). In certain embodiments where N:P is at least 2:1 to 20:1 or less, the P:A molar ratio can vary from at least 1:40 to about 40:1.

[0192] In certain embodiments where N:P is at least 2:1 to 20:1, the molar ratio of P:A can vary from at least 1:40 to about 1:10. In certain embodiments where N:P is at least 2:1 to 20:1, the molar ratio of P:A can vary from at least 1:25 to about 25:1. In certain embodiments where N:P is at least 2:1 to 20:1, the molar ratio of P:A can vary from at least 1:25 to about 1:10. In some cases where N:P is at least 2:1 to 20:1, the molar ratio of P:A can vary from at least 1:20 to about 20:1. In some cases where N:P is at least 2:1 to 20:1, the molar ratio of P:A can vary from at least 1:20 to about 1:10. In some cases where N:P is at least 2:1 to 20:1 or less, the molar ratio of P:A can vary from at least 1:10 to about 10:1. In some cases where 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 2:1. In some cases where 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 1:1.

[0193] 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 approximately 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 approximately 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 approximately 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 approximately 10:1:10, 10:1:15, 10:1:20, 10:1:25, 10:1:30, or 10:1:40.

[0194] 2.4.1.Hydrophilic uncharged moiety The hydrophilic uncharged portion of the polymer may be or include a polyalkylene polyol or polyalkylene oxypolyol portion, or a combination thereof. The hydrophilic uncharged portion of the polymer may be or include a polyalkylene glycol or polyalkylene oxyglycol portion. In certain embodiments, the polyalkylene glycol portion may be or include a polyethylene glycol portion and / or a monomethoxypolyethylene glycol portion. In certain preferred embodiments, the uncharged portion of the polymer may be or include polyethylene glycol. The hydrophilic uncharged portion of the polymer may be or include other biocompatible polymers such as polylactic acid.

[0195] 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 moieties 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).

[0196] The hydrophilic portion may have a weight-average molecular weight of approximately 500 Da to approximately 50,000 Da. In some embodiments, the hydrophilic portion has a weight-average molecular weight of approximately 1,000 Da to approximately 10,000 Da. In certain embodiments, the hydrophilic portion has a weight-average molecular weight of approximately 1,500 Da to approximately 7,500 Da. In certain embodiments, the hydrophilic portion has a weight-average molecular weight of approximately 3,000 Da to approximately 5,000 Da. In some cases, the hydrophilic portion has a weight-average molecular weight of 5,000 Da or approximately 5,000 Da.

[0197] 2.4.2. Anionic Polymer Portion The anionic polymer portion of the polymer may contain multiple functional groups that have a negative charge at physiological pH. A wide variety of anionic polymers are suitable for use in the methods and compositions described herein. However, such anionic polymers can be provided as components of polymers having a hydrophilic uncharged polymer portion and can form (e.g., reversible) charge:charge complexes with positively charged (e.g., bi-derivatized) chitosan-nucleic acid nanoparticles.

[0198] Examples of 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 consists of amino acids that have a charged side chain at physiological pH. For example, the anionic polymer portion of the polymer may be a polyglutamate polypeptide, a polyaspartate polypeptide, or a mixture thereof. Additional amino acids or their mimics 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.

[0199] In some cases, the anionic polymer may be an anionic carbohydrate polymer or may contain anionic carbohydrate polymers. Exemplary anionic carbohydrate polymers include, but are not limited to, glycosaminoglycans that have a negative charge at physiological pH. Exemplary anionic glycosaminoglycans include, but are not limited to, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparin sulfate, hyaluronic acid, or combinations thereof. In certain embodiments, the anionic polymer portion of the polymer may be hyaluronic acid or contain hyaluronic acid.

[0200] Additional or alternative anionic carbohydrate polymers may include polymers containing dextran sulfate.

[0201] In some cases, the polyanionic portion is or comprises 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, such as polyalkylene oxides and polyacrylic acid copolymers.

[0202] In some cases, the polyanion portion is or contains a polyanion selected from the group consisting of arginate, carrageenan, fercereran, pectin, xanthan gum, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, cellulose, oxidized cellulose, carboxymethylcellulose, crosmarmellose, synthetic polymers and copolymers containing pendant carboxyl groups, phosphate groups or sulfate groups, mainly negatively charged polyamino acids, and biocompatible polyphenol materials.

[0203] The anionic portion of the polymer may have a weight-average molecular weight of approximately 500 Da to approximately 5,000 Da. In some embodiments, the anionic portion has a weight-average molecular weight of approximately 500 Da to approximately 3,000 Da. In certain embodiments, the anionic portion has a weight-average molecular weight of approximately 500 Da to approximately 2,500 Da. In certain embodiments, the anionic portion has a weight-average molecular weight of approximately 500 Da to approximately 2,000 Da. In certain embodiments, the anionic portion has a weight-average molecular weight of approximately 500 Da to approximately 1,500 Da. In some embodiments, the anionic portion has a weight-average molecular weight of approximately 1,000 Da to approximately 5,000 Da. In some embodiments, the anionic portion has a weight-average molecular weight of approximately 1,000 Da to approximately 3,000 Da. In certain embodiments, the anionic portion has a weight-average molecular weight of approximately 1,000 Da to approximately 2,500 Da. In certain embodiments, the anionic moiety has a weight-average molecular weight of approximately 1,000 Da to approximately 2,000 Da. In some cases, the anionic moiety has a weight-average molecular weight of 1,500 Da or approximately 1,500 Da.

[0204] As used herein, “block copolymer,” “block copolymer,” etc., refer to copolymers containing distinct homopolymer regions. A binary block copolymer contains two distinct homopolymer regions. A ternary copolymer contains three distinct homopolymer regions. The three distinct regions may each be different (e.g., AAAA-BBBB-CCCC), or two regions may be the same (e.g., AAAA-BBBB-AAAA), similar (e.g., AAAA-BBBB-AAA), and “A,” “B,” and “C” represent different monomer subunits that form the copolymer. For example, “A” may represent the ethylene glycol monomer subunit of a polyethylene glycol homopolymer, and “B” may represent the glutamic acid subunit of a polyglutamic acid homopolymer. A block copolymer can be a linear (e.g., binary or ternary) block copolymer. Exemplary embodiments of linear binary and ternary block copolymers used in the present invention are listed below in the following limited list. [Table 1-1] [Table 1-2]

[0205] In one embodiment, the block copolymer is a PEG-polyglutamic acid polymer having the following structure, or comprises the same. [ka]

[0206] In one embodiment, the block copolymer is a PEG-polyaspartate polymer having the following structure, or comprises the same. [ka]

[0207] In one embodiment, the block copolymer is a PEG-hyaluronic acid polymer having the following structure, or comprises the same. [ka]

[0208] 2.5. How to Create As described above, those skilled in the art will understand that the polyplex polymer particles of the present invention can be produced in a variety of ways. For example, polyplex particles can be produced and then brought into contact with a polymer. In exemplary, non-limiting embodiments, polyplex particles are prepared by providing and combining functionalized chitosan and nucleotide raw materials. The raw material concentrations may be adjusted to suit various amino-to-phosphate (N / P) ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly in small batches, e.g., less than 2 mL, the functionalized chitosan and nucleotide raw materials may be mixed by gradually dropping the nucleotide raw material onto the functionalized chitosan raw material while vortexing the container. In other embodiments, the functionalized chitosan and nucleotide raw materials 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).

[0209] Similarly, polyplex particle raw materials (e.g., aqueous solutions containing the polyplex composition) can be provided (e.g., isolated from the above reaction mixture) and mixed with polymer raw materials (e.g., aqueous solutions containing the polymer). The raw material concentrations may be adjusted to suit various amino-to-anion ratios (N / A), amino-to-phosphorus ratios (N:P), N:P:A ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly in small batches, e.g., less than 2 mL, the raw materials may be mixed by gradually dropping the first raw material (e.g., polyplex) onto the second raw material (e.g., polymer) while vortexing the container. In other embodiments, the raw materials may be mixed by in-line mixing of two fluid streams. In other embodiments, the resulting polyplex:polymer complex 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).

[0210] In some embodiments, the polyplex polymer composition comprises a core-shell type particle composition, where the particles comprise a polyplex core and a non-covalently bonded (e.g., releaseable) polymer shell. As understood, one method for producing such a core-shell type composition comprises forming a polyplex and then combining it with polymer raw materials as described above.

[0211] Alternatively, the polyplex:polymer composition can be produced in a one-step method in which nucleic acids, derivatized chitosan, and multiple linear block copolymers comprising at least one polyanion (PA) anchor region and at least one hydrophilic polymer (e.g., PEG) tail region are mixed in appropriate ratios to form the polyplex:polymer composition. While not wishing to be bound by theory, the inventors believe that such a one-step method may produce smaller particle sizes that may be advantageous in certain indications where in vivo mucosal diffusion is limited.

[0212] 3. Powdered formulation The polyplex:polymer composition of the present invention comprises a powder. In a preferred embodiment, the present invention provides a dry powder polyplex:polymer composition. In a preferred embodiment, the dry powder polyplex:polymer composition is produced by dehydration (e.g., spray drying or freeze-drying) of the chitosan-nucleic acid polyplex dispersion of the present invention.

[0213] 4. Pharmaceutical preparations The present invention also provides "pharmaceutically acceptable" or "physiologically acceptable" formulations comprising the polyplex:polymer composition of the present invention. Such formulations can be administered in vivo to a subject to carry out a treatment method.

[0214] As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” preferably refer to carriers, diluents, excipients, etc., that can be administered to a subject without causing excessive 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, solvents, syrups, and elixirs. Liquid formulations may also be prepared by solid reconstitution.

[0215] Pharmaceutical formulations can be prepared from carriers, diluents, excipients, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for administration to the target. Such formulations may be contained in tablets (coated or uncoated), capsules (hard or soft), microbeads, emulsions, powders, granules, crystalline preparations, suspensions, syrups, or elixirs. Among other additives, auxiliary active compounds and preservatives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0216] Excipients may include salts, isotonic agents, serum proteins, buffers or other pH adjusters, antioxidants, thickeners, uncharged polymers, preservatives, or antifreeze agents. Excipients used in the compositions of the present invention may further include isotonic agents and buffers or other pH control agents. These excipients may be added to obtain a preferred pH range (about 6.0 to 8.0) and osmotic pressure (about 50 to 400 mmol / L). Examples of suitable buffers are acetates, borates, carbonates, citrates, phosphates, and sulfonated organic molecular buffers. Such buffers may be present in the composition at a concentration of 0.01 to 1.0% (w / v). The isotonic agent may be selected from those known in the art, for example, mannitol, dextrose, glucose, and sodium chloride, or other electrolytes. Preferably, the isotonic agent is glucose or sodium chloride. The isotonic agent may be used in an amount that gives the composition the same or similar osmotic pressure as the biological environment in which it is introduced. The concentration of the isotonic agent in the composition depends on the properties of the specific isotonic agent used and may be in the range of 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 isotonic agent is sodium chloride, it is preferably used in an amount of up to 1% w / v, particularly 0.9% w / v. The compositions of the present invention may further contain preservatives. Examples of preservatives include polyhexamethylene biguanidine, benzalkonium chloride, stable oxychloro complex (known as Purite®), phenyl 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%. Furthermore, the compositions of the present invention may also contain cryopreservatives. Preferred cryopreservatives are dextran, glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, glycerol and polyethylene glycol with a molecular weight of less than 100,000 g / mol, or mixtures thereof. Most preferred are glucose, trehalose, and polyethylene glycol.Typically, such cryopreservatives are present at a concentration of approximately 0.01–10%.

[0217] Pharmaceutical formulations can be formulated to be compatible with the intended route of administration. For example, for oral administration, a composition may be incorporated with excipients and used in the form of tablets, lozenges, capsules, such as gelatin capsules, or coatings, such as enteric coatings (Eudragit® or Sureteric®). Pharmacochemically compatible binders and / or auxiliary materials may be included in the oral formulation. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders, e.g., microcrystalline cellulose, tragacanth gum, or gelatin; excipients, e.g., starch or lactose; disintegrants, e.g., alginic acid, Primogel, or corn starch; lubricants, e.g., magnesium stearate or other stearates; lubricants, e.g., colloidal silicon dioxide; sweeteners, e.g., sucrose or saccharin; or flavorings, e.g., peppermint, methyl salicylate, or flavorings.

[0218] The formulation may also include a carrier to protect the composition from rapid degradation or elimination from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. For example, a time-delaying material such as glyceryl monostearate or glyceryl stearate may be used alone or in combination with a wax.

[0219] Suppositories and other rectally administerable formulations (e.g., those administerable by enema) are also being considered. Furthermore, regarding rectal delivery, see, for example, Song et al., Mucosal drug delivery: membranes, methods, 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.

[0220] Appropriate additional pharmaceutical formulations for administration are known in the art and applicable to the methods and compositions of the present invention (e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)).

[0221] 5. Administration In one embodiment, the use of a polyplex polymer composition provides long-term stability of the polyplex at physiological pH. This provides effective mucosal administration.

[0222] One of several routes of administration that involve contact with mucosal cells or tissues is possible, and the choice of a particular route will depend in part on the target mucosal cells or tissues. Syringes, endoscopes, cannulas, endotracheal tubes, catheters, and other items may be used for administration.

[0223] The dose or “effective dose” 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. However, prevention or suppression of the progression or worsening of the disorder or condition or symptoms is a desirable outcome. Therefore, in the case of 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 the condition treatable by the method of the present invention will depend on the condition and the desired outcome, which can be readily determined by those skilled in the art. The appropriate dose will depend on the condition being treated, the desired therapeutic effect, and the individual subject (e.g., bioavailability within the subject, sex, age, etc.). The effective dose can be determined by measuring the relevant physiological effect.

[0224] Veterinary applications are also intended by this invention. Accordingly, in one embodiment, the present invention provides a method for treating a non-human mammal, comprising administering the polyplex:polymer composition of the present invention to a non-human mammal in need of treatment.

[0225] 5.1. Oral administration This composition may be administered orally. Oral administration may involve swallowing, allowing the compound to enter the gastrointestinal tract. The composition of the present invention may also be administered directly to the gastrointestinal tract.

[0226] Formulations suitable for oral administration include solid formulations such as tablets, capsules, coated capsules containing particles or coated particles, liquids or powders, sweeteners (including liquid-filled formulations), tubes, multiply particles and nanoparticles, gels, films, ovules, and sprays.

[0227] Tablet dosage forms generally contain disintegrants. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant will make up 1% to 25% by weight of the dosage form, preferably 5% to 20% by weight.

[0228] Binders are generally used to give tablet formulations cohesiveness. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dicalcium phosphate dihydrate.

[0229] The tablets may optionally contain surfactants such as sodium lauryl sulfate and polysorbate 80, as well as flow promoters such as silicon dioxide and talc. If present, the surfactants may be present in an amount of 0.2% to 5% by weight of the tablet, and the flow promoters may be present in an amount of 0.2% to 1% by weight of the tablet.

[0230] The tablets generally also contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. The lubricant generally makes up 0.25% to 10% by weight of the tablet, preferably 0.5% to 3% by weight.

[0231] Other possible ingredients include antioxidants, colorants, flavorings, preservatives, and flavor enhancers.

[0232] The tablet blend may be compressed directly or by rollers to form tablets. The tablet blend or a part of the blend may alternatively be wet, dry, or melt granulated, melt solidified, or extruded before tableting. The final formulation may contain one or more layers, may or may not be coated, and may further be encapsulated.

[0233] The formulation of tablets is discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

[0234] Consumable oral films for human or veterinary use are typically flexible water-soluble or water-swellable thin film dosage forms, which may be rapidly dissolving or mucoadhesive, and typically contain a film-forming polymer, binder, solvent, humectant, plasticizer, stabilizer or emulsifier, viscosity modifier, and solvent. Some components of the formulation may perform multiple functions.

[0235] Also included in the present invention are multi-particle beads containing the composition of the present invention.

[0236] Other possible components include antioxidants, colorants, flavoring agents and flavor enhancers, preservatives, saliva stimulants, cooling agents, co-solvents (including oils), skin softeners, bulking agents, defoaming agents, surfactants, and taste modifiers.

[0237] The films according to the present invention are typically prepared by evaporation drying of an aqueous thin film coated on a peelable backing support or paper. This may be done in a drying oven or tunnel, typically a composite coater dryer, or by freeze drying or vacuuming.

[0238] Solid formulations for oral administration may be formulated to provide immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0239] Other suitable release techniques such as high energy dispersions and osmotic pressure and coated particles are known.

[0240] 5.2. Mucosal Administration The compositions of the present invention may also be administered to mucosal membranes. For example, the compositions can be administered to mucosal cells or tissues of the gastrointestinal tract including, but not limited to, mucosal cells or tissues of the small intestine and / or large intestine and / or colon. Other target mucosal cells or tissues include, but are not limited to, cells or tissues of the eye, airway epithelium, lung, vagina, and bladder.

[0241] Representative formulations for this purpose include solutions, gels, hydrogels, solvents, creams, foams, films, implants, sponges, fibers, powders, and microemulsions.

[0242] The compounds of the present invention can be administered to mucosal membranes by nasal or inhalation, usually in the form of dry powder from a dry powder inhaler (alone, as a mixture, e.g., as a dry blend with lactose, or as mixed component particles) without the use of a suitable propellant, or as an aerosol spray from a pressurized container, pump, spray, atomizer, or nebulizer.

[0243] Capsules, foaming agents, and cartridges used in inhalers or ventilators may be formulated to contain a powder mixture of the compounds of the present invention, a suitable powder base such as lactose or starch, and a performance modifier such as L-leucine, mannitol, or magnesium stearate.

[0244] Formulations for inhalation / intranasal administration may be formulated for immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0245] The compounds of the present invention may be administered rectally or vaginally, for example, in the form of suppositories, pessaries, or enemas. Cocoa butter is the conventional suppository base, but various alternatives may be used as needed.

[0246] Formulations for rectal / vaginal administration may be formulated to be immediate-release and / or controlled-release. Controlled-release formulations include delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, and programmed-release formulations.

[0247] The compounds of the present invention may also be administered directly to the eyes or ears, usually in the form of drops. Other formulations suitable for ophthalmic and ocular administration include ointments, biodegradable (e.g., absorbent gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particle systems. The formulations may also be delivered by iontophoresis.

[0248] Formulations for ocular / aural administration may be formulated to be immediate-release and / or controlled-release. Controlled-release formulations include delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, or programmed-release formulations.

[0249] 6.Therapeutic uses In one embodiment, the polyplex:polymer composition of the present invention may be used in therapeutic treatment. Such composition may, as it may be referred to herein, be called a therapeutic composition.

[0250] As discussed below, the therapeutic proteins of the present invention are produced by the polyplex:polymer composition of the present invention, which contains therapeutic nucleic acids. The use of the proteins described below refers to the use of the polyplex:polymer composition that affects the use of such proteins.

[0251] The therapeutic proteins intended for use in this invention possess a wide range of activities and find use in treating a wide range of disorders. The following descriptions of the activation of the therapeutic proteins of this invention and the indications treatable by the therapeutic proteins are illustrative and not intended to be exhaustive. The term "subject" refers to animals, preferably mammals, and particularly preferably humans.

[0252] A partial list of therapeutic proteins and target diseases is shown in Table 4. [Table 2-1] [Table 2-2]

[0253] In another embodiment, the therapeutic composition of the present invention comprises therapeutic nucleic acids that do not encode therapeutic proteins, such as therapeutic RNA. For example, by selecting therapeutic RNA that targets genes involved in the mechanisms of disease and / or undesirable cellular or physiological conditions, the composition may be used to treat a wide range of diseases and conditions. The composition is characterized in such a way that the range of therapeutic RNA used is not limited with respect to the scope of target selection. Accordingly, the composition finds use in any disease or condition involving suitable target mucosal tissue.

[0254] Specific non-limiting examples of therapeutic embodiments are described below. In some cases, therapeutic embodiments are intended to act on non-mucosal target tissues, cells, or organs. When the therapeutic effect is non-mucosal, it is understood that the cells or tissues into which the polyplex:polymer composition described herein comes into contact are mucosal, and the therapeutic effect is distal to the mucosal target. For example, mucosal cells can be transfected to produce and secrete hormones or other therapeutic agents.

[0255] 6.1. Hyperglycemia and weight Therapeutic proteins include insulin and insulin analogs. Diabetes mellitus is a debilitating metabolic disease caused by a lack of insulin production (type 1) or insufficient insulin production (type 2) from pancreatic β-cells (Unger, RH et al., Williams Textbook of Endocrinology Saunders, Philadelphia (1998)). Beta-cells are specialized endocrine cells that produce and store insulin for release after meals (Rhodes, et al. J. Cell Biol. 105:145 (1987)), and insulin is a hormone that facilitates the movement of glucose from the blood to the tissues where it is needed. Diabetic patients must have their blood glucose levels monitored frequently, and many require multiple insulin injections daily to survive. However, it is rare for such patients to reach ideal blood glucose levels with insulin injections (Turner, RC et al. JAMA 281:2005 (1999)). Furthermore, prolonged elevated insulin levels can lead to adverse side effects such as hypoglycemic shock and desensitization of the body's response to insulin. Therefore, diabetic patients still develop long-term complications such as cardiovascular disease, kidney disease, blindness, nerve damage, and impaired wound healing (UK Prospective Diabetes Study (UKPDS) Group, Lancet 352, 837 (1998)).

[0256] The disorders treatable by the methods of the present invention include hyperglycemic conditions such as insulin-dependent (type 1) or insulin-independent (type 2) diabetes mellitus, and physiological conditions or disorders associated with or resulting from hyperglycemia. Accordingly, hyperglycemic conditions treatable by the methods of the present invention also include histopathological changes associated with chronic or acute hyperglycemia (e.g., diabetes mellitus). Specific examples include pancreatic degeneration (β-cell destruction), renal tubular calcification, hepatic degeneration, eye damage (diabetic retinopathy), diabetic foot, ulcers of mucous membranes such as the mouth and gums, excessive bleeding, delayed blood clotting or wound healing, and an increased risk of coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity.

[0257] The composition is useful for reducing glucose, improving glucose tolerance, treating hyperglycemic conditions (e.g., diabetes), or treating physiological disorders associated with or resulting from hyperglycemic conditions. Such disorders include, for example, diabetic neuropathy (autonomic), nephropathy (kidney damage), skin infections and other skin disorders, deceleration or delay in the healing of injuries or wounds (e.g., leading to diabetic ulcers), eye damage (retinopathy, cataracts) that can lead to blindness, diabetic foot, and accelerated periodontitis. Such disorders also include an increased risk of developing coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity.

[0258] As used herein, the terms “hyperglycemic” or “hyperglycemia,” when used with respect to a subject's condition, mean transient or chronic abnormally high levels of glucose present in the subject's blood. The condition may be caused by a delay in glucose metabolism or absorption such that the subject exhibits an abnormal glucose tolerance or elevated glucose condition not normally found in a normal subject (e.g., a prediabetic subject with impaired glucose tolerance at risk of developing diabetes, or a diabetic subject). The fasting plasma glucose (FPG) level is less than about 110 mg / dl in normoglycemia, 110 - 126 mg / dl in the case of impaired glucose metabolism, and greater than about 126 mg / dl in diabetic patients.

[0259] Disorders treatable by protein production in the intestinal mucosal tissue include obesity or undesirable body weight. Leptin, cholecystokinin, PYY, and GLP-1 reduce hunger, increase energy expenditure, induce weight loss, or provide normal glucose homeostasis. Therefore, in various embodiments, the methods of the present invention for treating obesity or undesirable body weight or hyperglycemia include the use of therapeutic nucleic acids encoding leptin, cholecystokinin, PYY, or GLP-1. In another embodiment, therapeutic RNA targeting ghrelin is used. Ghrelin increases appetite and hunger. Therefore, in various embodiments, the methods of the present invention for treating obesity or undesirable body weight or hyperglycemia include the use of therapeutic RNA that targets ghrelin and reduces its expression. Treatable disorders typically include those associated with obesity, such as abnormally elevated serum / plasma LDL, VLDL, triglycerides, and cholesterol; plaque formation leading to narrowing or blockage of blood vessels; and an increased risk of hypertension / stroke and coronary heart disease.

[0260] As used herein, the terms “obesity” or “obesity” refer to a person whose weight is at least 30% greater than that of a normal person matched for age and sex. “Undesirable weight” refers to a person whose weight is between 1% and 29% greater than that of a normal person matched for age and sex, and a person who is normal in terms of weight and wishes to lose weight or prevent weight gain.

[0261] In one embodiment, the therapeutic protein of the present invention is a glucagon antagonist. Glucagon is a peptide hormone produced by β-cells in the pancreatic islets and is a major regulator of glucose metabolism (Unger RH & Orci LNEng.J.Med.304:1518(1981); Unger RHDiabetes 25:136(1976)). Similar to insulin, blood glucose concentration mediates glucagon secretion. However, in contrast to insulin, glucagon is secreted in response to a decrease in blood glucose levels. Therefore, the circulating concentration of glucagon is highest when fasting and lowest during meals. Elevated glucagon levels stimulate the liver to release glucose into the bloodstream, inhibiting insulin from promoting glucose storage. A specific example of a glucagon antagonist is [des-His1,des-Phe6,Glu9]glucagon-NH2. Streptozotocin-positive diabetic rats showed a 37% reduction in blood glucose levels within 15 minutes of an intravenous bolus (0.75 μg / g body weight) of this glucagon antagonist (Van Tine BA et al. Endocrinology 137:3316 (1996)). In another embodiment, the present invention provides a method for treating diabetes or hyperglycemia, comprising the use of therapeutic RNA that reduces the level of glucagon production from the pancreas.

[0262] In another embodiment, the therapeutic protein of the present invention useful for treating hyperglycemia or undesirable body weight (e.g., obesity) is glucagon-like peptide-1 (GLP-1). GLP-1 is a hormone released from L cells in the intestine during meals, which stimulates pancreatic β cells to increase insulin secretion. GLP-1 has additional activities that make it an attractive therapeutic agent for treating obesity and diabetes. For example, GLP-1 may reduce gastric emptying, suppress appetite, reduce glucagon concentration, increase β cell mass, stimulate glucose-dependent insulin biosynthesis and secretion, and increase the sensitivity of tissues to insulin (Kieffer TJ, Habener JF Endocrin. Rev. 20:876 (2000)). Therefore, regulated release of GLP-1 in the intestine in conjunction with meals may provide therapeutic benefits for hyperglycemia or undesirable body weight. GLP-1 analogs resistant to dipeptidyl peptidase IV (DPP IV) offer a longer duration of action and improved therapeutic efficacy. Therefore, GLP-1 analogs are preferred therapeutic polypeptides. In another embodiment, the present invention provides a method for treating diabetes or hyperglycemia, comprising the use of therapeutic RNA that reduces DPP IV levels.

[0263] In another embodiment, the therapeutic protein of the present invention, useful for treating hyperglycemia, is an antagonist to the hormone resistin. Resistin is an adipocyte-derived factor whose expression is elevated in diet-induced and genetic forms of obesity. Neutralizing circulating resistin improves blood glucose levels and insulin action in obese mice. Conversely, administration of resistin to normal mice impairs glucose tolerance and insulin action (Steppan CM et al. Nature 409:307 (2001)). Therefore, the production of a protein that antagonizes the biological effects of resistin in the gut may provide an effective treatment for insulin resistance and hyperglycemia associated with obesity. In another embodiment, the present invention provides a method for treating diabetes or hyperglycemia, comprising the use of therapeutic RNA that reduces the level of resistin expression in adipose tissue.

[0264] In another embodiment, the therapeutic polypeptide of the present invention useful for treating hyperglycemia or undesirable body weight (e.g., obesity) is leptin. Leptin is primarily produced by adipocytes, but also in small amounts in the stomach in response to food. Leptin transmits information about adipocyte metabolism and body weight to the appetite center in the brain, signaling a reduction in food intake (promoting satiety) and increasing the body's energy expenditure.

[0265] In another embodiment, the therapeutic polypeptide of the present invention, useful for treating hyperglycemia or undesirable body weight (e.g., obesity), is the C-terminal globular head domain of adipocyte complement-associated protein (Acrp30). Acrp30 is a protein produced by differentiated adipocytes. Administration of proteolytic cleavage products of Acrp30, consisting of the globular head domain, to mice leads to significant weight loss (Fruebis J. et al. Proc. Natl Acad. Sci USA 98:2005(2001)).

[0266] In another embodiment, the therapeutic polypeptide of the present invention useful for treating hyperglycemia or undesirable body weight (e.g., obesity) is cholecystokinin (CCK). CCK is a gastrointestinal peptide secreted from the intestines in response to certain nutrients in the gut. CCK release is thought to be proportional to the amount of food consumed and signals the brain to end the meal (Schwartz MWet. al. Nature 404:661-71 (2000)). Therefore, an increase in CCK may reduce the size of the meal and promote weight loss or weight stabilization (i.e., prevent or suppress the increase in weight gain).

[0267] Regarding PYY, for example, see le Roux et al., Proc Nutr Soc. 2005 May;64(2):213-6.

[0268] 6.2. Immune disorders In one embodiment, the therapeutic compositions of the present invention have immunomodulatory activity. For example, the therapeutic polypeptides of the present invention may be useful in treating deficiencies or disorders of the immune system by activating or inhibiting the proliferation, differentiation, or recruitment (chemotaxis) of immune cells. Immune cells develop through the hematopoietic process, which produces myeloid (platelets, erythrocytes, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The pathogenesis of these immunodeficiencies or disorders may be hereditary, somatic, e.g., cancer or some autoimmune disorders, acquired (due to chemotherapy or toxins), or infectious.

[0269] The therapeutic compositions of the present invention may be useful in treating hematopoietic cell deficiencies or disorders. For example, the therapeutic polypeptides of the present invention can be used to increase the differentiation or proliferation of hematopoietic cells, including pluripotent stem cells, for the purpose of treating diseases associated with a decrease in certain (or many) types of hematopoietic cells. Examples of immunodeficiency syndromes include, but are not limited to, blood protein disorders (e.g., agammaglobulinemia, abnormal gammaglobulinemia), ataxia telangiectasia, unclassifiable immunodeficiency, DiGeorge syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion disorder, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCID), Wiscott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria.

[0270] The therapeutic compositions of the present invention may also be useful in treating autoimmune disorders. Many autoimmune diseases result from the improper recognition of self as foreign by immune cells. This improper recognition leads to an immune response that results in the destruction of host tissue. Therefore, administration of the therapeutic compositions of the present invention that inhibit the immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, may be an effective treatment for preventing autoimmune disorders.

[0271] Examples of autoimmune disorders that can be treated with the present invention include, but are not limited to, Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmitis, bullous pemphigoid, pemphigus, polyglandular endocrine disorders, purpura, Reiter's disease, Stiffman syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pneumonia, Guillain's syndrome, insulin-dependent diabetes mellitus, Crohn's disease, ulcerative colitis, and autoimmune inflammatory eye diseases.

[0272] Similarly, allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems can also be treated with the therapeutic compositions of the present invention. Furthermore, these molecules can be used to treat anaphylaxis, hypersensitivity to antigen molecules, or blood type incompatibility.

[0273] The therapeutic compositions of the present invention may also be used to treat and / or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection results from the destruction of host immune cells in the transplanted tissue via an immune response. Similarly, immune responses are also involved in GVHD, in which case the exogenously transplanted immune cells destroy the host tissue. Administration of the therapeutic compositions of the present invention that inhibit the immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, may be an effective treatment for preventing organ rejection or GVHD.

[0274] Similarly, the therapeutic compositions of the present invention may also be used to modulate inflammation. For example, therapeutic polypeptides can suppress the proliferation and differentiation of cells involved in the inflammatory response. These molecules can be used to treat both chronic and acute inflammatory conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, pancreatitis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease (IBD), Crohn's disease, or conditions resulting from the overproduction of cytokines (e.g., TNF or IL-1). In one embodiment, a therapeutic RNA targeting TNFα is used in the composition to treat inflammation. In another preferred embodiment, a therapeutic RNA targeting IL-1 is used in the composition to treat inflammation. siRNA therapeutic RNA is particularly preferred. Examples of inflammatory disorders targeted for treatment in the present invention include, but are not limited to, chronic obstructive pulmonary disease (COPD), interstitial cystitis, and inflammatory bowel disease.

[0275] 6.3. Coagulation disorders In some embodiments, the therapeutic compositions of the present invention may also be used to modulate hemostasis (cessation of bleeding) or thrombolytic activity (thrombus formation). For example, by increasing hemostasis or thrombolytic activity, the therapeutic compositions of the present invention can be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiency), platelet disorders (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, the therapeutic compositions of the present invention that can decrease hemostasis or thrombolytic activity can be used to inhibit or dissolve coagulation. These therapeutic compositions may be important in the treatment of heart attacks (infarction), strokes, or scars. In one embodiment, the therapeutic polypeptide of the present invention is a thrombus-forming factor useful in the treatment of hemophilia or other coagulation / coagulation disorders (e.g., factor VIII, factor IX, or factor X).

[0276] 6.4. Overgrowth disorder In one embodiment, the therapeutic composition of the present invention is capable of regulating cell proliferation. Such therapeutic polypeptides can be used to treat hyperproliferative disorders, including neoplasms.

[0277] Examples of hyperproliferative disorders that can be treated with the therapeutic composition of the present invention include, but are not limited to, the abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nerves (central and peripheral systems), lymphatic system, pelvis, skin, soft tissues, spleen, rib cage, and genitourinary system.

[0278] Similarly, other hyperproliferative disorders can also be treated with the therapeutic compositions of the present invention. Examples of such hyperproliferative disorders include, but are not limited to, neoplasms located in the organ systems described above, hypergammaglobulinemia, lymphoproliferative disorders, abnormal proteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenström macroglobulinemia, Goucher disease, histiocytosis, and any other hyperproliferative disorders.

[0279] Delivery to the circulatory system provides access to therapeutic proteins in a wide variety of tissues. Alternatively, the therapeutic compositions of the present invention may stimulate the proliferation of other cells that can suppress hyperproliferative disorders.

[0280] For example, hyperproliferative disorders can be treated by increasing the immune response, particularly by increasing the antigenic properties of the hyperproliferative disorder, or by promoting the proliferation, differentiation, or recruitment of T cells. This immune response may be increased by enhancing an existing immune response or by initiating a new immune response. Alternatively, reducing the immune response may also be a method of treating hyperproliferative disorders, for example, by using chemotherapeutic agents.

[0281] 6.5. Infectious diseases In one embodiment, the therapeutic compositions of the present invention can be used to treat infectious diseases. For example, infectious diseases may be treated by increasing the immune response, particularly by increasing the proliferation and differentiation of B cells and / or T cells. The immune response may be increased by enhancing an existing immune response or by initiating a new immune response. Alternatively, the therapeutic compositions of the present invention may also inhibit infectious agents directly, without necessarily inducing an immune response.

[0282] Viruses are examples of infectious agents that can cause diseases or symptoms that can be treated with the therapeutic compositions of the present invention. Examples of viruses include, but are not limited to, the following DNA and RNA viridae: Arboviridae, Adenoviridae, Arteriviridae, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (e.g., Cytomegaloviridae, herpes simplex, herpes zoster), Mononegaviridae (e.g., Paramyxoviridae, Measles virus, Rhabdoviridae), Orthomyxoviridae (e.g., influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (e.g., smallpox or variola), Reoviridae (e.g., rotavirus), Retroviridae (e.g., HTLV-I, HTLV-II, lentivirus), and Togaviridae (e.g., rubivirus). Viruses that fall within these families can cause a variety of diseases or conditions, including, but are not limited to, arthritis, bronchiolitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic active, delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold, polio, leukemia, rubella, sexually transmitted infections, skin diseases (e.g., Kaposi's positivity, warts), and viremia. The therapeutic compositions of the present invention can be used to treat any of these conditions or diseases.

[0283] Similarly, bacterial or fungal pathogenic microorganisms that may cause disease or symptoms and can be treated with the therapeutic compositions of the present invention include, but are not limited to, the following Gram-negative and Gram-positive bacteriaceae and fungi: Actinomycetes (e.g., Corynebacterium, Mycobacterium, Nocardia), Bacillaceae (e.g., Anthrax bacilli, Clostridium), Bacteroides, Blastomycosis, Bordetella, Borrelia, Brucella, Candidiasis, Campylobacter Bacter, coccidioidomycosis, cryptococcosis, cutaneous mycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serracia, Yersinia), Erysipelothrix, Helicobacter, Legionnaires' disease, leptospirosis, Listeria, Mycoplasmales, Neisseriaceae (e.g., Acinetobacter, gonorrhea, meningococcus), Pasteurellosis (e.g., Actinobacillus, Haemophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, syphilis, and Staphylococcus. These bacteria or fungi may cause the following diseases or symptoms: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS-related infections), paronychia, prosthesis-related infections, Reiter's disease, respiratory tract infections (e.g., pertussis or sinusitis), sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria, leprosy, Johne's disease, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted infections, skin diseases (e.g., cellulitis, cutaneous mycoses), toxemia, urinary tract infections, wound infections, etc. The therapeutic compositions of the present invention can be used to treat any of these symptoms or diseases.

[0284] Furthermore, parasitic pathogens that cause diseases or symptoms that can be treated with the therapeutic compositions of the present invention include, but are not limited to, the following families: amoebiasis, babesiosis, coccidiosis, cryptosporidiosis, dientamoeviosis, necrotizing parasites, giardiasis, helminthiasis, leishmaniasis, theyleriosis, toxoplasmosis, trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but are not limited to, scabies, toxoplasmosis, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The therapeutic compositions of the present invention can be used to treat any of these symptoms or diseases.

[0285] 6.6. Playback The therapeutic compositions of the present invention can be used to differentiate, proliferate, and attract cells to promote the regeneration of mucosal tissue or tissue adjacent to target mucosal cells or tissue. (See Science 276:59-87 (1997)). Tissue regeneration can be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g., osteoporosis, osteoarthritis, periodontal disease, liver failure), surgery including cosmetic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

[0286] The therapeutic compositions of the present invention can promote the regeneration of various tissues, including, but are not limited to, organs (e.g., pancreas, liver, intestines, kidneys, skin, endothelium), muscles (smooth muscle, skeletal muscle, or cardiac muscle), blood vessels (including vascular endothelium), nerves, hematopoiesis, and skeletal (bone, cartilage, tendons, and ligaments) tissue. Preferably, regeneration occurs with or without minimal scarring. Regeneration may also include angiogenesis.

[0287] Furthermore, the therapeutic compositions of the present invention may increase the regeneration of tissues that are difficult to heal. For example, increased tendon / ligament regeneration would shorten the recovery time after injury. The therapeutic compositions of the present invention can also be used preventively to avoid injury. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Further examples of tissue regeneration in non-healing wounds include pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds.

[0288] Similarly, nerve and brain tissue can also be regenerated by proliferating and differentiating nerve cells using the therapeutic compositions of the present invention. Diseases that can be treated using this method include central and peripheral nervous system diseases, neuropathy, or mechanical and traumatic injuries (e.g., spinal cord injury, head injury, cerebrovascular disease, and seizures). Specifically, peripheral nerve injury, peripheral neuropathy (e.g., resulting from chemotherapy or other medical treatments), focal neuropathy, and diseases associated with central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can all be treated using the therapeutic compositions of the present invention. With respect to CNS disorders, many means of facilitating therapeutic access to brain tissue are known in the art, including methods of disrupting the blood-brain barrier and methods of conjugating therapeutic agents to a portion that provides transport to the CNS. In one embodiment, a therapeutic nucleic acid is engineered to encode a fusion protein, which comprises a transport portion and a therapeutic protein.

[0289] 6.7.Chemotaxis In one embodiment, the therapeutic composition of the present invention can modulate chemotaxis. For example, in one embodiment, the therapeutic polypeptide of the present invention has chemotactic activity. The chemotactic molecule attracts or recruits cells (e.g., monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells, and / or endothelial cells) to specific sites in the body, for example, sites of inflammation, infection, or hyperproliferation. The recruited cells can then fight off and / or heal specific injuries or abnormalities.

[0290] For example, the therapeutic polypeptides of the present invention may increase the chemotactic activity of specific cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorders by increasing the number of cells targeting specific locations in the body. For example, chemotactic molecules can be used to treat wounds and other injuries to tissues by attracting immune cells to the site of injury. The chemotactic molecules of the present invention may also attract fibroblasts, which can be used to treat wounds.

[0291] It is also intended that the therapeutic compositions of the present invention may inhibit chemotactic activity. These therapeutic compositions can also be used to treat disorders. Therefore, the therapeutic compositions of the present invention can be used as chemotactic inhibitors.

[0292] Pro- therapeutic proteins, which are activated near the target tissue, are particularly preferred for use.

[0293] Additional therapeutic polypeptides intended for use include, but are not limited to, growth factors for treating growth disorders or wasting syndromes (e.g., growth hormone, insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, acidic and basic fibroblast growth factors, transforming growth factor-β, etc.); and antibodies (e.g., human or humanized) that provide targeted passive immunity or protection against foreign antigens or pathogens (e.g., H. pylori) or that provide treatment for cancer, arthritis, or cardiovascular disease; cytokines, interferons (e.g., interferon (IFN), IFN-α2b and 2α, IFN-αN1, IFN-β1b, IFN-gamma), interleukins (e.g., IL-1 to IL-10), tumor necrosis factor (TNF-α, TNF-β), chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), polypeptide hormones, antimicrobial polypeptides (e.g., antimicrobial, antifungal, antiviral, and / or antiparasitic polypeptides) (Tide), enzymes (e.g., adenosine deaminase), gonadotrophins, chemotactin, lipid-binding proteins, filgastim (Neupogen), hemoglobin, erythropoietin, insulintropin, imiglucerase, salbramostim, tissue plasminogen activator (tPA), urokinase, streptokinase, phenylalanine hormone lyase, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), thrombopoietin (TPO), superoxide dismutase Sid dismutase (SOD), adenosine deamidase, catalase, calcitonin, endothelin, L-asparaginase pepsin, uricase, trypsin, chymotrypsin, elastase, carboxypeptidase, lactase, sucrase, endogenous factors, parathyroid hormone (PTH)-like hormones, soluble CD4, and antibodies and / or their antigen-binding fragments (e.g., FAb) (e.g., orthoclonal OKT-e (anti-CD3), GPIIb / IIa monoclonal antibodies). Furthermore, therapeutic RNAs targeting nucleic acids encoding such factors are being considered.

[0294] 6.8. Vaccines In one embodiment, the present invention provides a method for vaccinating a patient. The method comprises administering a composition of the present invention capable of generating a desired epitope. In a preferred embodiment, the composition comprises a therapeutic nucleic acid construct capable of expressing a protein containing the epitope.

[0295] 6.9. Cosmetic Uses In one embodiment, the present invention provides a DD-chitosan nucleic acid polyplex for cosmetic use. This cosmetic product contains the DD-chitosan nucleic acid polyplex in a formulation suitable for cosmetic use. [Examples]

[0296] Example 1: 1.General materials 1.1 Plasmid DNA Vectors [Table 3]

[0297] 1.2 Reagents [Table 4]

[0298] 1.3 Consumables [Table 5]

[0299] 1.4 Equipment [Table 6]

[0300] 2. General Procedure 2.1 Preparation of biderivatized chitosan According to US9623112B2, chitosan was double-derivatized with arginine and gluconic acid (DD-chitosan, DD-X).

[0301] 2.2 Preparation of double-derivatized chitosan and DNA polyplex DD-chitosan was polyplexed with plasmid DNA vectors in various amine-to-phosphate (N:P) molar ratios as needed, according to US9623112B2 and US8722646B2. Additional excipients such as sucrose, trehalose, or mannitol were included as needed. Various plasmid DNA vectors were tested as shown herein.

[0302] 2.3 Preparation of PEG-Polyglutamic Acid (PEG-PGA) Solution Generally, PEG-PGA was dissolved in water or an excipient solution at a concentration of up to 40 mg / mL as needed. The resulting PEG-PGA solution was then diluted to the required molar concentration of the required anion species (A, i.e., glutamic acid) to obtain the desired final ratio of amine-to-phosphate-to-anion molar ratio (N:P:A) for subsequent formulation.

[0303] 2.4 Preparation of PEG-Hyaluronic Acid (PEG-HA) Solution Generally, PEG-HA was partitioned into water at a concentration of 40-100 mg / mL and sonicated for 10 minutes. For subsequent formulation, the obtained PEG-HA solution was diluted to the required concentration of the anionic species (A, i.e., hyaluronic acid) in the required 10% trehalose to obtain the desired final ratio of amine-to-phosphate-to-anion molar ratio (N:P:A) and to achieve a final trehalose concentration of 5%.

[0304] 2.5 Preparation of PEG-Polyaspartic Acid (PEG-PAA) Solution Generally, PEG-PAA was dispensed into 5% trehalose at a concentration of 40-100 mg / mL and sonicated for 10-15 minutes. If necessary, the resulting PEG-PAA solution was diluted to the required concentration of the anionic species (A, i.e., aspartic acid) in the required 5% trehalose to obtain the desired final ratio of amine-to-phosphate-to-anion molar ratio (N:P:A) and to achieve a final trehalose concentration of 5% for subsequent formulation.

[0305] 2.6 Preparation of Trehalose Solution Generally, trehalose was dissolved in water at a maximum concentration of 0.2 g / mL, as needed. The resulting trehalose solution was filtered using a 0.2 μm filter. If necessary, the trehalose solution was diluted to the concentration required for subsequent formulation.

[0306] 2.7 Preparation of PAA solution in 50 mM Tris (pH 8) Generally, PAA was dissolved in 50 mM Tris (pH 8) at concentrations of 100 mg / mL or 20 mg / mL, as needed. If necessary, the PAA solution was then diluted with 50 mM Tris (pH 8) to the required concentration as needed.

[0307] 2.8 Preparation of 20 mM NaCl Dissolve 46.75 mg of NaCl in 40 mL of water. Filter the solution through a 0.2 μm filter.

[0308] 2.9 Preparation of simulated intestinal fluid (SIF) A FaSSIF V1 solution was prepared by dissolving FaSSIF V1 powder in water at a concentration of 2.24 mg / mL. The pH was confirmed to be approximately 6.6. A FaSSIF V2 solution was prepared by dissolving FaSSIF V2 powder in water at a concentration of 1.79 mg / mL. The pH was confirmed to be approximately 6.6.

[0309] 2.10 PEG-Polyanion (PEG-PA) PEG-PA is a general term for PEG conjugated with polyanions such as PEG-polyglutamic acid, PEG-hyaluronic acid, or PEG-polyaspartic acid.

[0310] 2.11 Preparation of PEGylated Polyplexes by Droplet Addition PEGylated polyplexes are prepared by adding an equal volume (0.1 mL) of polyplex solution to a diluted PEG-PA solution (0.1 mL).

[0311] 2.12 Preparation of PEGylated Polyplexes by In-Line Mixing PEGylated polyplexes are prepared by mixing an equal volume of diluted PEG-PA solution with the polyplex solution using an in-line mixing apparatus such as those described in US9623112B2 and US8722646B2.

[0312] 2.13 Stability of PEGylated polyplexes in 150 mmol PBS Mix 100 μL of PEGylated polyplex with 300 μL of 150 mmol PBS. Measure the particle diameter at 0 and 2 hours after mixing. Compare with a control of unPEGylated polyplex.

[0313] 2.14 percent supercoiled DNA Before measuring the proportion of supercoiled DNA, all DNA must be released from the polyplex or PEGylated polyplex by exposure to excess PAA. A 1 μL sample aliquot (targeting 0.1 mg / mL of DNA) was mixed with 10 μL of PAA (100 mg / mL in 50 mM Tris) and incubated at 37°C for 30 minutes. After release, the DNA was subjected to agarose gel electrophoresis.

[0314] 2.15 Total DNA in formulations measured by PicoGreen Before measuring DNA using the PicoGreen assay, total DNA must be released from the polyplex or PEGylated polyplex using excess PAA. A 10 μL sample aliquot (target DNA at 2 ug / mL) was mixed with 10 μL of PAA (20 mg / mL in 50 mM Tris) and incubated at 37°C for 30 minutes. After release, the DNA was digested using a suitable restriction enzyme (RE) to linearize the supercoiled DNA plasmid. In the PicoGreen assay, a 10 μL aliquot of the RE-digested sample was mixed with 190 μL of PicoGreen working solution (Qubit ds DNA buffer:Qubit ds DNA PicoGreen reagent 199:1), incubated for 2 minutes, and measured for fluorescence (excitation: 485, emission cutoff: 515 nm, emission: 535 nm). The results were quantified against a reference DNA standard curve.

[0315] 2.16 Free DNA To verify DNA capture by polyplex, 10 μL sample aliquots (1000 ng of DNA target) were mixed with 2 μL of 6X loading buffer and subjected to gel electrophoresis.

[0316] 2.17 Gel electrophoresis The prepared sample was placed in the sample lane. The supercoiled DNA ladder was placed in the standard lane. 2 μL of reference DNA (200 ng of DNA) + 8 μL of water + 2 μL of 6X loading buffer were placed in the reference lane. The samples were separated on a 0.8% agarose gel containing 1X SYBRSafe DNA Stain at 100V for 75 minutes. The gel was imaged using a GelDoc imaging system.

[0317] 2.18 Nanoparticle sizing of polyplexes and PEGylated polyplexes Particle size was measured using a Zetasizer Nano light scattering instrument. Generally, samples were either undiluted or diluted up to 20-fold with 10 mM NaCl and placed in disposable cuvettes or Zetasizer folded capillary cells (minimum 0.8 mL). The Zetasizer was programmed to incubate the sample at 25°C for up to 3 minutes before performing three 3-minute measurements. The Z-mean diameter and polydispersity (PDI) were reported as standard deviations (n=3). The Zetasizer was also programmed to account for the sample composition regarding viscosity and refractive index.

[0318] 2.19 Zeta potential of polyplexes and PEGylated polyplexes Zeta potential measurements were performed using the Zetasizer Nano light scattering instrument. Generally, undiluted samples were placed in Zetasizer fold capillary cells (minimum 0.8 mL), with the exception of PEGylated polyplexes diluted with 10 mM NaCl. Before repeating measurements, the Zetasizer was programmed to incubate the sample at 25°C for a maximum of 3 minutes (the number of repetitions was automatically determined by the Zetasizer software). Zeta potential values ​​were reported as standard deviations (n=3). The Zetasizer was also programmed to account for the final composition of the sample regarding viscosity and dielectric constant.

[0319] 2.20 Short-term stability For short-term stability studies, the formulations were freeze-dried as described and stored at appropriate temperatures (-20°C, 4°C, or room temperature). At appropriate times, the samples were rehydrated and analyzed as described.

[0320] 3. PEGylation of polyplexes using PEG-PGA (mPEG1K-b-PLE10) 3.1 Procedure DD-X-DNA(pVAX) polyplexes with the following N:P ratio and 5% trehalose as described above were prepared with a DNA concentration of 0.1 mg / mL. [Table 7]

[0321] Next, the PEG-PGA solution was mixed with the DD-X-DNA polyplex in a 1:1 volume ratio using dropwise mixing to obtain a PEGylated DNA polyplex with a concentration of 0.05 mg / mL, as shown below. The samples were tested for particle size, PDI, and zeta potential (the tested formulations were not frozen). [Table 8]

[0322] 3.2 Results Unfreeze-thawed PEGylated polyplexes prepared with mPEG1K-b-PLE10 yielded stable formulations with the following specific N:P:A ratios (unless otherwise indicated). [Table 9]

[0323] 4. PEGylation of polyplexes using PEG-PGA (mPEG1K-b-PLE10 and mPEG5K-b-PLE10) and PEG-HA (HA-202) 4.1 Procedure DD-X-DNA(pVax-opt-hIL10) polyplexes were prepared with the following N:P ratio and a DNA concentration of 0.25 mg / mL, containing 5% trehalose as described above. [Table 10]

[0324] Next, using dropwise mixing, a PEG-PGA solution or a PEG-HA solution was mixed with the DD-X-DNA polyplex in a 1:1 volume ratio to obtain a PEGylated DNA polyplex with a concentration of 0.125 mg / mL, as shown below. The samples were frozen and thawed, and tested for particle size, PDI, and zeta potential. [Table 11]

[0325] 4.2 Results The physicochemical results of freeze-thawed samples are provided in the table below. PEGylated polyplexes prepared with mPEG1K-b-PLE10 or HA-202 did not yield freeze-thaw stable formulations. PEGylated polyplexes prepared with mPEG5K-b-PLE10 were stable against freeze-thaw cycles. [Table 12]

[0326] 5. PEGylation of polyplexes using PEG-HA 5.1 Procedure DD-X-DNA(pVax-opt-hIL10) polyplexes were prepared with the following N:P ratio and a DNA concentration of 0.25 mg / mL, containing 5% trehalose as described above. [Table 13]

[0327] Next, using dropwise mixing, a PEG-HA solution in 5% trehalose was mixed with the DD-X-DNA polyplex in a 1:1 volume ratio to obtain a PEGylated DNA polyplex with a concentration of 0.125 mg / mL, as shown below. The sample was tested for particle size, PDI, and zeta potential. [Table 14] [Table 15]

[0328] 5.2 Results HA-201 (HA 10k-PEG2k) was insoluble in water. It formed a hydrogel and was therefore unusable. HA-202 (HA 50k-PEG2k) formed a viscous solution in water. Polyplex showed visible aggregation after PEGylation, and further testing was not performed.

[0329] 6. Preparation of PEG-PGA polyplex 6.1 Procedure Preparation of PEGylated Polyplexes As described herein, aliquots of N:P 7 polyplex (CMC-INT06-098A) were PEGylated using PEG-PGA. The resulting PEGylated polyplexes were tested for physicochemical properties and DNA capture. Formulations were prepared by adding the resulting N:P 7 polyplexes. [Table 16]

[0330] 6.2 Results The appearance, pH, particle size, PDI, zeta potential, and conductivity of the test specimen are as follows: [Table 17]

[0331] Free DNA and % supercoil are as follows: [Table 18]

[0332] 7. Concentrated PEGylated Polyplexes obtained by in-line mixing with PEG-PGA followed by TFF concentration 7.1 Procedure Concentrated PEGylated polyplexes obtained by in-line mixing with PEG-PGA followed by TFF concentration. A DD-X-DNA(pVax-PD-L1-Fc) polyplex with an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL was prepared using DNA containing 5% trehalose as described above. Next, a PEG-PGA solution in 5% trehalose was in-line mixed with the DD-X-DNA polyplex in a 1:1 volume ratio (each fluid flowing at 7 mL / min) to obtain a PEGylated polyplex with 0.125 mg / mL of DNA and an N:P:A ratio of 7:1:17.5. After incubation at room temperature for 60 minutes, the PEGylated polyplex was concentrated using a TFF (regenerated cellulose membrane, MWCO 10 kDa, inlet pressure 15 psig) using the process previously described in US8722646B2. Aliquots were collected in the TFF at nominal DNA concentrations of 0.25 mg / mL and 0.5 mg / mL. The final product was collected up to a nominal DNA concentration of approximately 1 mg / mL. The samples were divided into equal portions and stored at -80°C. After thawing, the samples were tested for particle size, PDI, zeta potential, free DNA, and % supercoiled DNA. A schematic diagram is shown in Figure 11. [Table 19]

[0333] 7.2 Results The particle size, PDI, zeta potential, free DNA, and % supercoil of the thawed test samples were found to be as follows: [Table 20]

[0334] 7.3 Conclusion PEGylated polyplexes can be concentrated to 0.125 mg / mL DNA to 1 mg / mL DNA using the TFF enrichment process, while maintaining colloidally stable nanoparticles.

[0335] 8. Concentration of PEGylated polyplexes by PEGylation of pre-concentrated polyplexes. 8.1 Procedure As previously described in US8722646B2, concentrated DD-X-DNA(pVax-PD-L1-Fc) polyplexes with a DNA concentration of 1 mg / mL and DNA having an N:P ratio of 7:1 and 9% sucrose were prepared. Next, a PEG-PGA solution in water was mixed in-line with the DD-X-DNA polyplexes in a 1:1 volume ratio (each fluid flowing at 7 mL / min) to obtain PEGylated polyplexes with a DNA concentration of 0.5 mg / mL and an N:P:A ratio of 7:1:7 or 7:1:17.5. After incubation at room temperature for 60 minutes, the PEGylated polyplexes were divided and stored at -80°C.

[0336] After thawing, the samples were tested for particle size, PDI, zeta potential, free DNA, and % supercoiled DNA. [Table 21]

[0337] 8.2 Results Physicochemical properties of original polyplex and PEGylated polyplex [Table 22]

[0338] 8.3 Conclusion DD-X-DNA polyplexes were PEGylated with 1 mg / mL of DNA at N:P:A ratios of 7:1:7 and 7:1:17.5, resulting in a final DNA concentration of 0.5 mg / mL. No visible agglutination was observed.

[0339] 9. PEGylation of polyplexes using PEG-PGA or PEG-PAA 9.1 Procedure As described herein, aliquots of N:P 7 polyplex (CMC-INT06-24C) were PEGylated using PEG-PGA or PEG-PAA. The resulting PEGylated polyplexes were tested for physicochemical properties and DNA capture. In addition to the N:P 7 polyplexes, the resulting formulations were produced. [Table 23]

[0340] 9.2 Results [Table 24] Samples A through F were tested for free DNA as described herein. The resulting gels did not show any visible free DNA.

[0341] 9.3 Conclusion Stable nanoparticles free of free DNA were formed using PEGylated polyplexes with either PEG-PGA (mPEG5K-b-PLE10) or PEG-PAA (mPEG5K-b-PLD10).

[0342] 10. Stability of PEGylated polyplexes in artificial intestinal fluid 10.1 Procedure Test item As described herein, aliquots of N:P 7 polyplex (CMC-INT06-24C) were PEGylated using PEG-PGA. The resulting PEGylated polyplexes were tested for stability in FaSSIF buffer. [Table 25]

[0343] Stability testing of PEGylated polyplexes at SIF1:3, v / v. Add 50 μL of Polyplex to 150 μL of artificial intestinal fluid. Mix thoroughly. Immediately dispense 50 μL aliquots of the suspension into DLS cuvettes, add 350 μL of water, and measure the particle size on DLS as described. Divide the remaining sample solution into three tubes. At the appropriate time, add 350 μL of water to each tube, mix thoroughly, and measure the particle size on DLS.

[0344] Stability testing of PEGylated polyplexes at SIF3:1, v / v. Add 75 μL of Polyplex to 25 μL of artificial intestinal fluid. Mix thoroughly. Immediately dispense 50 μL aliquots of the suspension into DLS cuvettes, add 350 μL of water, and measure the particle size using DLS as described. Divide the remaining sample solution into three tubes. At appropriate times, add 350 μL of water to each tube, mix thoroughly, and measure the particle size using DLS.

[0345] 10.2 Results Non-PEGylated polyplexes (represented as 7-1-0) aggregated immediately upon mixing with buffer (maximum on the scale). PEGylated polyplexes (using PEG-PGA, mPEG5K-b-PLE10) remained stable for 24 hours at a 1:3 (FaSSIF:PEGylated polyplex, v / v) ratio. PEGylated polyplexes (using PEG-PGA, mPEG5K-b-PLE10) remained stable for 1 hour at a 3:1 (FaSSIF:PEGylated polyplex, v / v) ratio.

[0346] 11. Stability of PEGylated polyplexes in FaSSIF buffer-free DNA Reference:CMC-INT06-EXP-072 11.1 Procedure To obtain the target N:P:A ratios shown in the table below, aliquots of N:P 7 polyplex (CMC-INT06-024C, DD-X-DNA(pVax-opt-hIL10) polyplex, 5% trehalose, 0.25 mg / mL) were PEGylated using PEG-PGA (in 5% trehalose) as described herein. The resulting PEGylated polyplexes were tested for stability in FaSSIF buffer according to the ratios shown in the table below. After 2 hours, the following physicochemical properties were determined: free DNA, pH, diameter, and PDI. [Table 26]

[0347] 11.2 Results [Table 27] Free DNA assay of FaSSIF-polyplex sample. As shown in Figure 5, no free DNA was observed. The lane descriptions are shown below. [Table 28]

[0348] 12. Preparation of PEGylated Polyplexes and In vitro Transfection 12.1 In vitro transfection reagents [Table 29]

[0349] 12.2 In vitro transfection consumables [Table 30]

[0350] 12.3 In vitro transfection equipment [Table 31]

[0351] 12.4 Procedure Test item To obtain the target N:P:A ratio shown in the table below, aliquots of N:P 7 polyplex (CMC-INT06-024C, DD-X-DNA(pVax-opt-hIL10) polyplex, 5% trehalose, 0.25 mg / mL) were PEGylated using PEG-PGA (mPEG5K-b-PLE10, in 5% trehalose) as described herein. The resulting PEGylated polyplexes were tested for particle size, PDI, and zeta potential before and after freezing / thawing. The thawed samples were tested for in vitro transfection. [Table 32]

[0352] 12.5 In vitro transfection procedure Cell preparation HEK293T cells (ATCC) from passage 19 were passed through a centrifuge to remove trypsin, and then resuspended twice in DMEM supplemented with 10% FBS. Cell viability and counting were performed using a Vi-Cell viable and dead cell autoanalyzer to verify cell number and viability. 5 Cells were diluted to a concentration of cells / mL and dispensed into 96-well clear-bottom TC plates (200 μL, 25,000 cells per well). The plates were incubated at room temperature for 15-20 minutes before being incubated overnight at 37°C / 5% CO2, and then used for transfection.

[0353] Dilution of Polyplex Unless otherwise provided at the target concentration, test samples were diluted to 0.125 mg / mL of DNA. Control samples (CMC-INT06-024 and CMC-JAN01-010) provided 0.25 mg / mL of DNA, diluted to 0.14 mg / mL with water. Next, all test and control samples were serially diluted with water, totaling 10-fold dilutions by 1 / 1.35. These serial dilutions were further diluted 17.7-fold in Opti-MEM, and 35.6 μL of this diluted polyplex was added twice to each well (giving a final dose range of 282 ng to 19 ng of polyplex for the control samples and 251 ng to 17 ng of polyplex for the test samples).

[0354] Cell transfection Transfection was performed as follows: First, the culture medium was removed from each HEK293T well, followed by the addition of 0.1 mL of Opti-MEM (pH 7.4), which was then removed. Polyplex samples diluted with Opti-MEM (see previous section) were added to each well and incubated at 37°C for 3 hours. After incubation, the medium was removed and replaced with 0.2 mL of complete medium, and re-incubated at 37°C. After 48 hours, the supernatant was removed and immediately used for the MSD assay. The remaining cells were lysed and total protein was measured.

[0355] Quantification of total protein The total protein in each well was determined using the DC® protein assay with BSA for a standard curve. After removing the supernatant for ELISA, the remaining cell layer was lysed in lysis buffer at 4°C for 10 minutes. The lysate was pipetted several times (while minimizing bubble formation) and then transferred to a V-bottom 96-well plate. The lysate was then clarified by centrifugation at 1000g at 4°C for 5 minutes.

[0356] Preparation of protein standard curves A stock of protein assay standard II (bovine serum albumin) was prepared in 5 mg / ml milli-Q water and stored at 4°C. A standard curve was prepared by performing a 1:2 serial dilution of the protein standard with the lysis buffer (the same buffer used to prepare the sample).

[0357] Lowry / DCTM Protein Assay Working reagent A' was prepared by adding 20 μl of reagent S to 1 ml of reagent A. 25 μl of working reagent A' was transferred to each well of a 96-well plate. 5 μl of cell lysate or standard solution was added to each well. Next, 200 μl of reagent B was added to each well. The plate was shaken for 5 seconds and incubated at room temperature for 15 minutes to allow color development. Absorbance at 750 nm was measured using a SpectraMAX plate reader. The absorbance of the standard was plotted against the standard concentration. Using SpectraMax software curve fitting analysis, a four-parameter algorithm provided the optimal curve fit to the standard curve. The software also interpolated the protein concentrations of the samples in the protein standard curve.

[0358] Quantification of hIL-10 protein by MSD assay The supernatant was centrifuged at 1000xg for 10 minutes and then appropriately diluted. Before use, all reagents for the assay were equilibrated to room temperature. Standards for the assay were prepared by reconstituting hIL-10 in Diluent 2 to a specific concentration, incubated at room temperature for 15 minutes, and then serially diluted fourfold with Diluent 2 to prepare seven doses of the standard curve. 50 μL of standard solution was added twice to two rows of the MSD plate. 25 μL of Diluent 2 and 25 μL of sample were added to the remaining wells. The plate was covered and incubated at room temperature with shaking at 300 rpm for 2 hours. The plate was washed and then 25 μL of diluted detection antibody was added. Incubated at room temperature with shaking at 300 rpm for 2 hours. The plate was washed and then 150 μL of 2X Read Buffer T was added. Read using a Meso Scale with barcode (VPLEX single spot is automatically assigned). Standard absorbances were plotted against standard concentrations, and the amount of hIL-10 in the samples was interpolated using Prism GraphPad software with a 4-parameter algorithm. Once the amount of hIL-10 in each well was determined, it was normalized against total protein determined from the DC® protein assay (i.e., ng hIL-10 per 1 mg of total protein). Dose-response curves were constructed in GraphPad Prism by plotting ng hIL-10 per 1 mg of total protein against the amount of transfected DNA (ng). The EC50 and the predicted maximum amount of hIL-10 per 1 mg of total protein were determined.

[0359] 12.6 Results All particles were stable and showed no aggregation. The zeta potential of all samples after PEGylation was reduced to -3mV. After freeze-thaw cycles, the polyplexes exhibited the same properties. In vitro transfection results were similar for both unPEGylated and PEGylated samples (Figure 6). [Table 33]

[0360] 13. PEGylation of polystyrene beads See: CMC-INT06-EXP-039 and CMC-INT06-EXP-055 13.1 Procedure PEGylation of orange polystyrene beads Generally: Transfer 1 mL of amine-modified polystyrene beads to a 7 mL glass bottle containing an electromagnetic stirring rod. Add 5 μL of 1 M HCl. Dissolve 70 mg of PEG NHS and 8 mg of EDC-HCl in 1 mL of water in a 1.5 mL Eppendorf tube until a clear solution is formed. Gradually add 0.7 mL of this solution to the polystyrene suspension. Stir the solution overnight at room temperature, protecting it from light. Purify and concentrate the PEGylated orange particles as described below. PEGylation of PS particles was confirmed using particle sizing and zeta potential measurement.

[0361] PEGylation of green polystyrene beads Generally: Transfer 2 mL of green amine-modified polystyrene beads to a 7 mL glass bottle containing an electromagnetic stirring rod. Add 10 μL of 1 M HCl. Dissolve 100 mg of PEG NHS and 5 mg of EDC-HCl in 1 mL of water in a 1.5 mL Eppendorf tube until a clear solution is formed. Gradually add 0.9 mL of this solution to the polystyrene suspension. Stir the solution overnight at room temperature while protecting it from light. Purify and concentrate the PEGylated green particles as described below. PEGylation of the PS particles was confirmed using particle sizing and zeta potential measurement.

[0362] PEGylation of blue polystyrene beads Generally: Transfer 1 mL of blue amine-modified polystyrene beads to a 7 mL glass bottle containing an electromagnetic stirring rod. Add 2 mL of water. Dissolve 270 mg of PEG NHS and 10.34 mg of EDC-HCl in 1 mL of water in a 1.5 mL Eppendorf tube until a clear solution is formed. Gradually add 0.9 mL of this solution to the polystyrene suspension. Stir the solution overnight at room temperature while protecting it from light. The PEGylated blue particles were purified and concentrated as described below. PEGylation of the PS particles was confirmed using particle sizing and zeta potential measurement.

[0363] Purification and concentration of PEGylated polystyrene The PEGylated PS particles were purified and concentrated as follows: Divide 300 μL of the reaction mixture equally into Amicon Ultra filters. Centrifuge at 5000 rpm for 10 minutes. Remove the liquid from the bottom and add 300 μL of water to the top of the filter (so that the beads remain on the filter). Wash the beads by repeating centrifugation and washing four times. Pool the beads from the different filters onto a single filter. Add 300 μL of water and centrifuge again. Suspend the green and orange beads in water to approximately 1.5 mL.

[0364] The hydrodynamic diameter of polystyrene particles is measured by mixing a 10 μL suspension with 390 μL of water. The zeta potential of polystyrene particles is measured by mixing a 20 μL particle suspension with 780 μL of 10 mM NaCl.

[0365] 13.2 Results A significant decrease in the zeta potential at the end of the reaction indicated that PEGylation had occurred. [Table 34]

[0366] 14. Preparation of RITC-DDX+DNA polyplex and PEGylated RITC-DD-X+DNA polyplex Reference materials: Presented below 14.1 RITC-DD-X Reagent [Table 35]

[0367] 14.2 RITC-DD-X equipment [Table 36]

[0368] 14.3 Procedure RITC labeled DD-X RITC was conjugated to DD-X based on a method derived from Carbohydrate Polymers 72(2008)616-624. In summary, DD-X stock (lot JAN03-009) was dissolved in water to obtain a concentration of 40 mM and pH 5.8. The DD-X solution was mixed with an equal volume of DMSO and stirred at room temperature for 1 hour, followed by bubbling with nitrogen gas for 15 minutes. In a separate container, RITC was dissolved in DMSO (target 3.7 mg / mL) and added dropwise to the DD-X / DMSO mixture (RITC / DMSO:DD-X / DMSO 1:8, v / v). The resulting mixture was stirred at room temperature for 65 hours and protected from light. RITC-labeled DD-X was purified by dialysis with water and then lyophilized. Unreacted RITC was extracted with methanol and washed with methanol on a Buchner funnel until the filtrate was colorless. The washed powder was collected and vacuum-dried overnight at room temperature. The final collected powder was pink in appearance. Conjugation of RITC to DD-X was confirmed by H-NMR spectroscopy: RITC-DD-X was dissolved in D2O + DCl / D2O (8 mg / mL) to obtain a pH of approximately 2.5.

[0369] RITC-DD-X+DNA Polyplex A 50 / 50 blend of RITC-DD-X (described above) and unlabeled DD-X was prepared and then used to create an N:P 20 polyplex (0.1 mg / mL DNA) with 5% trehalose containing GWiz-GFP plasmid DNA, following the drip method procedure described herein.

[0370] RITC-DD-X+DNA Polyplex A 33 / 67 blend of RITC-DD-X (above) and unlabeled DD-X was prepared and then used to create an N:P 7 polyplex (0.25 mg / mL DNA) with 5% trehalose containing pVax-opt-hIL10 plasmid DNA, following the in-line mixing procedure described herein.

[0371] PEGylated RITC-DD-X + DNA polyplex As described herein, aliquots of N:P 7 polyplex were PEGylated using PEG-PGA. The resulting PEGylated polyplexes were tested for particle size, PDI, and zeta potential. Formulations were prepared by adding the N:P 7 polyplexes to the PEGylated polyplexes. [Table 37]

[0372] 14.4 Results Particle size, PDI, and zeta potential results [Table 38] Polyplex CMC-INT06-075 D, E, and F aggregated.

[0373] 15. Evaluation of aggregation of various PS beads within type III mucin, and quantification of diffusion through 1 and 3 μm Transwells. Reference:CMC-INT06-EXP-042

[0374] 15.1 Mucin Diffusion Materials [Table 39]

[0375] 15.2 Mucus Diffusion Device [Table 40]

[0376] 15.3 Procedure Preparation of Type III Mucin Suspension Type III mucin suspensions were prepared by dispensing type III mucin into water to target concentrations of 5% w / w to 0.1% w / w, as needed.

[0377] Mucin aggregation test Test samples (RITC-labeled polyplex, amine-modified PS beads, or PEGylated PS beads) were mixed with type III mucin suspensions at various concentrations in microtubes to obtain the final target concentrations of the test samples and mucin. The sample-mucin samples were protected from light and mixed at room temperature at 30 rpm. At 30 and 60 minutes, the mixtures were divided equally onto Ibidi microslides and observed under an EVOS microscope for visible aggregation.

[0378] Evaluation of diffusion through a Transwell containing mucin Sample preparation. The test samples (RITC-labeled polyplex, amine-modified PS beads, or PEGylated PS beads) were mixed with a type III mucin suspension in a microtube to obtain the final target concentration of the test sample and a final mucin concentration of 0.4%. Before placing the samples into the Transwell insert, they were protected from light and mixed at 40 rpm for 1 hour.

[0379] Preparation of Transwell and diffusion of the sample The bottom of the Transwell was filled with 0.6 mL of 0.4% type III mucin. 0.1 mL of pre-mixed sample + mucin was placed in an HTS Transwell insert (equipped with a 1 μm or 3 μm PET membrane). A control was added without the 0.4% mucin. The reference (simulating 100% diffusion) was the pre-mixed sample + mucin without the Transwell insert (0.7 mL). After incubation at 37°C for 20 hours, the diffusion of the test samples into the bottom well was evaluated using a fluorescence plate reader (Envision).

[0380] 15.4 Results Mucin aggregation test As shown in Figure 7, both RITC-labeled polyplex and amine-modified PS beads showed aggregation in mucin after 1 hour of incubation. No visible aggregation was observed in the PEGylated PS beads.

[0381] Evaluation of diffusion through a Transwell containing mucin As shown in Figure 8, with 0.4% mucin, PEGylated PS beads exhibited the highest level of diffusion after 20 hours of incubation through either a 3 μm or 1 μm PET membrane, compared to amine-modified PS beads and RITC-labeled polyplex. This is attributed to the reduced aggregation of PEGylated PS beads in mucin.

[0382] 16. Diffusion of PEGylated polyplex formulations via type III mucin using Transwell repeats and the effect of Pluronics F127. 16.1 Materials [Table 41]

[0383] 16.2 Equipment [Table 42]

[0384] 16.3 Procedure Preparation of a 4% Pluronics F127 solution Pluronics F127 was dissolved in water to obtain a 4% w / w solution, which was then filtered through 0.22 μm.

[0385] Preparation of Type III Mucin Suspension A type III mucin suspension was prepared as described herein.

[0386] Mucin aggregation test Test samples (PEGylated RITC-DD-X+DNA polyplex, RITC-labeled polyplex, amine-modified PS beads, or PEGylated PS beads) were mixed with type III mucin suspensions at various concentrations in microtubes to obtain the final target concentrations of the test samples and mucin. The sample-mucin samples were protected from light and mixed at room temperature at 30 rpm. At 30 and 60 minutes, the mixtures were divided equally onto Ibidi microslides and observed under an EVOS microscope for visible aggregation.

[0387] Evaluation of diffusion through a Transwell containing mucin Sample preparation. Test samples (PEGylated RITC-DD-X+DNA polyplex, RITC-labeled polyplex) were mixed in microtubes with or without Pluronics F127 solution to obtain the final target concentration of the test samples and a final mucin concentration of 0.5%. Controls (amine-modified PS beads, PEGylated PS beads) were mixed with mucin alone to obtain a final mucin concentration of 0.5%. Before placing the samples in the Transwell insert, they were protected from light and mixed at 40 rpm for 1 hour.

[0388] Preparation of Transwell and diffusion of the sample The bottom of the Transwell was filled with 0.6 mL of 0.5% type III mucin. 0.1 mL of pre-mixed sample + mucin was placed in an HTS Transwell insert with a 3 μm PET membrane. The reference (simulating 100% diffusion) was the pre-mixed sample + mucin without the Transwell insert (0.7 mL). After incubation at 37°C for 20 hours, the diffusion of the test sample into the bottom well was evaluated using a fluorescence plate reader (Envision).

[0389] 16.4 Results PEGylation of PS beads PEGylation of amine-modified PS beads improved mucin diffusion by almost 10 times (Figure 9). Fluorescence microscopy showed that PEGylated PS beads did not aggregate with mucin, while unPEGylated PS beads aggregated significantly under the same conditions.

[0390] Diffusion of PEGylated polyplexes in mucin PEGylation of polyplex (N:P 7) increased mucin diffusion from 32% to 50-52%, resulting in approximately 60% better diffusion than unPEGylated polyplex (Figure 9). The tested N:P:A ratio was similarly applied to mucin diffusion. Fluorescence microscopy showed no significant morphological differences between PEGylated and unPEGylated polyplex.

[0391] The effect of Pluronics F127 on mucin diffusion The presence of Pluronics F127 improved mucin diffusion in both unPEGylated and PEGylated polyplexes by 10-15% (Figure 10).

[0392] 17. Lyophilization of PEGylated polyplexes prepared in 2.5 or 5% trehalose at various NPA ratios. Reference:CMC-INT06-EXP-103

[0393] 17.1 Freeze-dried materials [Table 43]

[0394] 17.2 Freeze drying equipment [Table 44]

[0395] 17.3 Procedure

[0396] A DD-X-DNA(pVax-PD-L1-Fc) polyplex with an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL containing 5% trehalose was prepared. Next, aliquots of the polyplex were PEGylated as described above (mPEG5K-b-PLE10). In addition to the N:P 7 polyplex, the resulting formulation was prepared and filled into 2 mL glass vials (filling volumes of 1 mL and 0.1 mL were tested). [Table 45]

[0397] The samples were freeze-dried using the following controlled freeze-drying cycle. Freezing: +20 to -40°C (1°C / min), then -40°C for 120 minutes. Primary drying: -32°C and 63 mTorr for 3800 minutes Secondary drying: P=63mTorr, shelf T -32~30℃ (0.2℃ / min), then 30℃ for 360 minutes.

[0398] At the end of secondary drying, the vials were purged with nitrogen gas, stoppered, and allowed to equilibrate at room temperature. Short-term stability tests were performed at room temperature (ambient humidity), room temperature in a desiccator, and 4°C. At the selected point, 1 mL of the sample was rehydrated with water and incubated for 15 minutes before analysis. The samples were analyzed for particle size, PDI, zeta potential, and free DNA as described herein.

[0399] 17.4 Results The freeze-dried solids showed no signs of disintegration or cracking. Due to insufficient volume to form a solid, a 0.1 mL sample was shaped into a ring. No free DNA was observed in the rehydrated sample.

[0400] Figure 13 shows the stability of freeze-dried PEGylated polyplexes at room temperature and 4°C for up to 4 weeks. The samples were rehydrated to their pre-freeze-dried volume.

[0401] 18. Screening of potential lyophilized excipients for in-line mixed PEG-PP by freeze-thaw cycles. 18.1 Excipient Screening Materials [Table 46]

[0402] 18.2 Procedure Preparation of mannitol solution Generally, mannitol was dissolved in water at a concentration of 0.125 g / mL. The resulting solution was filtered using a 0.2 μm filter. If necessary, the mannitol solution was diluted to the concentration required for subsequent formulation.

[0403] Preparation of Kollidon 12 (PVP2) solution Generally, PVP2 was dissolved in water at a concentration of 0.05 g / mL. The resulting solution was filtered using a 0.2 μm filter. If necessary, the PVP2 solution was diluted to the concentration required for subsequent formulation.

[0404] Preparation of PEG 4kDa solution Generally, PEG 4kDa was dissolved in water at a concentration of 0.05 g / mL. The resulting solution was filtered using a 0.2 μm filter. If necessary, the PEG 4kDa solution was diluted to the concentration required for subsequent formulation.

[0405] Preparation of pharmaceutical products A DD-X-DNA(pVax-PD-L1-Fc) polyplex with an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL was prepared, free from any of the other excipients mentioned above. Next, aliquots of the polyplex were PEGylated as described herein (mPEG5K-b-PLE10). [Table 47]

[0406] Aliquots of non-PEGylated polyplex (CMC-INT06-112A) or PEGylated polyplex (CMC-INT06-112B) were mixed with water or various excipients at different concentrations to obtain the final target excipient concentration and final DNA concentration of 0.1 mg / mL described below. [Table 48]

[0407] The obtained formulations were divided into equal portions, filled into cryovials, and frozen at -80°C or lyophilized as described herein. The thawed samples were analyzed for particle size, PDI, zeta potential, pH, % supercoiled DNA, and free DNA as described herein. The lyophilized samples were assigned separate sample IDs as presented below. [Table 49]

[0408] The freeze-dried samples were rehydrated with water to their initial concentrations before drying, and analyzed for particle size, PDI, zeta potential, pH, % supercoiled DNA, and free DNA as described herein.

[0409] 18.3 Results The unPEGylated polyplex (CMC-INT06-112 A1) in water alone underwent significant aggregation after freeze-thaw cycles. Therefore, no further testing was performed on this sample.

[0410] The PEGylated polyplex formulations in either water or excipients, or in the tested concentrations, were translucent, and other physicochemical properties remained unchanged from before the freeze-thaw cycle. [Table 50]

[0411] Appearance of freeze-dried solids. Freeze-dried solids of non-PEGylated polyplex (CMC-INT06-116A1) in water alone were inconsistent in appearance and disintegrated. Freeze-dried solids of PEGylated polyplex formulations (CMC-INT06-116 B1 to CMC-INT06-116 B11) in water, either an excipient or the tested concentrations were uniform, showed minimal shrinkage, and were crack-free.

[0412] Physicochemical properties of freeze-dried samples after rehydration The unPEGylated polyplex (CMC-INT06-116 A1) in water alone aggregated significantly after rehydration. Therefore, no further testing was performed on this sample. The PEGylated polyplex formulations in water, excipients, or the tested concentrations were translucent, and other physicochemical properties remained unchanged from before lyophilization. [Table 51-1] [Table 51-2]

[0413] 18.4 Conclusion PEGylated polyplexes with an N:P:A ratio of 7:1:17.5 using PGA(1.3k)-PEG(5k) prevented aggregation of the polyplexes after freeze-thaw or freeze-drying in the absence of excipients. Trehalose, mannitol, PEG 4kDa, or PVP 2kDa were not required to prevent aggregation of PEGylated polyplexes during freeze-thaw or freeze-drying, and they did not cause aggregation at the concentrations tested.

[0414] 19. Freeze-drying of concentrated and diluted PEGylated polyplexes in 19.5% trehalose. See: CMC-INT06-EXP-110 and CMC-INT06-128 19.1 Procedure DD-X-DNA(pVax-PD-L1-Fc) polyplexes were prepared with an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL for DNA with or without the aforementioned 5% trehalose. Next, aliquots of the polyplex were PEGylated (mPEG5K-b-PLE10) and concentrated using the TFF process as described above. In addition to the N:P 7 polyplex, the resulting formulations were produced and filled into 2 or 10 mL glass vials (filling volumes of 0.3 mL, 1 mL, and 2 mL were tested). [Table 52]

[0415] The samples were freeze-dried using the following controlled freeze-drying cycle. Freezing: +20 to -40°C (1°C / min), then -40°C for 120 minutes. Primary drying: -32°C and 63 mTorr for 3800 minutes Secondary drying: P=63mTorr, shelf T -30~30℃ (0.2℃ / min), then 30℃ for 6 hours.

[0416] At the end of the secondary drying, the vial was purged with nitrogen gas, stoppered, and allowed to equilibrate at room temperature. The sample was rehydrated with water to obtain various dilutions (1x or 10x). The sample was analyzed for particle size and PDI as described herein.

[0417] 19.2 Results The lyophilized sample, at a packing volume of 1 mL, was a solid with a firm appearance, slight shrinkage, and cracking. The lyophilized samples rehydrated to 1 mg / mL and 10 mg / mL were translucent in appearance with no visible aggregates. The lyophilized sample rehydrated to 25 mg / mL formed a viscous gel because there was not enough water to form a suspension (Figure 12). The lyophilized sample rehydrated to 50 mg / mL formed a paste because there was not enough water to form a suspension.

[0418] Lyophilized samples rehydrated to 10 mg / mL and 25 mg / mL were tested for nanoparticle size, PDI, and zeta potential. No significant aggregation was observed under any of the conditions at 4°C for at least 48 hours (Figure 12).

[0419] PEG-PP (various NPA ratios) prepared in 20.4.5% sucrose or water, freeze-dried. Reference:CMC-INT06-129 20.1 Procedure A DD-X-DNA(pVax-PD-L1-Fc) polyplex with an N:P ratio of 7:1 and a DNA concentration of 0.125 mg / mL was prepared, free from any of the other excipients mentioned above. Aliquots of the polyplex were then PEGylated as described herein (mPEG5K-b-PLE10). Additional test samples CMC-INT06-124A and CMC-INT06-124B have already been described herein. [Table 53]

[0420] The resulting formulations were filled into 2 mL glass vials (tested with 0.3 mL and 1 mL filling volumes) and lyophilized using the following controlled lyophilization cycle. Freezing: 20 to -40°C (1°C / min), then -40°C for 120 minutes. Primary drying: -35°C and 63 mTorr for 3660 minutes Secondary drying: P=63mTorr, shelf T -35~30℃ (0.2℃ / min), then 30℃ for 360 minutes.

[0421] At the end of the secondary drying process, the vials were purged with nitrogen gas, sealed, and allowed to equilibrate at room temperature. They were then stored at 4°C until use.

[0422] Short-term stability was tested at 4°C for samples without excipients (CMC-INT06-129A, CMC-INT06-129B, CMC-INT06-129C). 4.5% sucrose samples (CMC-INT06-129D and CMC-INT06-129E) were tested at time 0. At the selected time point, the samples were rehydrated with water to obtain the concentration before lyophilization. As described herein, the rehydrated samples were analyzed for particle size, PDI, and zeta potential.

[0423] 20.2 Results All formulations were efficiently freeze-dried. Formulations without cryoprotectants exhibited shrinkage / radial shrinkage; those with a low NPA ratio (i.e., NPA = 7:1:7) and small fill volume (0.3 mL) were more susceptible to static electricity (solids shifting upwards and sideways, breaking, etc.). Formulations containing 4.5% sucrose showed only limited shrinkage and had a glossy surface.

[0424] Description of freeze-dried samples [Table 54]

[0425] Appearance, particle size, PDI, and zeta potential of the rehydrated sample at time 0. [Table 55]

[0426] Figure 14 shows the stability of freeze-dried samples (particle size, PDI, and zeta potential of rehydrated samples) at 4°C for up to 4 weeks in the absence of excipients. The results showed no change in particle size or zeta potential, and either a slight increase or no increase in PDI.

[0427] The following table summarizes the current findings: [Table 56]

[0428] Example 2: The general methods, compositions, and procedures are as described in Example 1 above, unless otherwise specified.

[0429] 80 μL of reversibly PEGylated bi-derivative chitosan polyplex (DDX), in which PEG contains polyglutamic acid tails and the polyplex has an NPA ratio of 7:1:7 (medium PEGylated) or 7:1:17.5 (highly PEGylated), was administered to mouse bladders at 250 μg / mL. As a control, 80 μL of unPEGylated polyplex with an NP ratio of 7:1 was administered to mouse bladders. The administered formulations were incubated in the bladder for 1 hour, after which the bladder contents were collected for analysis. As shown in Figure 15, incubation in the bladder caused a high degree of aggregation of the unPEGylated polyplex. In contrast, neither the highly nor medium PEGylated polyplexes showed detectable aggregation after incubation in the bladder.

[0430] Example 3: The in vivo gene delivery efficiency of reversibly PEGylated and unPEGylated DDX particles was compared. UnPEGylated DDX particles of NP7, NP15, and NP20, as well as PEGylated DDX with NPA 7:1:3.5 (low PEGylation) and NPA 7:1:17.5 (high PEGylation), were tested. Reversibly PEGylated DDX particles were coated with PEG-polyglutamic acid as described in Examples 1 and 2. Each DDX formulation was tested at two different concentrations (125 μg / mL and 1,000 μg / mL). The formulations were injected by intracolonic infusion (ICI) at a dose of 3 x 150 μL. After 24 hours, colon samples were collected and mRNA expression of the transgene was analyzed. As shown in Figure 16, the PEGylated DDX particles achieved significantly improved gene delivery, as detected by increased copy number expression (greater than 10x). The percentage of PEGylated DDX formulations showing detectable gene expression also improved significantly.

[0431] In addition to mRNA expression, the expression of the protein encoded by the transgene was also evaluated. UnPEGylated DDX particles of NP15 (Gr.2) and NP20 (Gr.3), as well as PEGylated DDX of NPA 7:1:3.5 (Gr.7) and NPA 7:1:17.5 (Gr.5 and 6), were tested. Reversibly PEGylated DDX particles were coated with PEG-polyglutamic acid as described in Examples 1 and 2. Each DDX formulation was tested at 125 μg / mL. The formulations were injected by intracolonic infusion (ICI) at a dose of 3 x 150 μL. After 24 hours, cell samples were collected and the protein expression of the transgene was analyzed using a Meso Scale Discovery immunoassay. As shown in Figure 17, the expression of the transgene-encoded protein was significantly increased in mouse colons treated with PEGylated DDX particles compared to unPEGylated DDX particles.

[0432] Protein expression experiments were repeated using unPEGylated DDX particles with a 7:1 NP ratio and PEGylated DDX particles with a 7:1:17.5 NPA ratio, each formulated at 1000 μg / mL. Colon sections were administered using 3 x 150 μL of the formulation via an ICI procedure, collected 24 hours after administration, and human PD-L1-Fc protein was quantified using protein lysates with a Meso Scale Discovery immunoassay. As shown in Figure 18, the expression of the transgene-encoded protein was significantly increased in mouse colons treated with PEGylated DDX particles compared to unPEGylated DDX particles.

[0433] Example 4: PEGylated DDX nanoparticles were generated using the general methods and assayed physicochemical parameters outlined in the above examples. Double derivatized polyplexes with a final functionalization degree of 25% cation (arginine(R)) and 10% polyol (glucose(G)) were prepared in NP 20:1 (unPEGylated) and NPA 10:1:5 (PEGylated) ratios. A cryoprotectant (5% trehalose or 1% mannose) was selected based on the improved cryoprotection provided to the respective unPEGylated and PEGylated polyplexes. The formulations were frozen or lyophilized, and then thawed or rehydrated as necessary. Samples were tested for % supercoiled DNA, appearance, particle size, PDI, zeta potential, and pH.

[0434] As shown in Figure 19, in the case of non-PEG lyophilized formulations, % supercoiled DNA at t=0 was significantly lower than that of equivalent PEGylated formulations. % supercoiled DNA indicates the quality of the nucleic acid delivery material and should preferably be greater than 80%, more preferably greater than 90%.

[0435] Polyplexes were prepared with an NPA (PEGylation) ratio of 10:1:5. In these polyplexes, the final functionalization degree of cation / polyol was 28% R and 10% G, and the DNA concentration was 125 μg / mL. The cryoprotectant was 1% mannitol. The formulations were lyophilized and stored at 4°C and room temperature for up to 12 weeks. At t=0 (initial), 2 weeks, 4 weeks, 8 weeks, and 12 weeks, the samples were rehydrated to the original DNA concentration and tested for % supercoiled DNA, particle size, and PDI. As shown in Figure 20, the lyophilized PEGylated DDX formulations were stable for up to 12 weeks when stored at 4°C or room temperature (RT).

[0436] Polyplexes were prepared with an NPA (PEGylated) ratio of 10:1:5 and an NP (unPEGylated) ratio of 20:1. In these polyplexes, the final functionalization degree of cation / polyol was 28% R and 10% G, and the DNA concentration was 1000 μg / mL. A cryoprotectant (5% trehalose or 1% mannitol) was selected based on the improvement in cryoprotection provided to each unPEGylated and PEGylated polyplex. The formulations were lyophilized and then rehydrated to the indicated target DNA concentrations (c1000=1 mg / mL, c2000=2 mg / mL, c5000=5 mg / mL, c10,000=10 mg / mL). Samples were tested for particle size and PDI. As shown in Figure 21, the lyophilized PEGylated formulation was stably rehydrated to a maximum concentration of 10 mg / mL, while the non-PEGylated formulation could be stably rehydrated to 2 mg / mL. While a rehydration concentration of 2 mg / mL provided a useful gene delivery formulation, the unexpectedly large increase in the achievably stable DNA concentration (10 mg / mL) of the PEGylated formulation offers significant advantages in terms of the dosage required to achieve therapeutic or clinically relevant effects.

[0437] Polyplexes were prepared with an NPA (PEGylated) ratio of 10:1:5 and an NP (unPEGylated) ratio of 10:1. For these polyplexes, the final functionalization degree of cation / polyol was 28% R and 10% G. Formulations containing 1% mannitol (PEGylated polyplex) or 5% trehalose (unPEGylated polyplex) were administered to the bladder of mice at a DNA concentration of 125 / ml, incubated for 1 hour, and bladder contents were collected for analysis. Samples were tested for nanoparticle sizing by appearance and dynamic light scattering. As shown in Figure 22, the PEGylated polyplexes did not show detectable aggregation, while the unPEGylated polyplexes showed high levels of aggregation, indicated by increased size and polydispersity index (PDI) (shown on the left) and the appearance of white clumps in the images of the collected urine samples (shown on the right).

[0438] Polyplexes were prepared with an NPA (PEGylated) ratio of 10:1:5 and an NP (unPEGylated) ratio of 20:1. For these polyplexes, the final functionalization degree of cation / polyol was 28% R and 10% G. Preparations containing 1% mannitol (PEGylated polyplex) or 5% trehalose (unPEGylated polyplex) were assayed for sterile filtration suitability. 0.5 mL of unPEGylated (NPA 20:1) and PEGylated (NPA 10:1:5) DDX polyplex preparations at 1 mg DNA / mL were filtered through 0.2 μm pore filters (13 mm diameter, 1 cm) made of various membrane types: PES (polyethersulfone), PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene), and nylon. 2It was filtered through a filter with a surface area of []. Samples were tested for nanoparticle sizing, zeta potential, conductivity, pH, cation (arginine), polyol (glucose) content, and DNA content. As shown in Figure 23, the DNA concentration showed a significantly greater decrease (15 - 32%) after filtration of the non-PEGylated polyplex formulation compared to the PEGylated polyplex formulation (less than 10%). These results suggest that non-PEGylated polyplexes aggregate under the assay conditions.

[0439] To confirm the results shown in Figure 23, the generation of PEGylated and non-PEGylated DDX chitosan-DNA polyplexes was scaled up and the formulations were tested for bulk filtration properties. For these polyplexes, the final degree of functionalization of cation / polyol was 28% R and 10% G. Formulations containing 1% mannitol (PEGylated polyplex) or 5% trehalose (non-PEGylated polyplex) were assayed for compatibility with sterile filtration. 5 - 15 mL of non-PEGylated (NPA 20:1:0) and PEGylated (NPA10:1:5) DDX DNA polyplex formulations at a DNA concentration of 1 mg DNA / mL were filtered through a 0.8 / 0.2 μm pore PES filter stack (diameter 25 mm, surface area 2.8 cm 2 , each filter). Filtration was by a constant pressure Vmax filtration setup at 30 psig (schematic shown in Figure 24). Filtration properties were determined as the mass of the collected filtrate per unit surface area. Samples before and after filtration were tested for nanoparticle sizing, zeta potential, conductivity, pH, cation / polyol content, and DNA content.

[0440] As shown in Figure 24, the DNA concentration showed a significantly greater decrease (19%) after filtration of the non-PEGylated polyplex formulation compared to the PEGylated polyplex formulation (less than 10%). Furthermore, the non-PEGylated polyplex formulation had a flux of 1.375 g of polyplex / cm 2When supplied at the maximum concentration (of the membrane surface area), it clogged the filtration system. In contrast, PEGylated polyplex clogged all polyplex concentrations tested (maximum 5.35 g of polyplex / cm³). 2 The filter did not clog at a surface area of ​​5.35 g / cm³. 2 This suggests that PEGylated polyplex formulations exceeding a certain level remain filterable.

[0441] Polyplexes were prepared with the NPA (PEGylated) or NP (non-PEGylated) ratios and DNA concentration (c125) shown in Figure 25. The cation / polyol number ratio was 25% R and 10% G. The cryoprotectant was 1% mannitol (1% Man). The formulations were freeze-thawed (FT) or lyophilized and rehydrated (FD). Samples were tested for % supercoiled DNA nanoparticle size and zeta potential. As shown in Figure 25, the non-PEGylated DDX formulations precipitated after freeze / thaw and lyophilization / rehydration. % supercoiled DNA could not be measured in the precipitated samples. In contrast, the PEGylated DDX formulations (NPA 10:1:5 and 10:1:2.5) did not aggregate after freeze / thaw, while the 10:1:1 formulations showed some aggregation after freeze / thaw, but significantly less than the non-PEGylated material. After freeze-drying and rehydration, all tested PEGylated DDX formulations (NPA 10:1:5 to 10:1:1) showed no detectable agglutination.

[0442] PEGylated DDX polyplexes were prepared and delivered to the small intestine of dogs by direct intravenous infusion. As shown in Figure 26, mRNA copy numbers indicating gene delivery in dogs were detected 24 hours after delivery.

[0443] PEGylated polyplexes were mixed with acetate buffers in various ratios to simulate a low pH environment, and the pH of the resulting solutions was measured. The zeta potential of the particles was also monitored. When the pH decreased below the pKa of the anion anchor region of the polymer coating (approximately 4.25), the zeta potential increased dramatically, indicating the release of the polymer coating. See Figure 27.

[0444] Example 5: Reversibly PEGylated polyplexes containing bi-derivative chitosan (Arg: gluconic acid) with polyglutamic acid anchor regions of different sizes were prepared in an N:P:A ratio of 7:1:X (wherein X is 3.5, 9, or 14.5). PLE5 refers to a polyplex containing a PEG-polyglutamic acid (PEG-PLE) polymer, where the polyglutamic acid anchor region has a length of 5 glutamic acid amino acids. PLE10 refers to a length of 10 glutamic acid amino acids, and PLE25 refers to a length of 25 glutamic acid amino acids. The physicochemical parameters of the obtained PEGylated polyplexes are shown in the table below. [Table 57]

[0445] The obtained PEGylated polyplexes were assayed for particle size, and no significant size changes were observed among the prepared polyplexes. Figure 28. The polyplexes were dispersed in water, and the pH was measured. This showed that both a longer PLE length and a decreasing N:A ratio contributed to the higher pH of the aqueous dispersion. Figure 29. Similarly, zeta potential measurements showed that both a longer PLE anchor region and a decreasing N:A ratio decreased the zeta potential. Figure 30 (left). Furthermore, the inventors observed a stable state of this tendency to decrease the zeta potential of PLE25 polyplexes as N:P:A (adjusted to 7:1:X (X=9) to 7:1:X (X=14.5)), thereby demonstrating that maximum PEGylation is achieved with a smaller amount of polymer. Figure 30 (right).

[0446] PEGylated polyplexes were assayed using a polyaspartate (PAA) competitive assay. Nucleic acid accessibility was observed by mixing PAA at various concentrations in an aqueous buffer containing the PEGylated polyplexes. Nucleic acid accessibility was measured using a PicoGreen assay. Accessible nucleic acids bind to PicoGreen, and the resulting increase in fluorescence signal is detected. Fully released and / or fully accessible nucleic acids provide the greatest fluorescence signal. The EC50 of the PAA concentration required to achieve up to half the signal is a measure of how easily the particulate composition is destroyed by PAA contact, i.e., the stability of the tested polyplexes. The results indicate that PLE25 PEGylated polyplexes are somewhat less stable than other polyplexes. Figures 31-32.

[0447] PEGylated polyplexes were prepared as a c125 (125 μg / mL DNA) reaction mixture and then mixed with FaSSIF-V2 in a 1:2 volume ratio (excess FaSSIF). After mixing with FaSSIF-V2, DLS measurements were performed at 37°C, time zero (T0) and 30 minutes (T=30) to measure particle size and polydispersity (Figure 33), and the samples were also assayed for zeta potential and pH (Figure 34). Under the tested conditions, polyplexes with longer polyanion chain lengths (e.g., PLE25 was the most stable, PLE5 the least stable) and higher levels of chitosan amino-functionalization (e.g., N:P:A 14.5 was the most stable, N:P:A 3.5 the least stable) were more stable.

[0448] Example 6: The effects of polyanion species and molecular weight (MW) on PEGylated polyplexes were investigated. PLE (polyglutamic acid), PLD (polyaspartic acid, i.e., PAA), and HA (hyaluronic acid) were studied. A 4.51% trehalose solution was used as a storage stabilizer. PEG polyanion working solutions were prepared.

[0449] PEG-HA25 did not dissolve in the trehalose diluent and formed a gel-like species. When diluted 100x, the gel did not dissolve. PEG-HA25 was excluded from further analysis. The resulting c250 solution was mixed with PEG polyanion in a 1:1 v / v ratio by adding the PEG polyanion solution to the c250 solution while vortexing. Vortexing was continued for 10 seconds after adding the PEG polyanion. The following PEGylated polyplexes were produced. [Table 58]

[0450] The above compositions were incubated at ambient temperature for 1 hour and then further analyzed.

[0451] Separately, solutions of the biderivatized chitosan nucleic acid polyplex were diluted in a 4.51% trehalose solution from a nucleic acid concentration of 1000 μg / mL (c1000) to 125 μg / mL (c125). After freeze-thaw cycles (F / T), all samples were tested for appearance, pH, size, zeta potential, and free DNA content. The results are shown in the table and Figures 35-36 below. [Table 59-1] [Table 59-2] [Table 60] [Table 61]

[0452] Polyplex samples were analyzed by agarose gel electrophoresis to detect nucleic acids that did not form complexes. Figure 37. With the exception of the 7:1:30 N:P:A polyplex PEGylated with PEG-PLD50 (Figure 37, lower panel 6-8), all tested samples did not show any nucleic acids that did not form detectable complexes.

[0453] The results suggest the following: PEG-PLE10, PEG-PLD10, and PEG-PLD50 are compatible with DDX polyplexes at a 7:1 NP ratio for PEGylation. No precipitates were observed in any of the c125 PEGylated polyplex solutions after F / T. PEG-PLE10 and PEG-PLD10 behave similarly when mixed with polyplexes. The size of the polyplexes increased from 140 nm to 160 nm after PEGylation. The size reached a stable level of 160 nm at NPA ratios of 7:1:3.5, 7:1:5, 7:1:9, 7:1:15, and 7:1:30. The zeta potential decreases as the PEG polyanion ratio increases, but the decrease slows beyond 7:1:9, suggesting that PEGylation may reach saturation. In the case of PEG-PLD50, after PEGylation, the polyplex size was equivalent to the unPEGylated polyplex (7:1:0) up to an NPA ratio of 7:1:15. At an NPA of 7:1:30, the polyplex size increased to 440 nm after PEGylation. The zeta potential of the PEG-PLD50 PEGylated polyplex decreased as the PEG-PLD50 ratio increased. Free DNA was observed with PEG-PLD50 at 7:1:30, but not with other formulations. This suggests that the nucleic acids within the polyplex of PEG-PLD50 at 7:1:30 are loosely encapsulated, and / or that PLD-50 disrupts the binding of derivatized chitosan to the nucleic acids.

[0454] Example 6: The effects of non-covalent reversible PEGylation were compared with those of covalent PEGylation. As shown in Figure 38, both non-covalently PEGylated and covalently PEGylated polyplexes with titrable polyanion anchor regions are expected to have near-neutral zeta potentials under pH conditions that maintain the negative charge of the polyanion anchor region. Titrating the polyanion anchor region to a neutral or positive charge at a low pH (e.g., pH 2) should cause a detectable increase in the zeta potential of the non-covalently PEGylated polyplex. In contrast, covalently bonded PEG cannot be released, and its zeta potential should not change significantly.

[0455] As shown in Figure 39, two different covalent PEGylation strategies were studied. First, PEG-PLE was crosslinked to double-derivatized (DDX) chitosan to form a crosslinked PEG-chitosan raw material, and DNA was added to form a polyplex (PEG crosslinked to chitosan) (Figure 39, middle). Next, a reversibly PEGylated polyplex was formed, and EDC / NHS was added to covalently crosslink the PEG-PA polymer with the polyplex (PEG crosslinked to the polyplex) (Figure 39, bottom). Both covalently PEGylated polyplexes showed only slight changes in zeta potential after adjusting the pH to 6-2. In contrast, the zeta potential of the reversibly PEGylated polyplex increased significantly. The stability of the polyplexes was also tested by induction with free polyaspartic acid (PAA). PAA induction released nucleic acids in the polyplexes from both the reversibly PEGylated polyplex and the PEG crosslinked to the chitosan polyplex. In contrast, cross-linking after polyplex formation provided polyplexes that did not release nucleic acids after PAA induction under the tested conditions.

[0456] The transfection efficiency of polyplexes reversibly PEGylated with 7:1:X (where X is 9, 3, or 1) N:P:A was assayed and compared with that of unPEGylated polyplexes (7:1:0). The results showed that transfection efficiency was not reduced by reversible PEGylation. Figure 40.

[0457] The transfection efficiencies of reversibly PEGylated polyplexes and covalently PEGylated polyplexes were compared. As shown in Figure 41, reversibly PEGylated polyplexes maintained high transfection efficiencies, while both types of covalently PEGylated polyplexes showed low transfection efficiencies.

[0458] Example 7: The transfection efficiency of a reversibly PEGylated polyplex (1-step) formed by a one-step mixing of nucleic acid, chitosan, and PEG-polyanion polymer was compared with that of a reversibly PEGylated polyplex (2-step) formed by mixing a pre-formed chitosan DNA polyplex with PEG-polyanion polymer. mRNA expression was detected in colon tissue 24 hours after intracolonic delivery of the 1-step or 2-step PEGylated polyplex. mRNA and protein were detected in bladder tissue 24 hours after intrabladder delivery of the 1-step or 2-step PEGylated polyplex. The results shown in Figures 42-43 demonstrate that both the 1-step and 2-step polyplexes exhibit similar transfection efficiency under the tested conditions. While we do not wish to be bound by theory, it is generally assumed that smaller 1-step polyplexes may exhibit superior diffusion properties in high-viscosity mucosa.

[0459] Equal portions All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually shown to be incorporated by reference for any purpose. The above disclosure may encompass several distinct inventions having independent utility. Each of these inventions is disclosed in preferred forms, but the particular embodiments disclosed and illustrated herein should not be considered restrictively, as numerous variations are possible. The subject matter of the present invention includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions, and / or characteristics disclosed herein. The following claims point in particular to certain combinations and subcombinations that are considered novel and non-obvious. Inventions performed in other combinations and subcombinations of features, functions, elements, and / or characteristics may be claimed in this application, in an application claiming priority to this application, or in a related application. Such claims, whether relating to different inventions or the same invention, whether broader, narrower, equal to, or different in scope from the original claims, are also deemed to be included within the subject matter of the inventions of this disclosure.

Claims

1. A composition, the following: (a) A complex comprising an amino-functionalized chitosan and chitosan derivative nanoparticles containing at least one nucleic acid molecule, wherein the at least one nucleic acid molecule is non-covalently bonded to the chitosan derivative nanoparticle in an amino acid to phosphorus (N:P) molar ratio greater than 3:1, thereby forming a positively charged derivatized chitosan nucleic acid complex; and (b) An outer film comprising a plurality of linear block copolymers non-covalently bonded to the chitosan derivative nanoparticles, wherein the linear block copolymer comprises at least one polyanion (PA) anchor region and at least one polyethylene glycol (PEG) tail region, and the PEG-PA molecule is non-covalently bonded to the chitosan derivative nanoparticles; A composition comprising, wherein the composition comprises an amino acid to anion (N:A) molar ratio greater than about 1:100 and less than about 10:1, the PEG-PA molecule is PEG-polyglutamic acid (PEG-PGA) or PEG-polyaspartic acid (PEG-PAA), the weight-average molecular weight (Mw) of the PEG portion of the PEG-PA molecule is about 1,500 Da to about 7,500 Da, and the weight-average Mw of the PA portion of the PEG-PA molecule is about 500 Da to about 3,000 Da.

2. The composition according to claim 1, wherein the linear block copolymer is a binary block copolymer comprising a PA anchor region and a PEG tail region.

3. The composition according to claim 1, wherein the linear block copolymer is a ternary block copolymer comprising a central PA anchor region adjacent to two PEG tail regions, or, alternatively, a central PEG tail region adjacent to two PA anchor regions.

4. The composition according to claim 1, wherein the PA anchor region comprises a polypeptide, and the polypeptide is a load electric current.

5. The composition according to claim 1, wherein the PEG-PA molecule comprises a PEG-polyglutamic acid (PEG-PGA) molecule.

6. The composition according to any one of claims 1 to 4, wherein the PEG portion of the PEG-PA molecule contains a weight-average molecular weight (Mw) of about 5,000 Da.

7. The composition according to any one of claims 1 to 6, wherein the PA portion of the PEG-PA molecule contains a weight-average molecular weight (Mw) of about 1,000 Da to about 2,500 Da.

8. The composition according to any one of claims 1 to 6, wherein the PA portion of the PEG-PA molecule contains a weight-average Mw of about 1,500 Da.

9. The composition according to any one of claims 1 to 6, wherein the PA portion of the PEG-PA molecule comprises, for example, a linear polypeptide containing about 5 to about 25 acidic amino acids.

10. The composition according to any one of claims 1 to 9, wherein the N:P molar ratio is greater than about 3:1 and less than about 100:

1.

11. The composition according to claim 10, wherein the N:P molar ratio is greater than about 5:1 and less than about 20:

1.

12. The composition according to claim 11, wherein the N:P molar ratio is about 10:

1.

13. The composition according to any one of claims 1 to 12, wherein the N:A molar ratio is greater than about 1:75 and less than about 8:

1.

14. The composition according to any one of claims 1 to 13, wherein the N:A molar ratio is greater than about 1:5 and less than about 6:

1.

15. The composition according to any one of claims 1 to 14, wherein the N:P molar ratio is about 5:1 to about 30:1, and the phosphorus to anion (P:A) molar ratio is about 1:50 to about 1:

5.

16. The composition according to claim 15, wherein the N:P molar ratio is about 5:1 to 15:1, and the P:A molar ratio is about 1:50 to 1:

5.

17. The composition according to any one of claims 1 to 16, wherein the chitosan derivative nanoparticles contain a polyol of formula II or are functionalized with a polyol of formula II: 【Chemistry 1】 (In the formula, (a) R ２ is selected from H and hydroxyl, (b) R ３ is selected from H and hydroxyl, (c) X is C ２ -C ６ (Selected from alkylenes).

18. The composition according to claim 17, wherein the C2-C6 alkylene of X is optionally substituted with one or more hydroxyl substituents.

19. The composition according to any one of claims 1 to 18, wherein the chitosan derivative nanoparticles comprise a polyol of formula III: 【Chemistry 2】 (In the formula, -----Y is = O, R ２ is selected from H and hydroxyl, X is C ２ -C ６ Selected from alkylenes, 【Transformation 3】 (This represents the bond between the polyol and the derivatized chitosan.)

20. The composition according to claim 19, wherein the C2-C6 alkylene of X is optionally substituted with one or more hydroxyl substituents.

21. The composition according to any one of claims 1 to 20, wherein the amino-functionalized chitosan is functionalized with arginine, lysine, or ornithine, preferably with arginine.

22. The composition is (a) In an empty intestinal fluid for at least 24 hours, or (b) Disperse in mammalian urine at 37°C for at least 1 hour. A stable composition according to any one of claims 1 to 21.

23. The composition according to any one of claims 1 to 22, wherein the composition further comprises a surfactant, an excipient, and / or a storage stabilizer.

24. The composition according to claim 23, wherein the storage stabilizer is selected from the group consisting of monosaccharides, disaccharides, polysaccharides, and their reducing alcohols.

25. The composition according to claim 24, wherein the storage stabilizer is selected from trehalose and mannitol.

26. The composition according to claim 23, wherein the surfactant is poloxamer.

27. The composition according to claim 26, wherein the poloxamer is poloxamer 407.

28. The composition according to any one of claims 1 to 27, wherein the at least one nucleic acid comprises RNA.

29. The composition according to any one of claims 1 to 27, wherein the at least one nucleic acid comprises DNA.

30. The composition according to any one of claims 1 to 29, wherein the composition is stable at 4°C for 48 hours, or at least 48 hours, or for one week, in an aqueous dispersion containing the composition dispersed in purified water.

31. The composition according to claim 30, wherein the composition is stable in the aqueous dispersion after freezing / thawing and / or drying / rehydration.

32. The composition according to claim 31, wherein the drying includes spray drying, freeze-drying, spray freeze-drying, evaporation, or supercritical drying.

33. The composition according to claim 31 or 32, wherein the composition exhibits a polydispersity index of less than 0.2 in the aqueous dispersion at 4°C for 48 hours or 1 week.

34. The composition according to claim 1, The amino-functionalized chitosan is arginine-functionalized chitosan. The chitosan derivative nanoparticles contain glucose, The PEG-PA molecule contains PEG-PGA, The weight-average molecular weight of the PEG portion is approximately 5000 Da, and The weight-average molecular weight of the PGA portion is approximately 1500 Da, and The N:P molar ratio is approximately 10:1, and The N:A molar ratio is greater than approximately 1:5 and less than approximately 6:

1. composition.

35. A method for preparing any one of the compositions described in claims 1 to 34, wherein the method is (a) To provide a complex comprising an amino-functionalized chitosan and the chitosan derivative nanoparticles containing at least one nucleic acid, and (b) Mix the complex with a solution containing PEG-PA molecules to form a reaction mixture containing the composition. The method, including the method described above.

36. The method according to claim 35, wherein the complex of (a) is provided at a nucleotide concentration of 0.01 mg / mL to 25 mg / mL.

37. The method according to claim 36, wherein the complex of (a) is provided at a nucleotide concentration of 0.10 to 2 mg / mL.

38. The method according to claim 36 or 37, wherein (a) and (b) are mixed in a ratio of 1:10 to 10:1 (v / v).

39. The method according to claim 38, wherein (a) and (b) are mixed in a ratio of 1:5 to 5:1 (v / v).

40. The method according to any one of claims 35 to 39, wherein the method further comprises concentrating the reaction mixture by ultrafiltration, solvent sublimation, solvent evaporation, or tangential flow filtration.

41. The method according to claim 40, wherein the amino-functionalized chitosan comprises or is functionalized with a hydrophilic polyol.

42. The method according to any one of claims 35 to 41, wherein the method provides a reduction in mucosal adhesion compared to particles that do not contain the polymer component including PEG-PA.

43. A method for producing any one of the compositions of claims 1 to 34, wherein the method comprises simultaneously or sequentially mixing amino-functionalized chitosan, at least one nucleic acid, and a PEG-PA molecule to form a reaction mixture containing the composition.

44. The method according to claim 43, wherein the method comprises simultaneously combining the amino-functionalized chitosan, at least one nucleic acid, and the PEG-PA molecule to form a reaction mixture comprising the composition.

45. A composition according to any one of claims 1 to 34 for use in a method of transfecting cells with nucleic acids, wherein the method comprises bringing the cells into contact with the composition in vivo.

46. The composition according to claim 45, wherein the cells include or are derived from mucosal tissue.

47. The composition according to claim 46, wherein the mucosal tissue is lung tissue, nasal tissue, eye tissue, vaginal tissue, bladder tissue, or gastrointestinal tract tissue.

48. The composition according to claim 46, wherein the cells are intestinal cells.

49. The composition according to claim 46, wherein the cells are bladder cells.

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