Reversible coatings of chitosan-nucleic acid nanoparticles and methods of use thereof

Abstract

Described herein are chitosan-nucleic acid multicomplex compositions containing a reversibly bound polymer coating comprising a linear block copolymer having a polyanion anchoring region and at least one polyethylene glycol tail region. In some cases, the compositions exhibit improved stability and / or mucosal diffusion compared to uncoated particles. In some cases, the reversibly bound polymer coating does not interfere with or enhance transfection of the target cell or tissue as compared to uncoated particles.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of March 13, 2020, application number 202080035434.9, and invention name “Reversible coating of chitosan-nucleic acid nanoparticles and method of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 818,425 filed on March 14, 2019, U.S. Provisional Application No. 62 / 923,403 filed on October 18, 2019, and U.S. Provisional Application No. 62 / 924,131 filed on October 21, 2019, the contents of which are incorporated herein by reference in their entirety and for all purposes. Background Art

[0004] Chitosan is a deacetylated form of chitin. Chitosan 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, has been used as a DNA delivery vehicle to transfect cells. Due to the complexity and potential toxicity of viral vectors, much interest has been focused on the use of chitosan for non-viral delivery of nucleic acids.

[0005] A variety of chitosan / DNA complexes, including complexes between modified chitosan and nucleic acids, have been examined in an attempt to identify compositions that are well suited for gene transfection. See, e.g., WO 2010 / 088565; WO 2008 / 082282. These complexes have been found to differ in important physicochemical and biological properties, including solubility, aggregation tendency, complex stability, particle size, ability to release DNA, and transfection efficiency.

[0006] Chitosan-nucleic acid polyplexes with improved transfection efficiency have been developed by functionalizing the chitosan backbone with arginine and hydrophilic polyols. See, e.g., U.S. Pat. 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 the systemic circulation due to significant pH changes, enzyme activity, and complex mucosal interactions.

[0007] Since the dense network of mucin fibers can effectively capture foreign particles through adhesion and spatial interactions, and then quickly clear them, the mucosal barrier is a unique challenge for nanoparticle delivery systems. In fact, especially for oral drug delivery, the intestinal mucosa can be turned over in just fifty (50) minutes, resulting in very effective removal of the 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 can bind hydrophobic materials at high affinity, including periodic hydrophobic domains of many commonly used drug delivery materials such as poly (lactic acid-co-glycolic acid) (PLGA). These complex charge interactions plus steric hindrance have proven to be difficult to overcome in the absence of some form of shielding in nanoparticle drug delivery forms. However, on the contrary, traditional shielding methods usually produce nanoparticles with greatly reduced transfection ability.

[0008] Polyethylene glycol (PEG) has become a major component of various shielding strategies, based at least initially on its demonstrated ability to increase mucosal adhesion through interpenetrating network effects, thereby prolonging drug delivery. See, for example, Huckaby and Lai, Advanced Drug Delivery Reviews 124: 125-39 (2018). Unfortunately, however, these mucoadhesion strategies are not suitable for therapeutic agents that require intracellular delivery, such as nucleic acids, because enhanced mucoadhesion prevents particles from reaching the underlying epithelium or other relevant target tissues. Paradoxically, perhaps PEG coatings have also been used in mucus-inert methods that attempt to enhance particle diffusion. However, it is worth noting that recent findings and conclusions on these types of systems emphasize the importance of high PEG grafting density to minimize mucin affinity and enhance mucosal penetration. Same as above, Wang and Lai, Angew Chem 47: 9726-9729 (2008). These unusually dense and covalently bound PEGylation strategies are problematic for effective gene transfection.

[0009] Thus, there remains a need for new compositions and methods for in vivo gene transfer with improved mucosal penetration properties. Summary of the invention

[0010] Provided herein are compositions and methods for reducing mucosal adhesion and enhancing mucosal penetration of chitosan-nucleic acid nanoparticles, which use reversible (i.e., non-covalent binding) polymer coatings, the polymer coatings comprising a variety of linear block copolymers, the linear block copolymers having a negatively charged anchoring region and at least one (e.g., hydrophilic) uncharged tail region. In a preferred embodiment, the linear block copolymer is a diblock or triblock copolymer comprising at least one polyanion anchoring region and at least one PEG tail region. In some embodiments, the compositions described herein unexpectedly reduce mucosal adhesion and enhance mucosal penetration at a surface coating density lower than previously expected. In some embodiments, despite the presence of polyanions, the compositions described herein unexpectedly provide consistent physical and / or chemical stability. In some embodiments, despite including a reversible polymer coating, the compositions described herein unexpectedly provide consistent and / or comparable transfection efficiencies. In some embodiments, the compositions described herein are unexpectedly stable during long-term (e.g., months) storage in bulk and / or in a coated or encapsulated form. In some embodiments, the compositions described herein are unexpectedly stable when in contact with mucosal barriers and / or mucus-associated fluids (eg, urine or gastric or intestinal fluids).

[0011] In one aspect, the present invention provides chitosan compositions comprising chitosan derivative nucleic acid nanoparticles (polyplexes) that form complexes with a variety of polyanion-containing block copolymers (e.g., linear diblock and / or triblock copolymers), each copolymer comprising at least one polyanion anchoring 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 portion of an outer coating. The complex between the polyplex and the polyanion-containing block copolymer is typically formed by means of a reversible and non-covalent electrostatic interaction between the polyanion anchoring region of the polymer and the net positive charge of the uncoated polyplex. In some cases, the complex is reversible because the strength of the electrostatic interaction between the polyanion anchoring region of the polymer is reduced by increasing the ionic strength, and / or reducing the pH value to protonate the anion moiety in the polyanion region of the polymer, and all or a portion of the polyanion-hydrophilic polymer can be released from the complex.

[0012] An exemplary diblock 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 triblock copolymer molecule useful in the methods and compositions of the present invention is a "PEG-PA" copolymer molecule comprising a central PA anchor region flanked by two PEG tail regions [PEG-PA-PEG], or two PA anchor regions flanking a central PEG tail region [PA-PEG-PA].

[0013] In some embodiments, the present invention provides a complex comprising chitosan derivative nanoparticles and at least one nucleic acid molecule, wherein the chitosan derivative nanoparticles comprise amino-functionalized chitosan, wherein the at least one nucleic acid molecule is non-covalently bound to the chitosan derivative nanoparticles at an amino to phosphorus (N:P) molar ratio greater than 3:1, thereby forming a derivatized chitosan nucleic acid complex having a positive charge; and a plurality of linear block copolymers non-covalently bound to the chitosan derivative nanoparticles, wherein the block copolymers comprise at least one polyanion (PA) anchor region and at least one polyethylene glycol (PEG) tail region, and wherein the composition comprises an amino to anion (N:A) molar ratio greater than about 1:100 and less than about 10:1.

[0014] In some embodiments, the linear block copolymer is a diblock copolymer comprising a PA anchor region and a PEG tail region. In some embodiments, the linear block copolymer is a triblock copolymer comprising a central PA anchor region flanked by two PEG tail regions, or alternatively, a central PEG tail region flanked by two PA anchor regions.

[0015] In some embodiments, the PA anchor region comprises a polypeptide, wherein the polypeptide is negatively charged. In some embodiments, the PA anchor region of the PEG-PA molecule comprises a carbohydrate, wherein the carbohydrate is negatively charged. In some embodiments, the carbohydrate comprises a plurality of phosphate and / or sulfate moieties. In some embodiments, the carbohydrate comprises a plurality of carboxylate moieties. In some embodiments, the carbohydrate comprises a plurality of carboxylate moieties and a plurality of 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 an exemplary embodiment, the carbohydrate is a glycosaminoglycan.

[0016] In specific embodiments, the PEG-PA molecule comprises: a PEG-polyglutamic acid (PEG-PGA) molecule; a PEG-polyaspartic acid (PEG-PAA) molecule; or a PEG-hyaluronic acid (PEG-HA) molecule, or a combination thereof.

[0017] In some embodiments, the PEG tail region of the PEG-PA molecule comprises 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, 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 comprises 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.

[0018] 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, more preferably greater than about 5:1 and less than about 30:1, more preferably greater than about 5:1 and less than about 20:1, more preferably greater than about 5:1 and less than about 10:1. In some cases, the N:P molar ratio is from about 3:1 to about 30:1. In some cases, the N:P molar ratio is from about 3:1 to about 20:1. In some cases, the N:P molar ratio is from about 3:1 to about 10:1. In some cases, the N:P molar ratio is about 7:1.

[0019] 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, more preferably greater than about 1:25 and less than about 6:1, more preferably greater than about 1:10 and less than about 6:1, more preferably greater than about 1:5 and less than about 6:1.

[0020] In some embodiments, the N:P molar ratio is from about 1:8 to about 8:1, and the P:A molar ratio is from about 0.02 to about 0.2, more preferably, wherein the N:A molar ratio is from about 0.1 to about 5, more preferably from about 0.2 to about 2, more preferably from about 0.3 to about 1.5, more preferably from about 0.4 to about 1, more preferably, wherein the N:P:A ratio is about 7:1:7, about 7:1:12, or about 7:1:17.

[0021] In some embodiments, the N:P molar ratio is from about 1:8 to about 30:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 1:5 to about 20:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 1:2 to about 10:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, and more preferably from about 1:2.5 to about 1.

[0022] In some embodiments, the N:P molar ratio is from about 1:1 to about 30:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 1:1 to about 20:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 1:1 to about 15:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1. In some embodiments, the N:P molar ratio is from about 1:1 to about 10:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1.

[0023] In some embodiments, the N:P molar ratio is from about 2:1 to about 30:1, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, 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, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, 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, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, 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, and the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1.

[0024] In some embodiments, the amino-functionalized chitosan is arginine, lysine or ornithine functionalized, preferably arginine functionalized. 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 further functionalized with a polyol. In some embodiments, the amino-functionalized chitosan is further functionalized with a polyol.

[0025] In some embodiments, the composition is stable or at least stable for 1 hour, 24 hours, 48 ​​hours, 1 week, or 1 or 2 months in simulated intestinal fluid in the fasting state. In some embodiments, in an aqueous dispersion, such as a dispersion of the composition in purified water, at 4°C, the composition is stable or at least stable for 1 hour, 24 hours, 48 ​​hours, 1 week, or 1 or 2 months. In some embodiments, in purified water, at 4°C, the composition is stable or at least stable for 1 hour, 24 hours, 48 ​​hours, 1 week, or 1 or 2 months. In some embodiments, in urine, at 4°C, the composition is stable or at least stable for 1 hour, 24 hours, 48 ​​hours, 1 week, or 1 or 2 months. In some embodiments, the composition is stable because it is substantially free of (<10%) precipitated aggregates in simulated intestinal fluid and / or aqueous dispersion and / or urine and / or purified water after a specified time, such as 24 hours, 48 ​​hours, 1 week, or 1 or 2 months.

[0026] In some embodiments, in aqueous dispersions, the composition is stable after freezing / thawing and / or lyophilization / rehydration. In some embodiments, in aqueous solution, at 4° C., the composition exhibits a polydispersity index of less than 0.2 after or at least after 48 hours, 1 week, or 1 or 2 months. In some embodiments, in aqueous dispersions, the composition is stable after drying (e.g., lyophilization, spray drying, evaporation, supercritical drying, spray freeze drying, etc.) and then rehydration.

[0027] 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 at room temperature or at 37° C. for at least 1 hour. 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.).

[0028] In some embodiments, the composition transfects cells with a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is transcribed into a therapeutic protein. In some embodiments, the therapeutic nucleic acid inhibits the expression of an endogenous protein encoding gene. In some embodiments, the therapeutic nucleic acid inhibits the expression of an endogenous gene.

[0029] 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, a disaccharide, a polysaccharide or their reducing alcohols, more preferably, wherein the storage stabilizer is selected from trehalose and mannitol. In some embodiments, the surfactant comprises a poloxamer, more preferably, wherein the poloxamer is poloxamer 407.

[0030] In some embodiments, at least one nucleic acid comprises RNA. In some embodiments, at least one nucleic acid comprises DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Polyplex: polymer compositions and methods of making the compositions are shown.

[0032] Figure 2 An inverse relationship between the zeta potential and the degree of PEGylation is shown for certain 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 near neutral to slightly negative values.

[0033] Figure 3 The stability of the polyplex: polymer compositions after freeze-thaw is shown. The PEGylated polyplexes remained stable after freeze-thaw at the tested ratios of [N+] to [A-].

[0034] Figure 4 The stability of polyplex:polymer compositions in simulated intestinal fluid at different volume:volume ratios is shown. FaSSIF-V2 = Fasted State Simulated Intestinal Fluid V2.

[0035] Figure 5 The ability of the polyplex:polymer composition to retain complexed nucleic acids in simulated intestinal fluid is shown. FaSSIF-V1 = Fasted State Simulated Intestinal Fluid V1. FaSSIF-V2 = Fasted State Simulated Intestinal Fluid V2. PP = Polyplex.

[0036] Figure 6 In vitro transfection with PEGylated DD-chitosan-nucleic acid polyplexes is demonstrated.

[0037] Figure 7 Shown are methods and results of a mucus aggregation assay using fluorescence microscopy for the polyplex:polymer compositions described herein.

[0038] Figure 8 A method for conducting a mucosal permeation assay using a transwell diffusion assay for the polyplex:polymer compositions described herein is shown.

[0039] Fig. 9 Shown are the results of transwell diffusion assays of polyplex:polymer compositions described herein.

[0040] Fig.10 Shown are the results of a transwell diffusion assay of the polyplex:polymer compositions described herein in the presence of Poloxamer 407.

[0041] Fig.11 A scalable method for making the polyplex:polymer compositions described herein is shown.

[0042] Fig.12 The stability of the polyplex:polymer compositions described herein upon lyophilization and rehydration as well as at high concentration conditions is shown.

[0043] Fig.13 The stability of freeze-dried PEGylated polyplexes at room temperature and 4°C for up to 4 weeks was shown.

[0044] Fig.14 The stability of the polyplex:polymer compositions described herein when lyophilized in the absence of excipients and stored at 4°C for up to 4 weeks is shown.

[0045] Fig.15The stability of the polyplex:polymer compositions described herein when administered to the bladder of mice and recovered in subsequently collected urine is shown.

[0046] Fig.16 It is shown that the polyplex:polymer compositions described herein significantly improve in vivo gene delivery when delivered by intracolonic instillation (ICI) as measured by transgene mRNA expression. The polyplex formulation administered contained 150 μg / mL nucleic acid and was administered as 3×150 μL intracolonic instillations; colon sections were harvested 24 hours after administration and human PD-L1-Fc mRNA was quantified using cell lysates.

[0047] Fig.17 It is shown that the polyplex:polymer compositions described herein significantly improve in vivo gene delivery when delivered by intracolonic instillation (ICI) as measured by transgenic protein expression. The polyplex formulations administered contained 150 μg / mL nucleic acid and were administered as 3×150 μL intracolonic instillations; colon sections were harvested 24 hours after administration and human PD-L1-Fc protein was quantified using protein lysates using a custom Mesoscale Discovery immunoassay.

[0048] Fig.18 It is shown that the polyplex:polymer compositions described herein significantly improve in vivo gene delivery when delivered by intracolonic instillation (ICI) as measured by transgenic protein expression. The polyplex formulations administered contained 1000 μg / mL nucleic acid and were administered as 3×150 μL intracolonic instillations; colon sections were harvested 24 hours after administration and human PD-L1-Fc protein was quantified using protein lysates using a custom Mesoscale Discovery immunoassay.

[0049] Fig.19 Shown is the increase in % supercoiled DNA content of PEGylated DDX polyplexes compared to non-PEGylated DDX polyplexes.

[0050] Fig. 20 The storage stability of PEGylated DDX polyplexes is shown.

[0051] Fig.21 The rehydration properties of PEGylated and non-PEGylated DDX polyplex formulations are shown. The PEGylated DDX polyplex was able to stably rehydrate at a higher final concentration (10 mg / mL) compared to the non-PEGylated DDX polyplex (2 mg / mL).

[0052] Fig. 22Shown are the stabilities of the indicated DDX polyplex preparations when incubated in mammalian bladders.

[0053] Fig.23 Shown are the filtration rates of the indicated DDX polyplex preparations under the indicated conditions when tested at small scale.

[0054] Fig.24 Shown are the filtration rates of the indicated DDX polyplex preparations under the indicated conditions when tested at pilot scale.

[0055] Fig.25 Shown are the stability of the indicated DDX multiplex formulations after freeze-thaw (FT) and lyophilization / rehydration (FD) as indicated by nanoparticle size (nm), zeta potential (mV), and % supercoiled content (%SC).

[0056] Fig.26 In vivo transfection of the canine small intestine by delivering PEGylated doubly derivatized (DDX) chitosan DNA polyplexes directly to the small intestine is shown.

[0057] Fig. 27 It is shown that when the aqueous polyplex formulation is mixed with a low pH acetate buffer at a ratio sufficient to lower the pH to below the pKa of the PEG-polyglutamic acid polymer (approximately 4.25), the zeta potential (mv) increases significantly, indicating that the PEGylated polyplex is not coated at pH values ​​below 4.

[0058] Fig.28 Particle sizes of reversibly PEGylated polyplexes formed at various N:P:A ratios as indicated are shown.Different polyglutamic acid (PLE) polyanion anchor regions were tested: PLE5 (polyglutamic acid region of 5 glutamic acids), PLE10 (polyglutamic acid region of 10 glutamic acids), and PLE25 (polyglutamic acid region of 25 glutamic acids).

[0059] Fig.29 The pH values ​​of aqueous dispersions of the indicated reversibly PEGylated polyplexes are shown.

[0060] Fig.30 Zeta potential measurements of the indicated reversibly PEGylated polyplexes are shown.

[0061] Fig.31The results of polyaspartic acid (PAA) challenge of reversibly PEGylated polyplexes are shown. Nucleic acid release was monitored as a function of PAA concentration. Different polyglutamic acid (PLE) polyanion anchor regions of PEG-PLE were tested: PLE5 (polyglutamic acid region of 5 glutamic acids), PLE10 (polyglutamic acid region of 10 glutamic acids), and PLE25 (polyglutamic acid region of 25 glutamic acids) at different NPA ratios.

[0062] Fig.32 The PAA levels required for DNA release from PEGylated polyplexes are shown. Nucleic acids in PEGylated polyplexes made with PEG-PLE25 were more loosely bound than those in polyplexes made with PEG-PLE5 or PEG-PLE10.

[0063] Fig.33 Shown are the effects of fasted state simulated intestinal fluid (FaSSIF v2) on the particle size and polydispersity index (PDI) of the indicated PEGylated polyplexes.

[0064] Fig.34 Shown are the effects of fasted state simulated intestinal fluid (FaSSIF v2) on the zeta potential of the indicated PEGylated polyplex particles and the pH of the resulting aqueous dispersions.

[0065] Fig.35 The stability of PEGylated polyplexes (PLE10, PLD10 and PLD50) after freeze-thaw as measured by particle size is shown. The PEG-PLD50 polyplex at N:P:A of 7:1:30 exhibited an increase in particle size after freeze-thaw.

[0066] Fig.36 The stability of PEGylated polyplexes (PLE10, PLD10 and PLD50) after freeze-thaw is shown as measured by particle size (top) and zeta potential (middle). The PEG-PLD50 polyplexes at N:P:A of 7:1:30 show increased particle size and lower zeta potential after freeze-thaw. When the P:A ratio decreases (A increases) and when the number of anionic subunits increases (bottom), the indicated polyplexes show an increase in pH as aqueous dispersions.

[0067] Fig.37 Shown are the results of analysis of reversibly PEGylated polyplexes by detection of uncomplexed nucleic acids by agarose gel electrophoresis.

[0068] Fig.38 Schematic diagram showing the expected behavior of reversibly PEGylated (top row) and covalently PEGylated polyplexes (bottom row) at different pH values.

[0069] Fig.39 Shown are the solution behaviors of a reversibly PEGylated polyplex (top row) and two different covalently PEGylated polyplexes (middle and bottom rows) at two pH values, 2 and 6, and in response to a polyaspartic acid (PAA) challenge.

[0070] Fig.40 Shown is the transfection efficiency of the reverse PEGylated polyplexes compared to the non-PEGylated polyplexes.

[0071] Fig.41 The transfection efficiency of the reverse PEGylated polyplexes compared to the covalently PEGylated polyplexes is shown.

[0072] Fig.42 The transfection efficacy of reversibly PEGylated polyplexes produced in either a one-step or two-step approach following intracolonic delivery into mice is shown.

[0073] Fig.43 The transfection efficacy of the reversibly PEGylated polyplexes produced in either the one-step or two-step process after intravesicular administration into mice is shown. DETAILED DESCRIPTION

[0074] 1. Definition

[0075] Unless otherwise defined, all domain terms, notations and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for clarity and / or ease of reference, the terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein should not be interpreted as indicating that they are different from those commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and are commonly used by those skilled in the art using conventional methods, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and widely used molecular cloning techniques. Unless otherwise stated, when appropriate, the procedures including the use of commercially available kits and reagents are generally implemented according to the schemes and / or parameters specified by the manufacturer.

[0076] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0077] The term "about" indicates and encompasses a specified value and a range above and below that value. In certain embodiments, the term "about" indicates a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where indicated, the term "about" indicates a specified value ± one standard deviation of that value.

[0078] The term "combinations thereof" includes every possible combination of the elements referred to by the term.

[0079] In certain embodiments, "treating" or "treatment" of any disease or condition refers to improving a disease or condition present in a subject. In another embodiment, "treating" includes improving at least one physical parameter that may not be discernible to the subject. In yet another embodiment, "treating" includes regulating a disease or condition physically (e.g., stabilization of discernible symptoms) or physiologically (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treating" includes delaying or preventing the onset of a disease or condition.

[0080] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an antibody or composition that is effective for treating a disease or disorder when administered to a subject.

[0081] As used herein, the term "subject" refers to 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 suffers from cancer, an autoimmune disease or condition, and / or an infection that can be treated with the antibodies provided herein. In some embodiments, the subject is a person suspected of suffering from cancer, an autoimmune disease or condition, and / or an infection.

[0082] "Chitosan" is a partially or fully deacetylated form of chitin, a polymer of N-acetylglucosamine. Chitosan having any degree of deacetylation greater than 50% is used in the present invention.

[0083] Chitosan can be derivatized by functionalizing free amino groups at the deacetylation site. Derivatized chitosan described herein has many properties that are conducive to nucleic acid delivery vehicles, including: they effectively bind negatively charged nucleic acids and compound with them, they can form nanoparticles of controlled size, they can be absorbed by cells, and they can release nucleic acids at the appropriate time in cells. Chitosan has any final functionalization degree between 1% and 50%. (The functionalization percentage is determined relative to the number of free amino moieties on the chitosan polymer before functionalization or when there is no functionalization.) The degree of deacetylation and the final degree of functionalization give the functionalized chitosan derivatives a specific charge density.

[0084] The polyols according to the invention may have a backbone of 3, 4, 5, 6 or 7 carbons and may have at least 2 hydroxyl groups. Such polyols or combinations thereof may be used for conjugation to a chitosan backbone, such as chitosan that has been functionalized with a cationic moiety (e.g., a molecule containing an amino group, such as lysine, ornithine, a molecule containing a guanidinium group, arginine or a combination thereof).

[0085] The term "C2-C6 alkylene" as used herein refers to a straight or branched divalent hydrocarbon group optionally containing one or more carbon-carbon multiple bonds. For the avoidance of doubt, the term "C2-C6 alkylene" as used herein encompasses divalent groups of alkanes, alkenes and alkynes.

[0086] As used herein, the terms "peptide" and "polypeptide" are used interchangeably unless otherwise indicated.

[0087] 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 mimetics that retain the 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 substitution or modification of side chains or functional groups.

[0088] The term "acidic amino acid" refers to a naturally or non-naturally occurring amino acid having a side chain that is negatively charged in an aqueous buffer at pH 7. Non-limiting examples of acidic amino acids are aspartic acid and glutamic acid.

[0089] As known in the art, a peptide mimetic may have one or more peptide bonds substituted by a surrogate bond. As known in the art, part or all of the peptide backbone may also be substituted by a conformationally restricted cyclic alkyl or aryl substituent to limit the mobility of the functional amino acid side chains.

[0090] The polypeptide of the present invention can be produced by recognized methods, such as recombinant and synthetic methods well known in the art. The technology of synthesizing peptides is well known and includes the technology 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).

[0091] As used herein, "linear polypeptide" refers to a polypeptide lacking branching groups covalently attached to its constituent amino acid side chains. As used herein, "branched polypeptide" refers to a polypeptide comprising branching groups covalently attached to its constituent amino acid side chains.

[0092] As used herein, the "final functionalization degree" of cations or polyols refers to the percentage of cationic (e.g., amino) groups functionalized with cations (e.g., amino) or polyols on the chitosan backbone, respectively. Therefore, "α:β ratio", "final functionalization degree ratio" (e.g., Arg final functionalization degree: polyol final functionalization degree ratio), etc. can be used interchangeably with the terms "molar ratio" or "quantity ratio".

[0093] A dispersed system consists of particulate matter, called the dispersed phase, distributed throughout a continuous medium. A "dispersion" of chitosan nucleic acid polyplexes is a composition comprising hydrated chitosan nucleic acid polyplexes, wherein the polyplexes are distributed throughout a medium.

[0094] As used herein, a "pre-concentration" dispersion is a dispersion that has not been subjected to a concentration process to form a concentrated dispersion.

[0095] As used herein, "substantially free" of polyplex precipitates means that the composition is substantially free of particles observable upon visual inspection.

[0096] As used herein, physiological pH refers to a pH between 6 and 8.

[0097] "Chitosan nucleic acid polyplex" or its grammatical equivalents means a complex comprising a plurality of chitosan molecules and a plurality of nucleic acid molecules. In a preferred embodiment, the (eg doubly) derivatized chitosan is complexed with the nucleic acid.

[0098] As used herein, the term "polyethylene glycol" ("PEG") is intended to mean a polymer of ethylene oxide having repeating units of -(CH2CH2-O)- and the general formula HO-(CH2CH2-O)nH.

[0099] As used herein, the term "monomethoxypolyethylene glycol" ("mPEG") is intended to refer to a polymer of ethylene oxide having repeating units of -(CH2CH2-O)- and the general formula CH3O-(CH2CH2-O)nH, such as a PEG terminated at one end with a methoxy group.

[0100] 2. Composition

[0101] Provided herein are chitosan compositions comprising chitosan derivative nucleic acid nanoparticles (polycomplexes) coated with diblock and / or triblock copolymers, wherein a single polymer molecule comprises a negatively charged anchor region and one or more uncharged hydrophilic tail regions. Exemplary negatively charged anchor regions include polyanionic anchor regions comprising repeating units, wherein the repeating units include one or more negatively charged subunits, such as one or more acidic amino acids. Exemplary hydrophilic tail regions include, but are not limited to, PEG tail regions and derivatives thereof, polyvinyl alcohol tail regions and derivatives thereof, polyoxazoline tail regions and derivatives thereof, polysarcosine tail regions and derivatives thereof, poly(N-(2-hydroxypropyl) methacrylamide) (pHPMA) tail regions and derivatives thereof, and combinations thereof.

[0102] An exemplary polymer molecule useful in the methods and compositions of the invention is a "PEG-PA" polymer molecule comprising a polyethylene glycol (PEG) portion and a polyanion (PA) portion.

[0103] Chitosan

[0104] The chitosan component 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 referred to as double derivatized chitosan (DD-chitosan). Exemplary DD-chitosan is functionalized with a hydrophilic moiety (e.g., polyol) and a cationic functional group (e.g., amino). Exemplary chitosan derivatives are also described in, for example, US2007 / 0281904 and US2016 / 0235863, each of which is incorporated herein by reference.

[0105] In one embodiment, the doubly derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 50%. In one embodiment, the degree of deacetylation is at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. In a preferred embodiment, the doubly derivatized chitosan described herein comprises chitosan having a degree of deacetylation of at least 98%.

[0106] Chitosan derivatives described herein have an average molecular weight range that is soluble at neutral and physiological pH values, and for purposes of the present invention, include molecular weights within the range of 3-110 kDa. Embodiments described herein are characterized in that the average molecular weight of the derivatized chitosan is relatively low (<25 kDa, e.g., about 5 kDa to about 25 kDa), which can have desirable delivery and transfection characteristics, and is small in size and has good solubility. Derivatized chitosans with lower average molecular weights are generally more soluble than derivatized chitosans with higher molecular weights, and the former therefore produce nucleic acid / chitosan complexes that are more easily released nucleic acids and increase cell transfection. Many documents are devoted to optimizing all these parameters of chitosan-based delivery systems.

[0107] The skilled person will recognize that chitosan refers to a plurality of molecules having a structure of formula I, wherein n is any integer, and each R1 is independently selected from acetyl or hydrogen, wherein the degree to which R1 is selected from hydrogen is between 50% and 100%. In addition, chitosan referred to as having an average molecular weight of, for example, 3kD to 110kD generally refers to a plurality of chitosan molecules having a weight average molecular weight of, for example, 3kD to 110kD, respectively, wherein each chitosan molecule may have a different chain length (n+2). It is also well known that chitosan referred to as "n-polymer chitosan" does not necessarily include chitosan molecules of formula I, wherein each chitosan molecule has a chain length of n+2. In contrast, "n-polymer chitosan" as used herein refers to a plurality of chitosan molecules, each of which may have a different chain length, wherein the average molecular weight of the plurality of chitosan molecules is substantially similar to or equal to a chitosan molecule having a chain length of n. For example, a 24-mer chitosan may comprise a plurality of chitosan molecules, each molecule having a different chain length in the range of, for example, 7-50, but having a weight average molecular weight substantially similar to or equal to a chitosan molecule having a chain length of 24.

[0108] The double derivatized chitosan of the present invention can also be functionalized with polyols or hydrophilic functional groups such as polyols. Without wishing to be bound by theory, it is assumed that functionalization with hydrophilic groups such as polyols may help increase the hydrophilicity of chitosan (including arginine chitosan) and / or may provide hydroxyl groups. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticles is or comprises gluconic acid. See, for example, WO 2013 / 138930. In some embodiments, the hydrophilic functional group of the chitosan derivative nanoparticles is or comprises glucose. Additionally or alternatively, the hydrophilic functional group may comprise a polyol. See, for example, US2016 / 0235863. Exemplary polyols for chitosan functionalization are further described below.

[0109] Functionalized chitosan derivatives described herein include dual derivatized chitosan compounds, such as cation-chitosan-polyol compounds. Typically, the cation-chitosan-polyol compounds are functionalized with amino-containing moieties such as arginine, lysine, ornithine, or molecules containing guanidine salts, or combinations thereof. In certain embodiments, the cation-chitosan-polyol compounds have the following formula I structure:

[0110]

[0111] Where n is an integer from 1 to 650,

[0112] α is the final degree of functionalization of the cationic moiety (e.g., a molecule comprising an amino group, such as lysine, ornithine, a molecule comprising a guanidinium group, arginine, or a combination thereof),

[0113] β is the final degree of functionalization of the polyol; and

[0114] Each R 1 Independently selected from hydrogen, acetyl, cations (eg, arginine), and polyols.

[0115] Preferably, the doubly derivatized chitosan of the present invention may be functionalized with the cationic amino acid arginine.

[0116] In one embodiment, the chitosan derivative nanoparticles include chitosan coupled to gluconic acid with a final functionalization degree of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30% or more. In one embodiment, the chitosan derivative nanoparticles include chitosan coupled to glucose with a final functionalization degree of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30% or more. In one embodiment, the chitosan derivative nanoparticles include chitosan coupled to a cationic moiety (e.g., arginine) with a final functionalization degree of about 1% to about 25%. In one embodiment, the chitosan derivative nanoparticles include chitosan coupled to a cationic moiety (e.g., arginine) with a final functionalization degree of about 10% to about 40%.

[0117] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 10% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 20% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 25% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 25% to about 30%, preferably 28%.

[0118] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 15% to about 40%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 15% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 15% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 15% to about 28%.

[0119] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 10% to about 35%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 10% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 10% to about 28%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cationic moiety (e.g., arginine) having a final degree of functionalization of about 28%.

[0120] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled with gluconic acid having a final functionalization degree of about 2% to about 30%, about 5% to about 30%, about 7.5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled with gluconic acid having a final functionalization degree of about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 15%, or about 7.5% to about 12%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled with gluconic acid having a final functionalization degree of about 10%.

[0121] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a hydrophilic polyol having a final functionalization degree of about 2% to about 30%, about 5% to about 30%, about 7.5% to about 30%, about 5% to about 25%, about 5% to about 22%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a hydrophilic polyol having a final functionalization degree of about 7.5% to about 25%, about 7.5% to about 20%, about 7.5% to about 15%, or about 7.5% to about 12%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a hydrophilic polyol having a final functionalization degree of about 10%.

[0122] In one embodiment, the chitosan derivative nanoparticles include chitosan coupled to glucose having 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 include chitosan coupled to glucose having 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 include chitosan coupled to glucose having a final functionalization degree of about 10%.

[0123] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 2% to about 40% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 5% to about 40% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 7.5% to about 40% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 10% to about 40% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 15% or about 10%.

[0124] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 2% to about 35% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 5% to about 35% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 7.5% to about 35% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 10% to about 35% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 15% or about 10%.

[0125] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 10% to about 30% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 2% to about 30%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 12% to about 30% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 5% to about 25%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 14% to about 30% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 15% to about 30% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 15% or about 10%.

[0126] In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 25% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 15%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 28% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 7.5% to about 15%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 25% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 5% to about 20%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (eg, arginine) with a final functionalization degree of about 28% and a hydrophilic polyol (eg, glucose or gluconic acid) with a final functionalization degree of about 5% to about 20%.

[0127] In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 14% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 10%. In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 15% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 12%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to arginine having a final functionalization degree of about 14% and glucose having a final functionalization degree of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to arginine having a final functionalization degree of about 15% and glucose having a final functionalization degree of about 12%.

[0128] In a preferred embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to a cation (e.g., arginine) having a final functionalization degree of about 28% and a hydrophilic polyol (e.g., glucose or gluconic acid) having a final functionalization degree of about 10%. In another preferred embodiment, the chitosan derivative nanoparticles comprise chitosan coupled to arginine having a final functionalization degree of about 28% and glucose having a final functionalization degree of about 10%.

[0129] In some embodiments, when appropriate, DD-chitosan includes DD-chitosan derivatives, for example, DD-chitosans incorporating additional functionalizations, for example DD-chitosans to which ligands are attached. It will be understood that "derivatives" include a broad class of chitosan-based polymers, which include covalently modified N-acetyl-D-glucosamine and / or D-glucosamine units, and chitosan-based polymers incorporating other units or being connected to other parts. Derivatives are typically based on modifications of the hydroxyl or amine groups of glucosamine, for example, modifications under arginine-functionalized chitosan. Examples of chitosan derivatives include, but are not limited to, trimethylated chitosan, pegylated chitosan, thiolated chitosan, galactosylated chitosan, alkylated chitosan, chitosan incorporating PEI, chitosan modified by uronic acid, glycol chitosan, etc. For further teaching on chitosan derivatives, see, for example, "Non-viral Gene Therapy", K. Taira, K. Kataoka, T. Niidome (eds.), 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.

[0130] 2.2. Chitosan nucleic acid polyplex

[0131] Chitosan derivative nanoparticle compositions generally contain at least one nucleic acid molecule, and preferably a plurality of such nucleic acid molecules. Typical nucleic acid molecules include phosphorus as a component of the nucleic acid backbone, for example in the form of a plurality of phosphodiester or derivatives thereof (e.g., phosphorothioate). The ratio of cationically functionalized chitosan derivatives to nucleic acids can be characterized by a cation (+) to phosphorus (P) molar ratio, wherein (+) refers to the cation of the cationically functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, (+): (P) molar ratios are selected so that the chitosan derivative-nucleic acid complex has a positive charge in the absence of PEG-PA polymer molecules. Therefore, (+): (P) molar ratios are generally greater than 1. In preferred embodiments, (+): (P) molar ratios are greater than 1.5, at least 2, or greater than 2. In certain preferred embodiments, (+): (P) molar ratios are greater than 2.

[0132] In some cases, the (+):(P) molar ratio is or is about 3: 1. In some cases, the (+):(P) molar ratio is or is about 4: 1. In some cases, the (+):(P) molar ratio is or is about 5: 1. In some cases, the (+):(P) molar ratio is or is about 6: 1. In some cases, the (+):(P) molar ratio is or is about 7: 1. In some cases, the (+):(P) molar ratio is or is about 8: 1. In some cases, the (+):(P) molar ratio is or is about 9: 1. In some cases, the (+):(P) molar ratio is or is about 10: 1.

[0133] In some cases, the (+):(P) molar ratio is greater than 1 to at most about 20: 1, about 2 to at most about 20: 1, or about 2 to at most about 10: 1. In some cases, the (+):(P) molar ratio is greater than about 2 to at most about 20: 1, or greater than about 2 to at most about 10: 1. In some cases, the (+):(P) molar ratio is about 3 to at most about 20: 1, about 3 to at most about 10: 1, about 3 to at most about 8: 1, or about 3 to at most about 7: 1. In some cases, the (+):(P) molar ratio is about 3 to at most 20: 1, about 3 to at most about 10: 1, about 3 to at most about 8: 1, or about 3 to at most about 7: 1.

[0134] 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 to less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 2 to less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than or equal to 3 to less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than or equal to 5 to less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than or equal to 7 to less than or equal to 100:1. In some cases, the (+):(P) molar ratio may be greater than 2 to less than or equal to 50:1. In some cases, the (+):(P) molar ratio may be greater than or equal to 3 to less than or equal to 50:1. In some cases, the (+):(P) molar ratio may be greater than or equal to 5 to less than or equal to 50:1. In some cases, the (+):(P) molar ratio may be greater than or equal to 7 to less than or equal to 50: 1. In some cases, the (+):(P) molar ratio may be greater than or equal to 2 to less than or equal to 25: 1. In some cases, the (+):(P) molar ratio may be greater than or equal to 3 to less than or equal to 25: 1. In some cases, the (+):(P) molar ratio may be greater than or equal to 5 to less than or equal to 25: 1. In some cases, the (+):(P) molar ratio may be greater than or equal to 7 to less than or equal to 25: 1.

[0135] In some embodiments, the cationic functional group of the chitosan derivative nanoparticle is or comprises an amino group. Examples of such amino-functionalized chitosan derivative nanoparticles include, but are not limited to, nanoparticles containing chitosan functionalized with: guanidine or molecules comprising guanidine groups, lysine, ornithine, arginine, or combinations thereof. In a preferred embodiment, the cationic functional group is arginine. The ratio of amino-functionalized chitosan derivatives to nucleic acids can be characterized by an amino (N) to phosphorus (P) molar ratio, wherein (N) refers to the nitrogen atom of the amino group in the amino-functionalized chitosan derivative and (P) refers to the phosphorus of the nucleic acid backbone. Typically, the N:P molar ratio is selected so that the chitosan derivative-nucleic acid complex has a positive charge at a physiologically relevant pH value in the absence of a PEG-PA polymer molecule. Therefore, the N:P molar ratio is generally greater than 1. In a preferred embodiment, 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 n is greater than 2.

[0136] In some cases, the N:P molar ratio is or is about 3: 1. In some cases, the N:P molar ratio is or is about 4: 1. In some cases, the N:P molar ratio is or is about 5: 1. In some cases, the N:P molar ratio is or is about 6: 1. In some cases, the N:P molar ratio is or is about 7: 1. In some cases, the N:P molar ratio is or is about 8: 1. In some cases, the N:P molar ratio is or is about 9: 1. In some cases, the N:P molar ratio is or is about 10: 1.

[0137] In some cases, the N:P molar ratio is greater than 1 to at most about 20: 1, about 2 to at most about 20: 1, or about 2 to at most about 10: 1. In some cases, the N:P molar ratio is greater than about 2 to at most about 20: 1, or greater than about 2 to at most about 10: 1. In some cases, the N:P molar ratio is about 3 to at most about 20: 1, about 3 to at most about 10: 1, about 3 to at most about 8: 1, or about 3 to at most about 7: 1. In some cases, the N:P molar ratio is about 3 to at most 20: 1, about 3 to at most about 10: 1, about 3 to at most about 8: 1, or about 3 to at most about 7: 1.

[0138] 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 to less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than 2 to less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than or equal to 3 to less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than or equal to 5 to less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than or equal to 7 to less than or equal to 100:1. In some cases, the N:P molar ratio may be greater than 2 to less than or equal to 50:1. In some cases, the N:P molar ratio may be greater than or equal to 3 to less than or equal to 50:1. In some cases, the N:P molar ratio may be greater than or equal to 5 to less than or equal to 50:1. In some cases, the N:P molar ratio may be greater than or equal to 7 to less than or equal to 50:1. In some cases, the N:P molar ratio may be greater than 2 to less than or equal to 25: 1. In some cases, the N:P molar ratio may be greater than or equal to 3 to less than or equal to 25: 1. In some cases, the N:P molar ratio may be greater than or equal to 5 to less than or equal to 25: 1. In some cases, the N:P molar ratio may be greater than or equal to 7 to less than or equal to 25: 1.

[0139] In a preferred embodiment, the subject polyplex has an amine to phosphate (N / P) ratio of 2 to 100, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 5. Preferably, the N / P ratio is inversely proportional to the molecular weight of the chitosan, i.e., a lower molecular weight (e.g., doubly) derivatized chitosan requires a higher N / P ratio, and vice versa.

[0140] Nucleic acids of the present invention generally contain phosphodiester bonds, but in some cases include nucleic acid analogs that may have replacement backbones or other modifications or parts that are incorporated for any of a variety of purposes, such as stability and protection. Other nucleic acid analogs encompassed include nucleic acids with non-ribose backbones. In addition, naturally occurring nucleic acids, analogs, and mixtures of the two can be made. Nucleic acids can be single-stranded or double-stranded, or contain parts of double-stranded or single-stranded sequences. Nucleic acids include, but are not limited to, DNA, RNA, and hybrids, wherein nucleic acids include any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc. Nucleic acids include any form of DNA, any form of RNA, including triplexes, duplexes or single strands, antisense, siRNA, ribozymes, deoxyribozymes, polynucleotides, oligonucleotides, chimeras, microRNAs, and their derivatives. Nucleic acids include artificial nucleic acids, including but not limited to peptide nucleic acids (PNA), phosphorodiamidate morpholino oligonucleotides (PMO), locked nucleic acids (LNA), glycol nucleic acids (GNA) and threose nucleic acids (TNA). It should be understood that for artificial nucleic acids that do not contain phosphorus, an equivalent measure of the (+):P or N:P ratio can be estimated by the number of nucleotide (or nucleotide analog) bases.

[0141] In a preferred embodiment, the multimeric complex 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 multimeric complex 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.

[0142] In a preferred embodiment, the polyplex comprises 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 comprises 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.

[0143] Chitosan and (eg, doubly) derivatized chitosan nucleic acid polyplexes can be prepared by any method known in the art, including but not limited to those described herein.

[0144] Nucleic Acids

[0145] As described above, chitosan polyplexes can mean a variety of nucleic acids. In one embodiment, the nucleic acid component comprises a therapeutic nucleic acid. The subject (e.g., double) 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 are RNA molecules that can play a therapeutic role in mammalian cells. Therapeutic RNA includes, but is not limited to, messenger RNA, antisense RNA, siRNA, short hairpin RNA, microRNA, and enzyme-catalyzed RNA. Therapeutic nucleic acids include, but are not limited to, nucleic acids, protein-bound nucleic acids, ribozymes, deoxyribozymes, and small nucleotide molecules intended to form triplex molecules.

[0146] Many types of therapeutic RNA are known in the art. See, for example, Meng et al., Aτnew developing class of gene delivery: messenger RNA-based therapeutics, Biomater. Sci., 5, 2381-2392, 2017; Grimm et al., Therapeutic application of RNAi is mRNA targeting finally ready for prime time? J. Clin. Invest., 117: 3633-3641, 2007; Aagaard et al., RNAi therapeutics: Principles, prospects and challenges, Adv. Drug Deliv. Rev., 59: 75-86, 2007; Dorsett et al., siRNAs: Applications in functional genomics and potential as therapeutics, Nat. Rev. Drug Discov., 3: 318-329, 2004. These include double-stranded short interfering RNA (siRNA).

[0147] Therapeutic nucleic acids also include nucleic acids encoding therapeutic proteins, including cytotoxic proteins and prodrugs.

[0148] In a preferred embodiment, the nucleic acid component comprises a therapeutic nucleic acid construct. The therapeutic nucleic acid construct is a nucleic acid construct that can play a therapeutic role. The therapeutic nucleic acid construct may include nucleic acids encoding therapeutic proteins, and nucleic acids that produce transcripts as therapeutic RNA. Therapeutic nucleic acids can be used to achieve gene therapy by being used as a replacement or enhancement for defective genes, or to compensate for the lack of specific gene products by encoding therapeutic products. Therapeutic nucleic acids can also inhibit the expression of endogenous genes. Therapeutic nucleic acids can encode all or part of the translation products, and can play a role by recombination with DNA already present in the cell, thereby replacing the defective part of the gene. It can also encode a part of a protein and play its role by co-suppression of a gene product. In a preferred embodiment, the therapeutic nucleic acid is selected from those nucleic acids disclosed in US2011 / 0171314, which is explicitly incorporated herein by reference.

[0149] In a preferred embodiment, the therapeutic nucleic acid encodes a therapeutic protein selected from the group consisting of hormones, enzymes, cytokines, chemokines, antibodies, mitogenic factors, growth factors, differentiation factors, factors affecting angiogenesis, factors affecting blood clot formation, factors affecting blood sugar levels, factors affecting glucose metabolism, factors affecting lipid metabolism, factors affecting blood cholesterol levels, factors affecting blood LDL or HDL levels, factors affecting 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 encoding insulin, leptin, glucagon antagonists, GLP-1, GLP-2, growth hormone releasing peptide (Ghrelin), cholecystokinin, growth hormone, coagulation factors, PYY, erythropoietin, inflammation inhibitors, IL-10, IL-12, IL-17 antagonists, TNFα antagonists, growth hormone releasing hormone or parathyroid hormone.

[0150] 2.2.1.1. Expression control region

[0151] In a preferred embodiment, the multimeric complex of the invention comprises a therapeutic nucleic acid, which is a therapeutic construct comprising an expression control region operably linked to a coding region. The therapeutic construct produces a therapeutic nucleic acid, which itself may be therapeutic, or may encode a therapeutic protein.

[0152] In some embodiments, the expression control region of the therapeutic construct has constitutive activity. In many preferred embodiments, the expression control region of the therapeutic construct does not have constitutive activity. This provides dynamic expression of therapeutic nucleic acids. "Dynamic" expression refers to expression that changes over time. Dynamic expression can include several such low expression periods or no expression periods separated by detectable expression periods. In many preferred embodiments, the therapeutic nucleic acid is operably connected to a regulatable promoter. This provides adjustable expression of therapeutic nucleic acids.

[0153] Expression control regions contain regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which affect the expression of an operably linked therapeutic nucleic acid.

[0154] The expression control elements included herein can be from bacteria, yeast, plants or animals (mammals or non-mammals). The expression control region includes a full-length promoter sequence, such as a natural promoter and enhancer elements, and a subsequence or polynucleotide that retains all or part of the full length or non-variant function (e.g., retaining a certain amount of nutritional regulation or cell / tissue-specific expression). As used herein, the term "function" and its grammatical variants, when used to refer to a nucleic acid sequence, a subsequence or a fragment, means that the sequence has one or more functions of a natural nucleic acid sequence (e.g., a non-variant or unmodified sequence). As used herein, the term "variant" means a sequence substitution, deletion or addition, or other modification (e.g., a chemical derivative, such as a modified form resistant to nucleases).

[0155] As used herein, the term "operably linked" refers to the physical juxtaposition of the components described so as to allow them to function in their intended manner. In the example of an expression control element operably linked to a nucleic acid, the relationship causes the control element to regulate the expression of the nucleic acid. Typically, the expression control region that regulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream"). The expression control region may also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., in an intron). The expression control element may be located at a distance from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000 or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is typically located at the 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which may be located at the 5' or 3' of the transcribed sequence, or within the transcribed sequence.

[0156] Some expression control regions confer regulatable expression of operably linked therapeutic nucleic acids. A signal (sometimes referred to as a stimulus) can increase or decrease the expression of a therapeutic nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are generally referred to as inducible. Such expression control regions that decrease expression in response to a signal are generally referred to as inhibitory. Typically, the amount of increase or decrease conferred by these elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0157] Many regulatable promoters are known in the art. Preferred inducible expression control regions include those containing inducible promoters stimulated with small molecule compounds. In one embodiment, the expression control region is responsive to a chemical substance that can be delivered orally but is not normally present in food. Specific examples can be found in, for example, U.S. Patent Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941.

[0158] In one embodiment, the therapeutic construct further comprises an integration sequence. In one embodiment, the therapeutic construct comprises a single integration sequence. In another embodiment, the therapeutic construct comprises a first and a second integration sequence for integrating the therapeutic nucleic acid or a portion thereof into the target cell genome. In a preferred embodiment, the integration sequence is functional in combination with an integration means selected from the group consisting of Sailor, Sleeping Beauty, FLP, Cre, ΦC31, R, λ, and integration means for integrating viruses such as AAV, retroviruses, and lentiviruses.

[0159] In one embodiment, the subject composition comprises a non-therapeutic construct in addition to the therapeutic construct, wherein the non-therapeutic construct comprises a nucleic acid sequence encoding an integration means operably linked to a second expression control region. The second expression control region and the expression control region operably linked to the therapeutic nucleic acid may be the same or different. The encoded integration means are preferably selected from the group consisting of: Sailor, Sleeping Beauty, FLP, Cre, ΦC31, R, λ, and integration means for integrating viruses such as AAV, retroviruses, and lentiviruses.

[0160] For further teachings see WO 2008 / 020318, the entire contents of which are expressly incorporated herein by reference. In one embodiment, the nucleic acid of the (eg doubly) derivatized chitosan nucleic acid polyplex is an artificial nucleic acid.

[0161] Preferred artificial nucleic acids include, but are not limited to, peptide nucleic acids (PNA), phosphorodiamidate morpholino oligonucleotides (PMO), locked nucleic acids (LNA), glycol nucleic acids (GNA), and threose nucleic acids (TNA).

[0162] 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 catalytic RNA.

[0163] In some embodiments, the therapeutic nucleic acid is DNA.

[0164] In one embodiment, the therapeutic nucleic acid comprises a nucleic acid sequence encoding a therapeutic protein.

[0165] 2.3. Polyols

[0166] Chitosan derivative nanoparticles can be functionalized with polyols. The polyols useful in the present invention are generally hydrophilic. In some cases, chitosan derivative nanoparticles are functionalized with cationic components such as amino groups and with polyols. Such chitosan derivative nanoparticles functionalized with cationic moieties such as amino groups and polyols are referred to as "double derivatized chitosan nanoparticles".

[0167] In some embodiments, the chitosan derivative nanoparticles comprise a polyol of Formula II:

[0168]

[0169] in:

[0170] R 2 Selected from: H and hydroxyl;

[0171] R 3 is selected from: H and hydroxyl; and

[0172] X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxy substituents.

[0173] In some embodiments, the chitosan derivative nanoparticles are functionalized with a polyol of formula II, wherein R 2 Selected from: H and hydroxyl; R 3 is selected from: H and hydroxy; and X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxy substituents.

[0174] In some embodiments, the chitosan derivative nanoparticles comprise a polyol of Formula III:

[0175]

[0176] in:

[0177] -----Y is =O or -H2;

[0178] R 2 Selected from: H and hydroxyl;

[0179] R 3 Selected from: H and hydroxyl;

[0180] X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxy substituents; and

[0181] represents the bond between the polyol and the derivatized chitosan.

[0182] In one embodiment, the polyols according to the present invention with 3 to 7 carbons may have one or more carbon-carbon multiple bonds. In a preferred embodiment, the polyols according to the present invention contain carboxyl groups. In a further preferred embodiment, the polyols according to the present invention contain aldehyde groups. The skilled person will recognize that when the polyols according to the present invention contain aldehyde groups, such polyols encompass open-chain conformations (aldehydes) and cyclic conformations (hemiacetals).

[0183] Non-limiting examples of polyols include gluconic acid, threonic acid, glucose and threose. Examples of other such polyols that may have carboxyl and / or aldehyde groups or may be in sugar or acid form are described in more detail in U.S. Patent No. 10,046,066, the disclosure of which is expressly incorporated herein by reference. The skilled artisan will recognize that polyols are not limited to specific stereochemistry.

[0184] In a preferred embodiment, the polyol may be selected from the group consisting of: 2,3-dihydroxypropionic acid; 2,3,4,5,6,7-hexahydroxyheptanal; 2,3,4,5,6-pentahydroxyhexanal; 2,3,4,5-tetrahydroxyhexanal; and 2,3-dihydroxypropanal.

[0185] In a preferred embodiment, the polyol may be selected from the group consisting of D-glyceric acid, L-glyceric acid, L-glyceryl-D-mannoheptose, D-glyceryl-L-mannoheptose, D-glucose, L-glucose, D-fucose, L-fucose, D-glyceraldehyde and L-glyceraldehyde.

[0186] In some embodiments, the polyol may be a compound of Formula IV or Formula V:

[0187]

[0188] In a preferred embodiment, the polyol is a compound of formula IV. In some cases, the polyol of formula IV has been coupled to chitosan by reductive amination.

[0189] Hydrophilic polyols having carboxyl groups can be coupled to chitosan or cationically functionalized chitosan, such as amine-functionalized chitosan (e.g., Arg-coupled chitosan (Arg-chitosan)). In some embodiments, the polyols are coupled at a reaction pH of 6.0±0.3. At this pH, the carboxylic acid groups of the hydrophilic polyols may be attacked by uncoupled amines on the chitosan backbone according to a nucleophilic substitution reaction mechanism.

[0190] Hydrophilic polyols that are natural sugars can be coupled to chitosan, for example, cationically functionalized chitosan, such as amine functionalized chitosan (eg, Arg-coupled chitosan (Arg-chitosan)), using reductive amination followed by reduction with NaCBH3 or NaBH.

[0191] 2.4. Polymer:Polymer Complex Composition

[0192] Chitosan polymer complexes can be mixed with a variety of polymers, the polymers comprising a hydrophilic uncharged portion and a negatively charged (anionic) portion. As described above, the chitosan polymer complex is formulated to have a positive charge in the absence of a polymer containing an anionic portion or before being compounded with a polymer containing an anionic portion. Therefore, under suitable conditions, the polymer component will form a reversible charge: charge complex with the chitosan derivative nucleic acid polymer complex. In some embodiments, the polymer of the polymer component is unbranched. In some embodiments, the polymer is branched. In some cases, the polymer component comprises a mixture of branched and unbranched polymers.

[0193] In some embodiments, the polymer component is released from the chitosan polyplex after administration, after entering the cell, and / or after endocytosis. Without wishing to be bound by theory, it is hypothesized that the polyplex:polymer composition formed by complexing the polyplex and the polymer containing the anionic moiety can provide improved in vitro, solution, and / or in vivo stability without significantly affecting transfection efficiency. In some embodiments, the polyplex:polymer composition thus formed can provide reduced mucoadhesive properties compared to, for example, an otherwise identical polyplex without the polymer component.

[0194] In a preferred embodiment, the polyplex: polymer composition has a low net positive zeta potential, a neutral zeta potential, or a net negative zeta potential (about +10 mV to about -20 mV) at physiological pH. Such compositions can reduce aggregation under physiological conditions and reduce nonspecific binding to anionic components that are ubiquitous in the body. Such properties can enhance the migration of such compositions (e.g., enhanced mucus diffusion) to contact cells and enhance the intracellular release of nucleic acids.

[0195] In a preferred embodiment, the average hydrodynamic diameter of the polyplex: polymer particle composition is less than 1000nm, more preferably less than 500nm and most preferably less than 200nm. In certain embodiments, the average hydrodynamic diameter of the polyplex: polymer particle composition is 50nm to at most 1000nm, preferably 50nm to at most 500nm and most preferably 50nm to at most 200nm. In certain embodiments, the average hydrodynamic diameter of the polyplex: polymer particle composition is 50nm to at most 175nm, preferably 50nm to at most 150nm. In certain embodiments, the average hydrodynamic diameter of the polyplex: polymer particle composition is 75nm to at most 1000nm, preferably 75nm to at most 500nm and most preferably 75nm to at most 200nm. In certain embodiments, the average hydrodynamic diameter of the polyplex: polymer particle composition is 75nm to at most 175nm, preferably 75nm to at most 150nm. In certain embodiments, the average hydrodynamic diameter of the polyplex:polymer particle composition is greater than 100 nm and less than 175 nm.

[0196] In one embodiment, the polyplex:polymer composition has a % supercoiled DNA content of 80%, at least 80% or preferably 90%, more preferably at least 90%.

[0197] In one embodiment, the polyplex:polymer composition has an average zeta potential between +10 mV and -10 mV at physiological pH, and most preferably between +5 mV and -5 mV at physiological pH.

[0198] The polyplex:polymer composition is preferably homogeneous with respect to particle size. Thus, in a preferred embodiment, the composition has a low average polydispersity index ("PDI"). In a particularly preferred embodiment, the PDI of the dispersion of the polyplex:polymer composition is less than 0.5, more preferably less than 0.4, more preferably less than 0.3, still more preferably less than 0.25, and most preferably less than 0.2.

[0199] In some cases, the dispersion of the polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range after one or more freeze-thaw cycles. In some cases, the dispersion of the polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range after storage in solution at 4°C for at least 48 hours. In some cases, the dispersion of the polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range after storage in solution at 4°C for at least 1 week or 2 weeks.

[0200] In some cases, the dispersion of the polyplex: polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average 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 PDI, average zeta potential, supercoiled DNA % or average 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 PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range when concentrated (e.g., by ultrafiltration, such as tangential flow filtration) to a nucleic acid concentration of at least 250 μg / mL. In some cases, the dispersion of the polyplex: polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 1,000 μg / mL. In some cases, a dispersion of a polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 25,000 μg / mL. In some cases, a dispersion of a polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 2,000 μg / mL. In some cases, a dispersion of a polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, supercoiled DNA % or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 5,000 μg / mL. In some cases, a dispersion of a polyplex:polymer composition exhibits one or more of the above PDI, average zeta potential, % supercoiled DNA, or average particle size (nm) or size range when concentrated to a nucleic acid concentration of 125 μg / mL to about 10,000 μg / mL.

[0201] In general, as measured by PDI or average particle size, the polyplex: polymer compositions described herein exhibit favorable solution behavior (e.g., stability and / or lack of aggregation) even in the absence of excipients such as lyoprotectants, cryoprotectants, surfactants, rehydrating agents, or wetting agents. In some cases, as measured by PDI or average particle size, in physiological fluids or simulated physiological fluids, the polyplex: polymer compositions described herein exhibit favorable solution behavior (e.g., stability and / or lack of aggregation). For example, in some embodiments, when incubated in a mammalian bladder (e.g., and in contact with urine) and / or when incubated in the intestine (e.g., colon, small intestine, or large intestine, preferably colon), the polyplex: polymer compositions described herein are stable in simulated intestinal fluid, intestinal fluid, simulated urine, mammalian urine. In some embodiments, the polyplex: polymer composition is stable under one or more conditions described herein (e.g., in simulated intestinal fluid) for at least about 10 minutes, or about 10 minutes to about 1 hour, or stable for at least about 1 hour, or 1 hour to about 2 hours.

[0202] As described above, the polyplex: polymer compositions described herein are preferably substantially dimensionally stable in the composition. In a preferred embodiment, 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, and most preferably 48 hours at room temperature. In a particularly preferred embodiment, the composition of the present invention comprises polyplex: polymer particles whose average diameter increases by less than 25% over at least 24 hours or at least 48 hours at room temperature.

[0203] The polyplex: polymer particles of the subject composition are preferably substantially dimensionally stable under cooling conditions. In a preferred embodiment, 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, and most preferably 48 hours at 2-8°C.

[0204] Polyplexes of the subject composition: The polymer particles are preferably substantially dimensionally stable under freeze-thaw conditions. In a preferred embodiment, the composition of the present invention comprises a polyplex whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% at -20 to -80°C for 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours.

[0205] In a preferred embodiment, the composition has a nucleic acid concentration greater than 0.5 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 0.6 mg / ml, more preferably at least 0.75 mg / ml, more preferably at least 1.0 mg / ml, more preferably at least 1.2 mg / ml, and most preferably at least 1.5 mg / ml, and is substantially free of precipitated polyplexes. In another preferred embodiment, the composition has a nucleic acid concentration greater than 2 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 2.5 mg / ml, more preferably at least 5 mg / ml, more preferably at least 10 mg / ml, more preferably at least 15 mg / ml, and most preferably about 25 mg / ml, and is substantially free of precipitated polyplexes. In some embodiments, the composition has a nucleic acid concentration of 0.5 mg / mL to about 25 mg / mL and is substantially free of precipitated polyplexes. In some embodiments, the composition has a nucleic acid concentration of ≤ about 25 mg / mL and is substantially free of precipitated polyplexes. The composition can be hydrated. In a preferred embodiment, the composition is substantially free of uncomplexed nucleic acids.

[0206] In a preferred embodiment, the polyplex:polymer particle composition is isotonic. Achieving isotonicity while maintaining polyplex stability is highly desirable when formulating pharmaceutical compositions, and these preferred compositions are well suited for pharmaceutical formulation and therapeutic applications.

[0207] In certain embodiments, the polyplex: polymer particle composition may be uncoated to release all or part of, for example, a PEG polymer coating by lowering the pH. In certain embodiments, the polymer coating is released by incubating the particles at a pH lower than the pKa of the polyanion anchoring region of the polymer. For example, where the polymer coating is polyglutamic acid, the polymer coating may be released by incubating the particles at a pH lower than the pKa of polyglutamic acid (e.g., a pH less than about 4.25). In certain embodiments, the polymer coating may be released by incubating the particles at a pH lower than the pKa of the polyanion anchoring region of the polymer coating by at least 0.25 pH units or at least 0.5 pH units.

[0208] In certain embodiments, the polyplex:polymer particle compositions may be uncoated to release all or a portion of, for example, a PEG polymer coating by subjecting the particles to high ionic strength.

[0209] Without wishing to be bound by theory, it is hypothesized that certain physiological conditions can promote partial (e.g., >5%), major (>50%), extensive (e.g., >90%), or complete uncoating of the reversibly PEGylated chitosan DNA polyplexes described herein. For example, low pH conditions in certain subcellular compartments (e.g., endosomes, early endosomes, late endosomes, or lysosomes) can promote release of the polymer coating. As another example, certain extracellular conditions can promote partial (e.g., >5%), major (>50%), extensive (e.g., >90%), or complete (100%) uncoating of the reversibly PEGylated chitosan DNA polyplexes described herein. In some cases, high ionic strength and / or acidic pH conditions typically encountered at certain locations in the digestive tract can promote partial (e.g., >5%), major (>50%), extensive (e.g., >90%), or complete (100%) uncoating of the reversibly PEGylated chitosan DNA polyplexes described herein.

[0210] In certain embodiments, the PEGylated polyplex described herein is formulated for delivery to cells, tissues, or body compartments (e.g., intestines, small intestines, large intestines, colons, lungs, or bladders) such that the polyplexes remain PEGylated, thereby facilitating transfection of target cells. In some embodiments, the PEGylated polyplexes described herein partially (e.g., >5%), mostly (>50%), extensively (e.g., >90%), or completely (100%) release the polymer coating 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., intestines, small intestines, large intestines, colons, lungs, or bladders) such that the PEGylated polyplexes described herein partially (e.g., >5%), mostly (>50%), extensively (e.g., >90%), or completely (100%) release the polymer coating after delivery to cells, tissues, or body compartments (e.g., intestines, small intestines, large intestines, colons, lungs, or bladders).

[0211] It should be understood that the anionic charge density and / or pKa of the anionic anchoring region of the polymer can be adjusted to promote or inhibit release under the expected conditions. Similarly, it should be understood that the pH value, volume and ionic strength of the preparation and other conditions can be adjusted to promote or inhibit release under the expected conditions. For example, in order to be delivered to the intestinal tract through the gastric environment of the low pH value, the PEGylated polycomplex preparation can be enteric-coated and / or delivered in a buffer to increase the pH value of the gastric environment. Optimized reversible PEGylated particle compositions and preparations can be identified by measuring stability and transfection efficiency using the assay described herein.

[0212] Compositions comprising chitosan polyplexes complexed with polymers containing anionic moieties can be characterized by the ratio of cationic functional groups (+) of the (e.g., doubly) derivatized chitosan polyplexes to the anionic moieties (-) of the polymer (referred to as the "(+):(-) molar ratio"). The (+):(-) molar ratio can vary from greater than about 1:100 to less than about 10:1.

[0213] In certain embodiments, the (+):(-) molar ratio may be from greater than about 1:75 to less than about 8:1. In some cases, the (+):(-) molar ratio may be from greater than 1:10 to less than 10:1. In some cases, the (+):(-) molar ratio may be from or about 1:10 to or about 10:1. In some cases, the (+):(-) molar ratio may be from or about 1:8 to or about 8:1. In certain embodiments, the (+):(-) molar ratio may be from greater than 1:50 to less than about 10:1. In some cases, the (+):(-) molar ratio may be from greater than 1:25 to less than about 10:1. In some cases, the (+):(-) molar ratio may be from greater than 1:10 to less than about 7:1. In some cases, the (+):(-) molar ratio may be from greater than 1:8 to less than about 7:1. In some cases, the (+):(-) molar ratio may be from greater than 1:8 to less than about 6:1.

[0214] In certain embodiments, where the cationic functional groups of the (e.g., double) derivatized chitosan polyplex are amino moieties, the composition comprising the chitosan polyplex complexed with the polymer containing anionic moieties can be characterized by the ratio of the amino (N) moieties of the (e.g., double) derivatized chitosan polyplex to the anionic (A) moieties of the polymer (referred to as the "N:A molar ratio"). The N:A molar ratio can vary from greater than about 1:100 to less than about 10:1.

[0215] In certain embodiments, the N:A molar ratio may be from greater than about 1:75 to less than about 8:1. In some cases, the N:A molar ratio may be from greater than 1:10 to less than 10:1. In some cases, the N:A molar ratio may be from or about 1:10 to or about 10:1. In some cases, the N:A molar ratio may be from or about 1:8 to or about 8:1. In certain embodiments, the N:A molar ratio may be from greater than 1:50 to less than about 10:1. In some cases, the N:A molar ratio may be from greater than 1:25 to less than about 10:1. In some cases, the N:A molar ratio may be from greater than 1:10 to less than about 7:1. In some cases, the N:A molar ratio may be from greater than 1:8 to less than about 7:1. In some cases, the N:A molar ratio may be from greater than 1:8 to less than about 6:1.

[0216] Additionally or alternatively, a composition comprising a chitosan polyplex complexed with a polymer containing anionic moieties can be characterized by a three-component ratio of cationic functional groups (+) of the (e.g., doubly) derivatized chitosan polyplex to phosphorus atoms (P) of the nucleic acid to anionic moieties (-) of the polymer (referred to as the "(+):P:(-) molar ratio").

[0217] In certain embodiments, the molar ratio of (+):(-) may vary from at least 1:40 to about 40:1, where (+):P is at least 2:1 to at most 20:1. In certain embodiments, the molar ratio of (+):(-) may vary from at least 1:40 to about 1:10, where (+):P is at least 2:1 to at most 20:1. In some embodiments, the molar ratio of (+):(-) may vary from at least 1:25 to about 25:1, where (+):P is at least 2:1 to at most 20:1. In some embodiments, the molar ratio of (+):(-) may vary from at least 1:25 to about 1:10, where (+):P is at least 2:1 to at most 20:1. In some cases, the molar ratio of (+):(-) may vary from at least 1:20 to about 20:1, where (+):P is at least 2:1 to at most 20:1. In some cases, the molar ratio of (+):(-) may vary from at least 1:20 to about 1:10, where (+):P is at least 2:1 to at most 20:1. In some cases, the molar ratio of (+):(-) may vary from at least 1:10 to about 10:1, where (+):P is at least 2:1 to at most 20:1. In some cases, the molar ratio of (+):(-) may vary from at least 1:25 to about 2:1, where (+):P is at least 2:1 to at most 20:1. In some cases, the molar ratio of (+):(-) may vary from at least 1:20 to about 1:1, where (+):P is at least 2:1 to at most 20:1.

[0218] In certain preferred embodiments, (+):P:(-) is 3:1:3.5 to 3:1:17.5. In certain preferred embodiments, (+):P:(-) is 5:1:3.5 to 5:1:17.5. In certain preferred embodiments, (+):P:(-) is 7:1:3.5 to 7:1:17.5. In certain preferred embodiments, (+):P:(-) is about 3:1:3.5, 3:1:7, 3:1:10, 3:1:15, 3:1:17.5, or 3:1:20. In certain preferred embodiments, (+):P:(-) is about 5:1:3.5, 5:1:7, 5:1:10, 5:1:15, 5:1:17.5, or 5:1:20. In certain preferred embodiments, (+):P:(-) is about 7:1:3.5, 7:1:7, 7:1:10, 7:1:15, 7:1:17.5, or 7:1:20. In certain preferred embodiments, (+):P:(-) is about 10:1:10, 10:1:15, 10:1:20, 10:1:25, 10:1:30, or 10:1:40.

[0219] Those skilled in the art will appreciate that the amino-functionalized chitosan polyplex particles complexed with the polymer containing anionic moieties can be characterized by the three-component ratio of amino functional groups (N) of the (e.g., double) derivatized chitosan polyplex to the phosphorus atoms (P) of the nucleic acid to the anionic moieties (A) of the polymer (referred to as the "N:P:A molar ratio"). In certain embodiments, the molar ratio of P:A can vary from at least 1:40 to about 40:1, with N:P being at least 2:1 to at most 20:1.

[0220] In certain embodiments, the molar ratio of P:A may vary from at least 1:40 to about 1:10, where N:P is at least 2:1 to at most 20:1. In certain embodiments, the molar ratio of P:A may vary from at least 1:25 to about 25:1, where N:P is at least 2:1 to at most 20:1. In certain embodiments, the molar ratio of P:A may vary from at least 1:25 to about 1:10, where N:P is at least 2:1 to at most 20:1. In some cases, the molar ratio of P:A may vary from at least 1:20 to about 20:1, where N:P is at least 2:1 to at most 20:1. In some cases, the molar ratio of P:A may vary from at least 1:20 to about 1:10, where N:P is at least 2:1 to at most 20:1. In some cases, the molar ratio of P:A can vary from at least 1:10 to about 10: 1, with an N:P of at least 2:1 to at most 20: 1. In some cases, the molar ratio of P:A can vary from at least 1:25 to about 2: 1, with an N:P of at least 2:1 to at most 20: 1. In some cases, the molar ratio of P:A can vary from at least 1:20 to about 1: 1, with an N:P of at least 2:1 to at most 20: 1.

[0221] In certain preferred embodiments, N:P:A is between 3:1:3.5 and 3:1:17.5. In certain preferred embodiments, N:P:A is between 5:1:3.5 and 5:1:17.5. In certain preferred embodiments, N:P:A is between 7:1:3.5 and 7:1:17.5. In certain preferred embodiments, N:P:A is between 10:1:10 and 10:1:40. In certain preferred embodiments, N:P:A is about 3:1:3.5, 3:1:7, 3:1:10, 3:1:15, 3:1:17.5, or 3:1:20. In certain preferred embodiments, N:P:A is about 5:1:3.5, 5:1:7, 5:1:10, 5:1:15, 5:1:17.5, or 5:1:20. In certain preferred embodiments, N:P:A is about 7:1:3.5, 7:1:7, 7:1:10, 7:1:15, 7:1:17.5, or 7:1:20. In certain embodiments, N:P:A is about 10:1:10, 10:1:15, 10:1:20, 10:1:25, 10:1:30, or 10:1:40.

[0222] 2.4.1. Hydrophilic uncharged part

[0223] The hydrophilic uncharged portion of the polymer can be or include a polyalkylene polyol or a polyalkyleneoxy polyol portion or a combination thereof. The hydrophilic uncharged portion of the polymer can be or include a polyalkylene glycol or a polyalkyleneoxy glycol portion. In certain embodiments, the polyalkylene glycol portion is or includes a polyethylene glycol portion and / or a monomethoxy polyethylene glycol portion. In certain preferred embodiments, the uncharged portion of the polymer is or includes polyethylene glycol. The hydrophilic uncharged portion of the polymer can be or include other biocompatible polymers, such as polylactic acid.

[0224] In addition to PEG, several hydrophilic uncharged entities are known in the art. See, for example: Lowe et al., Antibiofouling polymer interfaces: poly(ethylene glycol) and other promising candidates, Polym. Chem., 6, 198-212, 2015; and Knop et al., Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives. Angewandte Chemie International Edition, 49(36), 6288-6308, 2010. Examples of hydrophilic uncharged portions of polymers are, but are not limited to: poly(glycerol), poly(2-methacryloyloxyethylphosphorylcholine), poly(sulfobetaine methacrylate) and poly(carboxybetaine methacrylate), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline) and poly(vinylpyrrolidone).

[0225] The hydrophilic portion may have a weight average molecular weight of about 500Da to about 50,000Da. In some embodiments, the hydrophilic portion has a weight average molecular weight of about 1,000Da to about 10,000Da. In certain embodiments, the hydrophilic portion has a weight average molecular weight of about 1,500Da to about 7,500Da. In certain embodiments, the hydrophilic portion has a weight average molecular weight of about 3,000Da to about 5,000Da. In some cases, the hydrophilic portion has a weight average molecular weight of 5,000Da or about 5,000Da.

[0226] 2.4.2. Anionic polymer part

[0227] The anionic polymer portion of the polymer can contain multiple negatively charged functional groups at physiological pH. A variety of anionic polymers are suitable for use in the methods and compositions described herein, provided that such anionic polymers can be provided as a component of a polymer having a hydrophilic uncharged polymer portion and can form (e.g., reversible) charge:charge complexes with positively charged (e.g., dual) derivatized chitosan-nucleic acid nanoparticles.

[0228] Exemplary anionic polymers include, but are not limited to, polypeptides with a net negative charge at physiological pH. In some cases, a polypeptide or a portion thereof is composed of amino acids with a negatively charged side chain at physiological pH. For example, the anionic polymer portion of the polymer can be a polyglutamic acid polypeptide, a polyaspartic acid polypeptide, or a mixture thereof. Additional amino acids or their mimetics can be incorporated into the polyanionic polypeptide. For example, glycine and / or serine can be incorporated to increase flexibility or reduce secondary structure.

[0229] In some cases, the anionic polymer can be or include an anionic carbohydrate polymer. Exemplary anionic carbohydrate polymers include, but are not limited to, glycosaminoglycans that are negatively charged at physiological pH. Exemplary anionic glycosaminoglycans include, but are not limited to, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparan sulfate, hyaluronic acid, or a combination thereof. In certain embodiments, the anionic polymer portion of the polymer is or includes hyaluronic acid.

[0230] Additional or alternative anionic carbohydrate polymers may include polymers comprising dextran sulfate.

[0231] In some cases, the polyanionic portion is or comprises a polyanion selected from the group consisting of polymethacrylic acid and salts thereof, copolymers of polyacrylic acid and salts thereof, methacrylic acid and salts thereof, and copolymers of acrylic acid and / or methacrylic acid and salts thereof, such as polyoxyalkylene, polyacrylic acid copolymers.

[0232] In some cases, the polyanionic moiety is or comprises a polyanion selected from the group consisting of alginate, carrageenan, furcellan, pectin, xanthan gum, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate, cellulose, oxidized cellulose, carboxymethyl cellulose, cross-linked carboxymethyl cellulose, synthetic polymers and copolymers containing pendant carboxyl, phosphate or sulfate groups, predominantly negatively charged polyamino acids, and biocompatible polyphenolic materials.

[0233] The anionic portion of the polymer can have a weight average molecular weight of about 500Da to about 5,000Da. In some embodiments, the anionic portion has a weight average molecular weight of about 500Da to about 3,000Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 500Da to about 2,500Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 500Da to about 2,000Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 500Da to about 1,500Da. In some embodiments, the anionic portion has a weight average molecular weight of about 1,000Da to about 5,000Da. In some embodiments, the anionic portion has a weight average molecular weight of about 1,000Da to about 3,000Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 1,000Da to about 2,500Da. In certain embodiments, the anionic portion has a weight average molecular weight of about 1,000Da to about 2,000Da. In some cases, the weight average molecular weight of the anionic moiety is or is about 1,500 Da.

[0234] As used herein, "block copolymer", "block co-polymer", etc. refer to copolymers containing different homopolymer regions. A diblock copolymer contains two different homopolymer regions. A triblock copolymer contains three different homopolymer regions. The three different regions can be different from each other (e.g., AAAA-BBBB-CCC C), or the two regions can be the same (e.g., AAAA-BBBB-AAAA), similar (e.g., AAAA-BBBB-AAA), where "A", "B" and "C" represent different monomer subunits that form the copolymer formed. For example, "A" can represent the ethylene glycol monomer subunit of a polyethylene glycol homopolymer, and B can represent the glutamic acid subunit of a polyglutamic acid homopolymer. The block copolymer can be a linear (e.g., two or three) block copolymer. Exemplary embodiments of linear diblock and triblock copolymers for use in the present invention include those listed in the following non-exhaustive list:

[0235]

[0236]

[0237]

[0238] *K: molecular weight of PEG (kDa)

[0239] ##Number of subunits

[0240] In one embodiment, the block copolymer is or comprises a PEG-polyglutamic acid polymer having the structure:

[0241]

[0242] In one embodiment, the block copolymer is or comprises a PEG-polyaspartic acid polymer having the structure:

[0243]

[0244] In one embodiment, the block copolymer is or comprises a PEG-hyaluronic acid polymer having the structure:

[0245]

[0246] 2.5. Preparation method

[0247] As described above, it will be appreciated by those skilled in the art that the polyplex: polymer particles of the present invention can be produced by a variety of methods. For example, polyplex particles can be generated and then contacted with a polymer. In an exemplary non-limiting embodiment, polyplex particles are prepared by providing functionalized chitosan and nucleotide raw materials and combining them. The raw material concentration can be adjusted to accommodate various amino to phosphate ratios (N / P), mixing ratios, and target nucleotide concentrations. In some embodiments, particularly small batches, such as batches of less than 2 mL, the functionalized chitosan and nucleotide raw materials can be mixed by slowly dripping the nucleotide raw materials into the functionalized chitosan raw material while vortexing the container. In other embodiments, the functionalized chitosan and nucleotide raw materials can be mixed by mixing two fluid streams in a pipeline. In other embodiments, the resulting polyplex dispersion can be concentrated by means known in the art, such as ultrafiltration (e.g., tangential flow filtration (TFF)) or solvent evaporation (e.g., freeze-drying or spray drying). A preferred method for forming a polyplex is disclosed in WO2009 / 039657, which is expressly incorporated herein by reference in its entirety.

[0248] Similarly, a polyplex particle feedstock (e.g., an aqueous solution comprising a polyplex composition) can be provided (e.g., separated from the above reaction mixture) and combined with a polymer feedstock (e.g., an aqueous solution comprising a polymer). The feedstock concentration can be adjusted to accommodate various amino to anion ratios (N / A), amino to phosphorus (N:P) ratios, N:P:A ratios, mixing ratios, and target nucleotide concentrations. In some embodiments, particularly small batches, such as batches of less than 2 mL, the feedstocks can be mixed by slowly dripping the first feedstock (e.g., polyplex) into the second feedstock (e.g., polymer) while vortexing the container. In other embodiments, the feedstocks can be mixed by mixing two fluid streams in a pipeline. In other embodiments, the resulting polyplex: polymer dispersion can be concentrated by means known in the art, such as ultrafiltration (e.g., tangential flow filtration (TFF)) or solvent evaporation (e.g., freeze-drying or spray drying).

[0249] In some embodiments, the polyplex: polymer composition comprises a core-shell particle composition, wherein the particle comprises a polyplex core and a non-covalently bound (e.g., releasable) polymer shell. It should be understood that one method for making such a core-shell composition includes forming a polyplex and then combining it with a polymer feedstock as described above.

[0250] Alternatively, the polyplex: polymer composition can be prepared in a one-step process, wherein nucleic acids, derivatized chitosan, and a plurality of 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. Without wishing to be bound by theory, the inventors hypothesize that such a one-step process can produce smaller particle sizes, which may be advantageous in certain indications where mucosal diffusion is limited in vivo.

[0251] 3. Powdered preparations

[0252] The polyplex:polymer composition of the present invention includes powders. 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 prepared by dehydrating (e.g., spray drying or freeze drying) the chitosan-nucleic acid polyplex dispersion of the present invention.

[0253] 4. Pharmaceutical preparations

[0254] The present invention also provides a "pharmaceutically acceptable" or "physiologically acceptable" formulation comprising the polyplex: polymer composition of the present invention. Such formulations can be administered to a subject in vivo to implement a therapeutic method.

[0255] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients, etc. that can be administered to a subject, preferably without excessive adverse side effects (e.g., nausea, abdominal pain, headache, etc.). Such administration formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Liquid formulations include suspensions, solutions, syrups, and elixirs. Liquid formulations can be prepared by solid reconstitution.

[0256] Pharmaceutical preparations can be made of carriers, diluents, excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are compatible with administration to a subject. Such preparations can be contained in tablets (coated or uncoated), capsules (hard or soft), microbeads, emulsions, powders, granules, crystals, suspensions, syrups or elixirs. Supplementary active compounds and preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc.

[0257] Excipients may include salts, isotonic agents, serum proteins, buffers or other pH control agents, antioxidants, thickeners, uncharged polymers, preservatives or cryoprotectants. Excipients used in the compositions of the present invention may also include isotonic agents and buffers or other pH control agents. These excipients may be added to obtain a preferred pH range (about 6.0-8.0) and osmotic pressure (about 50-400mmol / L). Examples of suitable buffers are acetates, borates, carbonates, citrates, phosphates and sulfonated organic molecule buffers. Such buffers may be present in the composition at a concentration of 0.01% to 1.0% (w / v). Isotonic agents may be selected from any of those isotonic agents known in the art, such as mannitol, dextrose, glucose and sodium chloride, or other electrolytes. Preferably, isotonic agents are glucose or sodium chloride. Isotonic agents may be used in an amount that gives the composition the same or similar osmotic pressure as the biological environment into which it is introduced. The concentration of the isotonic agent in the composition will depend on the nature of the particular isotonic agent used, and may range from about 0.1% to 10%. When glucose is used, it is preferably used in a concentration of 1 to 5% w / v, more particularly 5% w / v. When the isotonic agent is sodium chloride, it is preferably used in an amount up to 1% w / v, particularly 0.9% w / v. The compositions of the invention may also contain a preservative. Examples of preservatives are polyhexamethylene-biguanide, benzalkonium chloride, stabilized oxychloride complexes (such as those known as ), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens and thimerosal. Typically, such preservatives are present in a concentration of about 0.001 to 1.0%. In addition, the composition of the present invention may also contain a cryopreservative. Preferred cryopreservatives are glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran with a molecular weight preferably less than 100,000 g / mol, glycerol and polyethylene glycol with a molecular weight less than 100,000 g / mol or mixtures thereof. Most preferred are glucose, trehalose and polyethylene glycol. Typically, such cryopreservatives are present in a concentration of about 0.01 to 10%.

[0258] The pharmaceutical preparation can be formulated to be compatible with its intended route of administration. For example, for oral administration, the composition can be combined with excipients and administered in the form of tablets, lozenges, capsules (e.g., gelatin capsules), or coatings (e.g., enteric coatings ( or )) in the form of. Pharmaceutically compatible binders and / or adjuvant materials may be included in oral formulations. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or other stearates; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate or flavorings.

[0259] The formulation may also include carriers to protect the composition from rapid degradation or elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate, alone or in combination with a wax, may be employed.

[0260] Suppositories and other rectal administrable formulations (e.g., those that can be administered by enema) are also contemplated. For further information on rectal delivery, see, e.g., Song et al., Mucosal drug delivery: membranes, methodologies, and applications, Crit. Rev. Ther. Drug. Carrier Syst., 21: 195-256, 2004; Wearley, Recent progress in protein and peptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. Carrier Syst., 8: 331-394, 1991.

[0261] Additional pharmaceutical agents suitable for administration are known in the art and are suitable for use with the methods and compositions of the invention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Company, Lancaster, Pa., (1993)).

[0262] 5. Application

[0263] In one embodiment, the use of a polyplex:polymer composition provides for prolonged stability of the polyplex at physiological pH. This provides for effective mucosal administration.

[0264] Any of a variety of administration routes that contact mucosal cells or tissues are possible, and the choice of a particular route will depend in part on the target mucosal cells or tissues.Syringes, endoscopes, cannulas, cannulas, catheters, and other items can be used for administration.

[0265] The dose or "effective amount" used to treat a subject is preferably sufficient to improve one, several or all symptoms of the disorder to a measurable or detectable degree, but preventing or inhibiting the progression or worsening of the condition or disorder, or the symptoms is a satisfactory result. Therefore, in the case of a disorder or condition that can be treated by expressing a therapeutic nucleic acid in a target tissue, the amount of therapeutic RNA or therapeutic protein produced to improve the disorder that can be treated by the methods of the present invention will depend on the disorder and the desired result and can be easily determined by a skilled person. The appropriate amount will depend on the disorder being treated, the desired therapeutic effect, and the individual subject (e.g., bioavailability in the subject, sex, age, etc.). The effective amount can be determined by measuring the relevant physiological effects.

[0266] Veterinary applications are also contemplated by the present invention. Thus, in one embodiment, the present invention provides a method of treating a non-human mammal comprising administering to the non-human mammal in need of treatment a polyplex:polymer composition of the present invention.

[0267] 5.1. Oral administration

[0268] The subject compositions can be administered orally. Oral administration may involve swallowing, thereby allowing the compound to enter the gastrointestinal tract. The compositions of the present invention can also be administered directly to the gastrointestinal tract.

[0269] Formulations suitable for oral administration include solid preparations such as tablets, capsules, coated capsules containing microparticles or coated microparticles, liquids or powders, lozenges (including liquid fills), chewables, multi- and nanoparticles, gels, films, vaginal suppositories and sprays.

[0270] Tablet dosage forms typically contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, cross-linked polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl substituted hydroxypropyl cellulose, starch, pregelatinized starch, and sodium alginate. Typically, the disintegrant will comprise 1 to 25 weight %, preferably 5 to 20 weight %, of the dosage form.

[0271] Binders are generally used to impart cohesiveness to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinyl pyrrolidone, 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 dihydrated calcium phosphate.

[0272] Tablets may also optionally contain surfactants, such as sodium lauryl sulfate and polysorbate 80, and glidants, such as silicon dioxide and talc. When present, surfactants may comprise 0.2% to 5% by weight of the tablet, and glidants may comprise 0.2% to 1% by weight of the tablet.

[0273] Tablets also typically contain lubricants, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and a mixture of magnesium stearate and sodium lauryl sulfate. Lubricants typically account for 0.25 to 10% by weight of the tablet, preferably 0.5 to 3% by weight.

[0274] Other possible ingredients include antioxidants, coloring agents, flavoring agents, preservatives and taste masking agents.

[0275] Tablet blends can be tableted directly or by roller compression. Tablet blends or portions of blends can alternatively be wet-, dry-, or melt-granulated, melt-congealed, or extruded prior to tableting. The final formulation can contain one or more layers and can be coated or uncoated; it can even be encapsulated.

[0276] The formulation of tablets is discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

[0277] Consumable oral films for human or veterinary use are typically flexible, water-soluble or water-swellable thin film dosage forms that are rapidly dissolving or mucoadhesive and typically comprise a film-forming polymer, a binder, a solvent, a humectant, a plasticizer, a stabilizer or emulsifier, a viscosity modifier, and a solvent. Certain components of the formulation may perform more than one function.

[0278] The present invention also includes multiparticulate beads comprising the composition of the present invention.

[0279] Other possible ingredients include antioxidants, colorants, flavorings and flavor enhancers, preservatives, salivary stimulants, cooling agents, co-solvents (including oils), emollients, fillers, anti-foaming agents, surfactants and taste masking agents.

[0280] Films according to the invention are typically prepared by evaporative drying of an aqueous film coated on a peelable backing carrier or paper. This can be done in a drying oven or flue, typically a combined coating dryer, or by freeze drying or vacuuming.

[0281] Solid preparations for oral administration can be formulated to be immediate and / or modified release. Modified release preparations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0282] Other suitable release technologies such as high energy dispersions and osmotic and coated particles are known.

[0283] 5.2. Mucosal administration

[0284] The compositions of the present invention can also be applied to mucosa. For example, the compositions can be applied 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 eye, airway epithelium, lung, vagina and bladder cells or tissues.

[0285] Typical formulations for this purpose include liquids, gels, hydrogels, solutions, creams, foams, films, implants, sponges, fibers, powders and microemulsions.

[0286] The compounds of the invention can be administered to the mucosa intranasally or by inhalation, typically in dry powder form (alone, as a mixture, e.g., with lactose, or as granules of mixed components) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, sprayer, nebulizer or atomizer, with or without a suitable propellant.

[0287] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as I-leucine, mannitol or magnesium stearate.

[0288] Formulations for inhaled / intranasal administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0289] The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary or enema. Cocoa butter is a traditional suppository base but various substitutes may be used as appropriate.

[0290] Formulations for rectal or vaginal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, or programmed release.

[0291] The compounds of the present invention can also be typically applied directly to the eyes or ears in the form of drops. Other formulations suitable for use in the eyes and ears include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and microparticle systems. The preparations can also be delivered by iontophoresis.

[0292] Formulations for ocular / aural administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted- and programmed-release.

[0293] 6. Therapeutic Applications

[0294] In one embodiment, the polyplex:polymer compositions of the invention can be used for therapeutic treatments. Such compositions are sometimes referred to herein as therapeutic compositions.

[0295] The therapeutic proteins of the invention, as discussed below, are produced from the polyplex:polymer compositions of the invention comprising therapeutic nucleic acids. Use of the subject proteins, as described below, refers to use of the subject polyplex:polymer compositions to effect use of such proteins.

[0296] The therapeutic proteins contemplated for use in the present invention have a variety of activities and can be used to treat a variety of diseases. The following description of therapeutic protein activities and indications that can be treated with the therapeutic proteins of the present invention is exemplary and is not intended to be exhaustive. The term "subject" refers to an animal, preferably a mammal, and particularly preferably a human.

[0297] Table 4 shows a partial list of therapeutic proteins and target diseases.

[0298] Table 4.

[0299]

[0300]

[0301]

[0302] In another embodiment, therapeutic composition of the present invention comprises therapeutic nucleic acid, for example therapeutic RNA, that does not encode therapeutic protein.For example, by selecting the therapeutic RNA of the gene relevant to the disease mechanism and / or undesirable cell or physiological disorder of targeting, the subject composition can be used for treating multiple diseases and illness.The subject composition has such feature, and the therapeutic RNA used is not subject to the restriction of target selection range.Therefore, the subject composition can be used for any disease or illness related to suitable target mucosal tissue.

[0303] The specific non-limiting examples of treatment embodiments are described below. In some cases, treatment embodiments are intended to act on non-mucosal target tissues, cells or organs. If the therapeutic effect is non-mucosal, it should be understood that the cells or tissues contacted with the polyplex described herein: polymer composition are mucosal and the therapeutic effect is away from the mucosal target. For example, mucosal cells can be transfected to produce and secrete hormones or other therapeutic agents.

[0304] 6.1. Hyperglycemia and body weight

[0305] Therapeutic proteins include insulin and insulin analogs. Diabetes is a debilitating metabolic disease caused by the lack of insulin (type 1) or insufficiency (type 2) produced by pancreatic β-cells (Unger, RH et al., Williams Textbook of Endocrinology Saunders, Philadelphia (1998)). β-cells are specialized endocrine cells that can make and store insulin for release after meals (Rhodes et al., J. Cell Biol. 105: 145 (1987)), and insulin is a hormone that promotes the transfer of glucose from the blood to tissues that need it. Diabetic patients must frequently monitor blood sugar levels, and many patients need multiple injections of insulin every day to survive. However, these patients rarely achieve ideal blood sugar levels by injecting insulin (Turner, RC et al. JAMA 281: 2005 (1999)). In addition, the long-term increase in insulin levels can lead to harmful side effects, such as hypoglycemic shock and the body's insensitivity to insulin response. Therefore, diabetic patients still suffer from 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)).

[0306] Disorders that can be treated by the methods of the present invention include hyperglycemic disorders, such as insulin-dependent (type 1) or non-insulin-dependent (type 2) diabetes, and physiological disorders or disorders associated with or caused by hyperglycemic states. Therefore, hyperglycemic disorders that can be treated by the methods of the present invention also include histopathological changes associated with chronic or acute hyperglycemia (e.g., diabetes). Specific examples include pancreatic degeneration (β-cell destruction), tubular calcification, liver degeneration, eye damage (diabetic retinopathy), diabetic foot, mucosal ulcers such as the mouth and gums, excessive bleeding, blood coagulation or delayed wound healing, and increased risk of coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity.

[0307] The subject composition can be used to reduce glucose, improve glucose tolerance, treat hyperglycemic state (such as diabetes) or be used to treat physiological conditions associated with or caused by hyperglycemic state. Such conditions include, for example, diabetic neuropathy (autonomic neuropathy), nephropathy (kidney injury), skin infection and other skin diseases, slow or delayed healing of injuries or wounds (such as causing diabetic carbuncle), eye damage (retinopathy, cataract) that can cause blindness, diabetic foot and accelerated periodontitis. Such conditions also include an increased risk of suffering from coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension and obesity.

[0308] As used herein, the term "hyperglycemia" or "hyperglycemia" when used to refer to a condition of a subject means a temporary or chronic abnormally high level 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 glucose intolerance or a glucose elevation state not usually found in a normal subject (e.g., a glucose-intolerant subdiabetic subject at risk for diabetes, or a diabetic subject). A normal blood sugar fasting blood glucose (FPG) level is less than about 110 mg / dl, for impaired glucose metabolism, between about 110 and 126 mg / dl, and for a diabetic, greater than about 126 mg / dl.

[0309] Diseases that can be treated by producing proteins in intestinal mucosal tissue also include obesity or poor weight. Leptin, cholecystokinin, PYY and GLP-1 can reduce hunger, increase energy consumption, induce weight loss or provide normal glucose homeostasis. Therefore, in various embodiments, the method for treating obesity or poor weight or hyperglycemia of the present invention includes the use of therapeutic nucleic acids encoding leptin, cholecystokinin, PYY or GLP-1. In another embodiment, a therapeutic RNA targeting growth hormone releasing peptide is used. Growth hormone releasing peptide increases appetite and hunger. Therefore, in various embodiments, the method for treating obesity or poor weight or hyperglycemia of the present invention includes the use of therapeutic RNA targeting growth hormone releasing peptide to reduce its expression. Treatable diseases also include those diseases that are usually associated with obesity, such as abnormally elevated serum / plasma LDL, VLDL, triglycerides, cholesterol, plaque formation that causes vascular narrowing or obstruction, increased risk of hypertension / stroke, coronary heart disease, etc.

[0310] As used herein, the term "obesity" or "obesity" refers to a subject whose body weight is at least 30% greater than that of a normal subject matched for age and sex. "Abnormal weight" refers to a subject whose body weight is 1%-29% greater than that of a matched normal subject and a subject whose body weight is normal but whose weight gain is desired to be reduced or prevented.

[0311] 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 and OrciL.N.Eng.J.Med.304:1518(1981); Unger RH Diabetes 25:136(1976)). Like insulin, blood glucose concentration mediates glucagon secretion. However, in contrast to insulin, glucagon is secreted in response to reduced blood glucose. Therefore, the circulating concentration of glucagon is highest during fasting and lowest during meals. Elevated glucagon levels promote glucose storage to reduce insulin and stimulate the liver to release glucose into the blood. A specific example of a glucagon antagonist is [des-His1, des-Phe6, Glu9] glucagon-NH2. In streptozotocin diabetic rats, intravenous injection of this glucagon antagonist reduced blood glucose levels by 37% (0.75 μg / g body weight) within 15 minutes (Van Tine B.A. et al. Endocrinology 137:3316 (1996)). In another embodiment, the present invention provides a method for treating diabetes or hyperglycemia, the method comprising using therapeutic RNA to reduce the level of glucagon produced by the pancreas.

[0312] In another embodiment, the therapeutic protein of the present invention that can be used to treat hyperglycemic disorders or unhealthy body weight (e.g., obesity) is glucagon-like peptide-1 (GLP-1). GLP-1 is a hormone released from intestinal L cells during meals that stimulates pancreatic β-cells and increases insulin secretion. GLP-1 has additional activities that make it an attractive therapeutic agent for the treatment of obesity and diabetes. For example, GLP-1 reduces gastric emptying, suppresses appetite, reduces glucagon concentration, increases β-cell mass, stimulates insulin biosynthesis and secretion in a glucose-dependent manner, and may increase tissue sensitivity to insulin (Kieffer TJ, Habener JF Endocrin. Rev. 20: 876 (2000)). Therefore, regulating the release of GLP-1 in the intestine simultaneously with meals can provide therapeutic benefits for hyperglycemic disorders or unhealthy body weight. GLP-1 analogs that are resistant to dipeptidyl peptidase IV (DPP IV) can provide longer duration of action and improved therapeutic value. Therefore, GLP-1 analogs are preferred therapeutic polypeptides. In another embodiment, the invention provides a method of treating diabetes or hyperglycemia comprising using a therapeutic RNA to reduce the level of DPP IV.

[0313] In another embodiment, the therapeutic protein of the present invention that can be used to treat hyperglycemic disorders is an antagonist of 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 and insulin action in obese mice. In contrast, administration of resistin in normal mice impairs glucose tolerance and insulin action (Steppan CM et al. Nature 409:307 (2001)). Therefore, the production of proteins in the intestine that antagonize the biological effects of resistin can provide an effective treatment for obesity-related insulin resistance and hyperglycemic disorders. In another embodiment, the present invention provides a method for treating diabetes or hyperglycemia, the method comprising using therapeutic RNA to reduce the expression level of resistin in adipose tissue.

[0314] In another embodiment, a therapeutic polypeptide of the invention that can be used to treat hyperglycemic conditions or undesirable body weight (e.g., obesity) is leptin. Although leptin is primarily produced by adipocytes, it is also produced in small amounts in the stomach in a meal-dependent manner. Leptin transmits information about adipocyte metabolism and body weight to the appetite center in the brain, where it signals to reduce food intake (promote satiety) and increase energy expenditure by the body.

[0315] In another embodiment, the therapeutic polypeptide of the invention that can be used to treat hyperglycemia or undesirable body weight (e.g., obesity) is the C-terminal globular head domain of adipocyte complement-related protein (Acrp30). Acrp30 is a protein produced by differentiated adipocytes. Administration of an Acrp30 proteolytic cleavage product consisting of the globular head domain to mice resulted in significant weight loss (Fruebis J. et al. Proc. Natl Acad. Sci USA 98: 2005 (2001)).

[0316] In another embodiment, the therapeutic polypeptide of the invention that can be used to treat hyperglycemia or unhealthy body weight (e.g., obesity) is cholecystokinin (CCK). CCK is a gastrointestinal peptide secreted from the intestine in response to specific nutrients in the intestine. The release of CCK is proportional to the amount of food consumed, and it is believed that it signals the brain to stop eating (Schwartz M.W. et al. Nature 404:661-71 (2000)). Therefore, elevated CCK can reduce meal size and promote weight loss or weight stability (i.e., prevent or inhibit weight gain).

[0317] For PYY, see, e.g., le Roux et al., Proc Nutr Soc. 2005 May;64(2):213-6.

[0318] 6.2. Immune disorders

[0319] In one embodiment, the therapeutic composition of the present invention has immunomodulatory activity. For example, by activating or inhibiting the proliferation, differentiation or mobilization (chemotaxis) of immune cells, the therapeutic polypeptide of the present invention can be used to treat defects or disorders of the immune system. Immune cells develop through the hematopoietic process, producing myeloid (platelets, erythrocytes, neutrophils and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The causes of these immune deficiencies or disorders may be genetic, somatic (e.g., cancer or certain autoimmune disorders), acquired (e.g., by chemotherapy or toxins) or infectious.

[0320] The therapeutic compositions of the present invention can be used to treat defects or disorders of hematopoietic cells. 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, in an attempt to treat those disorders 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, dysgammaglobulinemia), ataxia-telangiectasia, common variable immunodeficiency, DiGeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphocytopenia, phagocytic cell bactericidal dysfunction, severe combined immunodeficiency (SCID), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

[0321] The therapeutic composition of the present invention can also be used to treat autoimmune diseases. Many autoimmune diseases are caused by immune cells inappropriately identifying themselves as foreign substances. This inappropriate recognition can cause an immune response, leading to destruction of host tissues. Therefore, the use of the therapeutic composition of the present invention that inhibits immune responses, particularly T cell proliferation, differentiation or chemotaxis, may be an effective therapy for preventing autoimmune disorders.

[0322] Examples of autoimmune diseases that can be treated by the present invention include, but are not limited to, Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, multiple sclerosis, myasthenia gravis, neuritis, eye inflammation, bullous pemphigoid, pemphigus, polyendocrine disease, purpura, Reiter's Disease, stiff-man syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune lung inflammation, Guillain-Barre Syndrome, insulin-dependent diabetes mellitus, Crohn's disease, ulcerative colitis, and autoimmune inflammatory eye diseases.

[0323] Similarly, allergic reactions and disorders, such as asthma (particularly allergic asthma) or other respiratory problems, can also be treated by the therapeutic compositions of the present invention. In addition, these molecules can be used to treat allergic reactions, hypersensitivity reactions to antigenic molecules or blood incompatibility.

[0324] 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 occurs when host immune cells destroy transplanted tissues through an immune response. Similarly, GVHD also involves an immune response, but in this case, foreign transplanted immune cells destroy host tissues. Administration of a therapeutic composition of the present invention that inhibits immune responses, particularly T cell proliferation, differentiation or chemotaxis, may be an effective therapy for preventing organ rejection or GVHD.

[0325] Similarly, the therapeutic composition of the present invention can also be used to regulate inflammation. For example, therapeutic polypeptides can inhibit the proliferation and differentiation of cells involved in inflammatory responses. These molecules can be used to treat inflammatory diseases, chronic and acute diseases, 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 caused by excessive production of cytokines (e.g., TNF or IL-1). In one embodiment, therapeutic RNA targeting TNFα is used in the subject composition to treat inflammation. In another preferred embodiment, therapeutic RNA targeting IL-1 is used in the subject composition to treat inflammation. siRNA therapeutic RNA is particularly preferred. The inflammatory conditions of interest for treatment of the present invention include, but are not limited to, chronic obstructive pulmonary disease (COPD), interstitial cystitis, and inflammatory bowel disease.

[0326] 6.3. Coagulation disorders

[0327] In some embodiments, the therapeutic compositions of the present invention can also be used to regulate hemostasis (stop bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostasis or thrombolytic activity, the therapeutic compositions of the present invention can be used to treat coagulation disorders (e.g., afibrinogenemia, factor deficiency), platelet disorders (e.g., thrombocytopenia), or wounds caused by trauma, surgery, or other causes. Alternatively, the therapeutic compositions of the present invention that can reduce hemostasis or thrombolytic activity can be used to inhibit or dissolve coagulation. These therapeutic compositions may be important in the treatment of heart attacks (infarctions), strokes, or scars. In one embodiment, the therapeutic polypeptide of the present invention is a coagulation factor that can be used to treat hemophilia or other coagulation / coagulation disorders (e.g., Factor VIII, IX, or X).

[0328] 6.4. Hyperproliferative disorders

[0329] In one embodiment, the therapeutic compositions of the invention are capable of modulating cell proliferation. Such therapeutic polypeptides can be used to treat hyperproliferative disorders, including neoplasms.

[0330] Examples of hyperproliferative disorders that may be treated by the therapeutic compositions of the invention include, but are not limited to, neoplasms located in the abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eyes, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system.

[0331] Similarly, other hyperproliferative disorders may also be treated by the therapeutic compositions of the present invention. In addition to neoplasms located in the organ systems listed above, examples of such hyperproliferative disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative diseases, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disorder.

[0332] Delivery to the circulatory system provides access for therapeutic proteins to a variety of tissues. Alternatively, the therapeutic compositions of the invention may stimulate proliferation of other cells that may inhibit hyperproliferative disorders.

[0333] For example, hyperproliferative disorders can be treated by increasing the immune response, particularly increasing the antigenicity of the hyperproliferative disorder or by causing T cell proliferation, differentiation or mobilization. Such immune responses can be increased by enhancing existing immune responses or by initiating new immune responses. Alternatively, reducing the immune response can also be a method of treating hyperproliferative disorders, for example using chemotherapeutic agents.

[0334] 6.5. Infectious diseases

[0335] In one embodiment, the therapeutic composition of the present invention can be used to treat infectious diseases. For example, infectious diseases can be treated by increasing the immune response, particularly increasing the proliferation and differentiation of B cells and / or T cells. The immune response can be increased by enhancing an existing immune response or by initiating a new immune response. Alternatively, the therapeutic composition of the present invention can also directly inhibit the infectious agent, without necessarily causing an immune response.

[0336] Viruses are an example of infectious agents that can cause diseases or symptoms that can be treated by the therapeutic compositions of the present invention. Examples of viruses include, but are not limited to, the following DNA and RNA virus families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (e.g., cytomegalovirus, herpes simplex, herpes zoster), Mononegavirus (e.g., Paramyxoviridae), Viruses, such as rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella, rubella Viruses belonging to these families can cause a variety of diseases or symptoms, including but 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's Lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold, poliomyelitis, leukemia, rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. The therapeutic composition of the present invention can be used to treat any of these symptoms or diseases.

[0337] Similarly, bacterial or fungal agents that can cause disease or symptoms and can be treated by the therapeutic compositions of the present invention include, but are not limited to, the following Gram-negative and Gram-positive bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, losis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatomycosis, Enterobacteriaceae (Klebsiella, Sal monella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmas, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurella cea) infections (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal.These bacteria or fungi can cause the following diseases or symptoms, including but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (such as AIDS-related infections), paronychia, prosthesis-related infections, Reiter's disease, respiratory tract infections (such as pertussis or empyema), sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (such as cellulitis, dermatophytosis), toxemia, urinary tract infections, wound infections. The therapeutic composition of the present invention can be used to treat any of these symptoms or diseases.

[0338] In addition, parasites that can be treated by the therapeutic composition of the present invention and cause diseases or symptoms include, but are not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitism, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but not limited to, scabies, trichophytosis, eye infections, intestinal diseases (such as dysentery, giardiasis), liver disease, lung disease, opportunistic infections (such as 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.

[0339] 6.6. Regeneration

[0340] The therapeutic composition of the present invention can be used to differentiate, proliferate and attract cells to promote the regeneration of mucosal tissue or tissue adjacent to the target mucosal cells or tissue. (See Science 276: 59-87 (1997)) Tissue regeneration can be used to repair, replace or protect tissues 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 injury.

[0341] The therapeutic compositions of the present invention can promote regeneration of a variety of tissues, including but not limited to organ (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, bone or heart), vascular (including vascular endothelium), neural, hematopoietic and skeletal (bone, cartilage, tendon and ligament) tissue. Preferably, regeneration will produce little scarring, or occur without scarring. Regeneration can also include angiogenesis.

[0342] In addition, the therapeutic compositions of the present invention can increase the regeneration of difficult to heal tissues. For example, increasing tendon / ligament regeneration will speed up recovery time after injury. The therapeutic compositions of the present invention can also be used prophylactically to avoid injury. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Another example of tissue regeneration of non-healing wounds includes pressure sores, ulcers associated with vascular insufficiency, surgery, and traumatic wounds.

[0343] Similarly, it is also possible to regenerate nerve and brain tissue by using the therapeutic composition of the present invention to proliferate and differentiate nerve cells. Diseases that can be treated using this method include central and peripheral nervous system diseases, neuropathy or mechanical and traumatic conditions (such as spinal cord diseases, head trauma, cerebrovascular diseases and stroke). Specifically, diseases related to peripheral nerve injury, peripheral neuropathy (such as caused by chemotherapy or other drug therapies), local neuropathy and central nervous system diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Drager syndrome) can be treated using the therapeutic composition of the present invention. For CNS disorders, a variety of methods known in the art to promote the treatment of brain tissue, including methods for destroying the blood-brain barrier, and methods for coupling therapeutic agents with parts that provide transport to CNS. In one embodiment, therapeutic nucleic acids are engineered to encode fusion proteins that include transport moieties and therapeutic proteins.

[0344] 6.7. Chemotaxis

[0345] 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. Chemotactic molecules attract or mobilize cells (e.g., monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to specific parts of the body, such as inflammation, infection or hyperproliferation sites. The mobilized cells can then fight back and / or heal specific trauma or abnormalities.

[0346] For example, the therapeutic polypeptides of the present invention can increase the chemotactic activity of specific cells. These chemotactic molecules can then treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells that target specific parts of the body. For example, chemotactic molecules can treat wounds and other tissue trauma by attracting immune cells to the injured area. The chemotactic molecules of the present invention can also attract fibroblasts that can be used to treat wounds.

[0347] It is also contemplated that the therapeutic compositions of the present invention can inhibit chemotactic activity. These therapeutic compositions can also be used to treat disorders. Thus, the therapeutic compositions of the present invention can be used as chemotaxis inhibitors.

[0348] Particularly preferred for use are therapeutically enhancing proteins that are activated in the vicinity of the target tissue.

[0349] Additional therapeutic polypeptides contemplated for use include, but are not limited to, growth factors (e.g., growth hormone, insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, acidic and basic fibroblast growth factor, transforming growth factor-β, etc.) to treat growth disorders or wasting syndrome; and antibodies (e.g., human or humanized) to provide passive immunity or protection of a subject against foreign antigens or pathogens (e.g., Helicobacter pylori (H. Pylori)), or to provide treatment for cancer, arthritis, or cardiovascular disease; cytokines, interferons (e.g., interferons (IFN), IFN-α2b and 2α, IFN-αN1, IFN-β1b, IFN-γ), interleukins (e.g., IL-1 to IL-10), tumor necrosis factor (TNF-αTNF-β), chemokines, granulocyte macrophage colony stimulating factor (GM-CSF), polypeptide hormones, antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral and / or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase, cytokines ... enzymes), gonadotropins, chemokines, lipid binding proteins, filgastim (Neupogen), hemoglobin, erythropoietin, insulinotropin, imiglucerase, sarbramostim, tissue plasminogen activator (tPA), urokinase, streptokinase, phenylalanine ammonia lyase, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), thrombopoietin (TPO), superoxide dismutase (SOD), adenosine deamidase, catalase, calcitonin, endothelin, L-asparaginase pepsin, uricase, trypsin, chymotrypsin, elastase, carboxypeptidase, lactase, sucrase, intrinsic factor, parathyroid hormone (PTH)-like hormone, soluble CD4, and antibodies and / or antigen-binding fragments thereof (e.g., FAb) (e.g., orthoclone OKT-e (anti-CD3), GPIIb / IIa monoclonal antibody). Also contemplated are therapeutic RNAs that target nucleic acids encoding these factors.

[0350] 6.8. Vaccines

[0351] 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 producing a desired epitope. In a preferred embodiment, the composition comprises a therapeutic nucleic acid construct capable of expressing a protein comprising the epitope.

[0352] 6.9. Cosmetic Application

[0353] In one embodiment, the present invention provides a DD-chitosan nucleic acid polyplex for cosmetic use. The subject cosmetic comprises a DD-chitosan nucleic acid polyplex in a formulation suitable for cosmetic use.

[0354] Example

[0355] Embodiment 1:

[0356] 1. General materials

[0357] 1.1 Plasmid DNA vector

[0358]

[0359] 1.2 Reagents

[0360]

[0361]

[0362] 1.3 Consumables

[0363]

[0364]

[0365] 1.4 Equipment

[0366]

[0367] 2. General Procedures

[0368] 2.1 Preparation of double-derivatized chitosan

[0369] According to US 9623112 B2, chitosan is doubly derivatized with arginine and gluconic acid (DD-chitosan, DD-X).

[0370] 2.2 Preparation of dual-derivatized chitosan and DNA polyplexes

[0371] According to US9623112B2 and US8722646B2, DD-chitosan was polymerized with plasmid DNA vectors at various amine to phosphate (N:P) molar ratios as required. Additional excipients such as sucrose, trehalose or mannitol were included as required. Various plasmid DNA vectors were tested as indicated herein.

[0372] 2.3 Preparation of PEG-polyglutamic acid (PEG-PGA) solution

[0373] Typically, PEG-PGA can be dissolved in water or excipient solution at a concentration of up to 40 mg / mL as desired. The resulting PEG-PGA solution is diluted to the desired molar concentration of the anionic 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.

[0374] 2.4 Preparation of PEG-hyaluronic acid (PEG-HA) solution

[0375] Typically, PEG-HA is dispensed in water at a concentration of 40 to 100 mg / mL and sonicated for 10 minutes. The resulting PEG-HA solution is diluted in 10% trehalose to the desired concentration of anionic species (A, i.e., hyaluronic acid) required for the subsequent formulation to obtain the desired final ratio of amine to phosphate to anion molar ratio (N:P:A) and to a final concentration of trehalose of 5%.

[0376] 2.5 Preparation of PEG-polyaspartic acid (PEG-PAA) solution

[0377] Typically, PEG-PAA was dispensed in 5% trehalose at a concentration of 40 to 100 mg / mL and sonicated for 10 to 15 minutes. When necessary, the resulting PEG-PAA solution was diluted in 5% trehalose to the desired concentration of anionic species (A, i.e., aspartic acid) required for the subsequent formulation to obtain the desired final ratio of amine to phosphate to anion molar ratio (N:P:A) and to a final trehalose concentration of 5%.

[0378] 2.6 Preparation of trehalose solution

[0379] Typically, trehalose is dissolved in water at a concentration of up to 0.2 g / mL as needed. The resulting trehalose is filtered using a 0.2 um filter. If necessary, the trehalose solution is diluted to the desired concentration required for subsequent formulations.

[0380] 2.7 Preparation of PAA solution in 50 mM Tris pH 8

[0381] Typically, PAA was dissolved in 50 mM Tris pH 8 at a concentration of 100 mg / mL or 20 mg / mL as required. If necessary, the PAA solution was diluted in 50 mM Tris pH 8 to the desired concentration required for subsequent needs.

[0382] 2.8 Preparation of 20 mM NaCl

[0383] Dissolve 46.75 mg of NaCl in 40 mL of water. Filter the solution through a 0.2 um filter.

[0384] 2.9 Preparation of simulated intestinal fluid (SIF)

[0385] FaSSIF V1 solution was prepared by dissolving FaSSIF V1 powder in water at a concentration of 2.24 mg / mL. The pH value was confirmed to be approximately 6.6. FaSSIF V2 solution was prepared by dissolving FaSSIF V2 powder in water at a concentration of 1.79 mg / mL. The pH value was confirmed to be approximately 6.6.

[0386] 2.10 PEG-polyanion (PEG-PA)

[0387] PEG-PA is a general term for PEG conjugated to a polyanion such as PEG-polyglutamic acid, PEG-hyaluronic acid, or PEG-polyaspartic acid.

[0388] 2.11 Preparation of PEGylated Polyplexes by Instillation

[0389] The PEGylated polyplex was prepared by dropping an equal volume of the polyplex solution (0.1 mL) into the diluted PEG-PA solution (0.1 mL).

[0390] 2.12 Preparation of PEGylated Polyplexes by In-line Mixing

[0391] The PEGylated polyplex was prepared by mixing equal volumes of the diluted PEG-PA solution with the polyplex solution using an in-line mixing device as described, for example, in US9623112B2 and US8722646B2.

[0392] 2.13 Stability of PEGylated Polyplexes in 150 mmol PBS

[0393] Mix 100uL of PEGylated polyplex in 300uL of 150mmol PBS. Measure particle size diameter at 0 and 2 hours of mixing. Compare with non-PEGylated polyplex control.

[0394] 2.14 Percentage of supercoiled DNA

[0395] Before measuring the percentage of supercoiled DNA, the total DNA must be released from the polyplexes or PEGylated polyplexes by subjecting them to an excess of PAA. A 1 μL sample aliquot (target 0.1 mg / mL DNA) was combined with 10 μL PAA (100 mg / mL in 50 mM Tris), mixed and incubated at 37°C for 30 minutes. After release, the DNA was subjected to agarose gel electrophoresis.

[0396] 2.15 Determination of total DNA in the preparation by Picogreen

[0397] Before measuring DNA using the PicoGreen assay, total DNA must be released from polyplexes or PEGylated polyplexes using excess PAA. A 10 μL sample aliquot (target 2ug / mL DNA) is combined with 10 μL PAA (20mg / mL in 50mM Tris), mixed and incubated at 37°C for 30 minutes. After release, the DNA is digested with an appropriate restriction enzyme (RE) to linearize the supercoiled DNA plasmid. For the picogreen assay, a 10uL aliquot of the RE digested sample is mixed with 190uL picogreen working solution (Qubit ds DNA buffer: Qubit ds DNA Picogreen reagent 199:1), incubated for 2 minutes, and fluorescence is measured (excitation: 485, emission cutoff: 515nm, emission: 535nm). The results are quantified based on a reference DNA standard curve.

[0398] 2.16 Free DNA

[0399] To verify capture of DNA into polyplexes, a 10 μL sample aliquot (target 1000 ng DNA) was mixed with 2 μL of 6X loading buffer and subjected to gel electrophoresis.

[0400] 2.17 Gel electrophoresis

[0401] The sample lane was loaded with samples prepared as described. The standard lane was loaded with supercoiled DNA ladder. The reference lane was loaded with 2 μL reference DNA (200 ng DNA) + 8 μL water + 2 μL 6X loading buffer. The samples were resolved on a 0.8% agarose gel containing 1X SYBRSafe DNA stain at 100 V for 75 minutes. The gel was imaged with the GelDoc imaging system.

[0402] 2.18 Nanoparticle size determination of polyplexes and PEGylated polyplexes

[0403] Particle size measurements were performed using a Zetasizer Nano light scattering instrument. Typically, samples were either undiluted or diluted up to 20 times in 10 mM NaCl and loaded into a disposable cuvette or Zetasizer folded capillary cell (minimum 0.8 mL). The Zetasizer was programmed to incubate the sample at 25°C for up to 3 minutes, followed by three replicates of the 3-minute measurement. Z-average diameter and polydispersity (PDI) were reported as standard deviations (n=3). The Zetasizer was also programmed to take into account the composition of the sample in terms of viscosity and refractive index.

[0404] 2.19 Zeta potential of polyplexes and PEGylated polyplexes

[0405] Zeta potential measurements were performed using a Zetasizer Nano light scattering instrument. Typically, undiluted samples were loaded into a Zetasizer folded capillary cell (minimum 0.8 mL), except for PEGylated polyplexes, which were diluted in 10 mM NaCl. The Zetasizer was programmed to incubate the sample at 25°C for up to 3 minutes and then repeat the measurement (the number of repeats was automatically determined by the Zetasizer software). Zeta potential values ​​were reported as standard deviations (n=3). The Zetasizer was also programmed to take into account the final composition of the sample in terms of viscosity and dielectric constant.

[0406] 2.20 Short-term stability

[0407] For short-term stability studies, formulations were freeze-dried as described and stored at appropriate temperatures (-20°C, 4°C, or room temperature). At appropriate times, samples were rehydrated and analyzed as described.

[0408] 3. PEGylation of the Polyplexes Using PEG-PGA (mPEG1K-b-PLE10)

[0409] 3.1 Procedure

[0410] As previously described, DD-X-DNA (pVax) polyplexes were generated with 5% trehalose at the N:P ratios shown below and a DNA concentration of 0.1 mg / mL DNA.

[0411]

[0412] The PEG-PGA solution was then mixed with the DD-X-DNA polyplex at a 1:1 volume ratio by dropwise mixing to produce PEGylated polyplex at 0.05 mg / mL DNA as shown below. The samples were tested for particle size, PDI and zeta potential (test formulations were not frozen).

[0413]

[0414]

[0415] 3.2 Results

[0416] Non-freeze-thawed PEGylated polyplexes made with mPEG1K-b-PLE10 produced stable formulations at certain N:P:A ratios shown below (unless otherwise indicated).

[0417]

[0418] * Aggregated samples

[0419] 4. PEGylation of Polyplexes Using PEG-PGA (mPEG1K-b-PLE10 and mPEG5K-b-PLE10) and PEG-HA (HA-202)

[0420] 4.1 Procedure

[0421] As previously described, DD-X-DNA (pVax-opt-hIL10) polyplexes were generated with 5% trehalose at the N:P ratios shown below and a DNA concentration of 0.25 mg / mL DNA.

[0422]

[0423] The PEG-PGA solution or PEG-HA solution was then mixed with the DD-X-DNA polyplex at a 1:1 volume ratio by dropwise mixing to produce PEGylated polyplexes at 0.125 mg / mL DNA as shown below. The samples were cooled and thawed and then tested for particle size, PDI, and zeta potential.

[0424]

[0425] *N:A ratio settings: mPEG1K-b-PLE10 at 1:92, mPEG5K-b-PLE10 at 1:8.5, and HA-202 at 1:15.

[0426] 4.2 Results

[0427] The physicochemical results of the freeze-thaw samples are provided in the table below.PEGylated polyplexes made with mPEG1K-b-PLE10 or HA-202 did not result in freeze-thaw stable formulations.

[0428] PEGylated polyplexes made with mPEG5K-b-PLE10 are stable to freeze-thaw.

[0429]

[0430]

[0431] * Aggregated samples

[0432] ** Zeta potential of PEGylated samples was measured on fresh samples (not freeze-thawed).

[0433] 5. PEGylation of Polyplexes Using PEG-HA

[0434] 5.1 Procedure

[0435] As previously described, DD-X-DNA (pVax-opt-hIL10) polyplexes were generated with 5% trehalose at the N:P ratios shown below and a DNA concentration of 0.25 mg / mL DNA.

[0436]

[0437] A solution of PEG-HA in 5% trehalose was then mixed with DD-X-DNA polyplexes at a 1:1 volume ratio by drop mixing to produce PEGylated polyplexes at 0.125 mg / mL DNA as shown below. The samples were tested for particle size, PDI, and zeta potential.

[0438]

[0439]

[0440]

[0441] 5.2 Results

[0442] HA-201 (HA 10k-PEG 2k) was insoluble in water. It formed a hydrogel and was not used any more. HA-202 (HA 50k-PEG2k) formed a viscous solution in water. The polyplex showed visible aggregation after PEGylation and was not tested further.

[0443] 6. Preparation of PEG-PGA Polyplex

[0444] 6.1 Procedure

[0445] Preparation of PEGylated polyplexes

[0446] As described herein, an aliquot of N:P 7 polyplex (CMC-INT06-098A) was PEGylated using PEG-PGA. The resulting PEGylated polyplex was tested for physicochemical properties and DNA capture. The resulting formulations other than N:P7 polyplex were produced.

[0447]

[0448] 6.2 Results

[0449] The test samples’ appearance, pH, particle size, PDI, zeta potential and conductivity were:

[0450] Entity ID Appearance Particle size (nm) PDI Zeta potential (mV) pH CMC-INT06-098A Transparent / Translucent 132 0.192 31.4 6.09 CMC-INT06-098B Transparent / Translucent 136 0.196 16.2 5.96 CMC-INT06-098C Transparent / Translucent 136 0.173 Not measured 6.14 CMC-INT06-098D Transparent / Translucent 137 0.172 Not measured 6.03 CMC-INT06-098E Transparent / Translucent 139 0.150 4.5 6.2 CMC-INT06-098F Transparent / Translucent 139 0.157 Not measured 6.25

[0451] The free DNA and supercoil % are:

[0452] Entity ID Free DNA Supercoil content (%) CMC-INT06-098A Invisible 94 CMC-INT06-098B Invisible 93 CMC-INT06-098C Invisible 91 CMC-INT06-098D Invisible 92 CMC-INT06-098E Invisible 93 CMC-INT06-098F Invisible 93

[0453] 7. Concentrated PEGylated polyplexes by in-line mixing with PEG-PGA followed by TFF concentration

[0454] 7.1 Procedure

[0455] Concentrated PEGylated polyplexes obtained by in-line mixing with PEG-PGA followed by TFF concentration

[0456] As previously described, DD-X-DNA (pVax-PD-L1-Fc) polyplexes were produced with 5% trehalose at an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL DNA. A solution of PEG-PGA in 5% trehalose was then mixed with the DD-X-DNA polyplex at a 1:1 volume ratio by in-line mixing (each fluid flow at 7 mL / min) to produce a PEGylated polyplex of 0.125 mg / mL DNA and a N:P:A ratio of 7:1:17.5. After incubation at room temperature for 60 minutes, the PEGylated polyplex was concentrated by TFF (regenerated cellulose membrane, MWCO 10 kDa, 15 psig inlet pressure) using the method previously described in US8722646B2. During TFF, aliquots were taken at nominal DNA concentrations of 0.25 mg / mL and 0.5 mg / mL. The nominal DNA concentration of the collected final product was at most about 1 mg / mL. The samples were aliquoted and stored at -80° C. After thawing, the samples were tested for particle size, PDI, zeta potential, free DNA and % supercoiled DNA. Fig.11 A schematic diagram is provided.

[0457]

[0458] 7.2 Results

[0459] The particle size, PDI, zeta potential, free DNA and % supercoiled of the thawed test samples were found to be:

[0460]

[0461] 7.3 Conclusion

[0462] Through the TFF concentration process, the PEGylated polyplexes were able to be concentrated from 0.125 mg / mL DNA to 1 mg / mL DNA while maintaining colloid-stable nanoparticles.

[0463] 8. Concentrated PEGylated polyplexes obtained by PEGylation of pre-concentrated polyplexes

[0464] 8.1 Procedure

[0465] As previously described in US8722646B2, concentrated DD-X-DNA (pVax-PD-L1-Fc) polyplexes were produced with 9% sucrose at a N:P ratio of 7:1 and a DNA concentration of 1 mg / mL DNA. Then a solution of PEG-PGA in water was mixed with the DD-X-DNA polyplex at a volume ratio of 1:1 by in-line mixing (each fluid flow at 7 mL / min) to produce 0.5 mg / mL DNA and a 7:1:7 N:P:A ratio or a 7:1:17.5 N:P:A ratio of PEGylated polyplexes. After incubation at room temperature for 60 minutes, the PEGylated polyplexes were aliquoted and stored at -80°C.

[0466] After thawing, samples were tested for particle size, PDI, zeta potential, % free DNA and supercoiled DNA.

[0467]

[0468] 8.2 Results

[0469] Physicochemical properties of the original polyplexes and PEGylated polyplexes

[0470]

[0471] 8.3 Conclusion

[0472] DD-X-DNA polyplexes of 1 mg / mL DNA were PEGylated at N:P:A ratios of 7:1:7 and 7:1:17.5, giving a final DNA concentration of 0.5 mg / mL with no visible aggregation.

[0473] 9. PEGylation of Polyplexes with PEG-PGA or PEG-PAA

[0474] 9.1 Procedure

[0475] As described herein, an aliquot of N:P 7 polyplex (CMC-INT06-24C) was pegylated using PEG-PGA or PEG-PAA. The resulting pegylated polyplexes were tested for physicochemical properties and DNA capture. The resulting formulations other than N:P 7 polyplexes were produced.

[0476]

[0477] 9.2 Results

[0478]

[0479] Samples AF were tested for free DNA as described herein. The resulting gel showed no visible free DNA.

[0480] 9.3 Conclusion

[0481] Polyplexes PEGylated with PEG-PGA (mPEG5K-b-PLE10) or PEG-PAA (mPEG5K-b-PLD 10) formed stable nanoparticles without free DNA.

[0482] 10. Stability of PEGylated Polyplexes in Simulated Intestinal Fluid

[0483] 10.1 Procedure

[0484] Test Items

[0485] An aliquot of N:P 7 polyplex (CMC-INT06-24C) was PEGylated using PEG-PGA as described herein. The resulting PEGylated polyplex was tested for stability in FaSSIF buffer.

[0486]

[0487] Testing the stability of PEGylated polyplexes in SIF 1:3 v / v

[0488] Add 50uL of polyplex to 150uL of simulated intestinal fluid. Stir thoroughly. Immediately aliquot 50uL of suspension into DLS cuvettes and add 350uL of water and measure particle size by DLS as described. Divide the remaining sample solution into three tubes. At appropriate time points, add 350uL of water to the tubes, mix thoroughly, and measure particle size by DLS.

[0489] Testing the stability of PEGylated polyplexes in SIF 3:1 v / v

[0490] Add 75uL of polyplex to 25uL of simulated intestinal fluid. Stir thoroughly. Immediately aliquot 50uL of suspension into DLS cuvettes and add 350uL of water and measure particle size by DLS as described. Divide the remaining sample solution into three tubes. At appropriate time points, add 350uL of water to the tubes, mix thoroughly, and measure particle size by DLS.

[0491] 10.2 Results

[0492] The unpegylated polyplex (denoted as 7-1-0) aggregated immediately after mixing with the buffer (largest size). The pegylated polyplex (with PEG-PGA, mPEG5K-b-PLE10) remained stable for 24 hours at a ratio of 1:3 (FaSSIF:pegylated polyplex, v / v). The pegylated polyplex (with PEG-PGA, mPEG5K-b-PLE10) remained stable for 1 hour at a ratio of 3:1 (FaSSIF:pegylated polyplex, v / v).

[0493] 11. Stability of PEGylated polyplexes in FaSSIF buffer - free DNA

[0494] Reference: CMC-INT06-EXP-072

[0495] 11.1 Procedure

[0496] As described herein, aliquots of N:P 7 polyplexes (CMC-INT06-024C, DD-X-DNA (pVax-opt-hIL10) polyplexes, 5% trehalose, 0.25 mg / mL) were PEGylated using PEG-PGA (in 5% trehalose) to achieve the target N:P:A ratios in the table below. The stability of the resulting PEGylated polyplexes in FaSSIF buffer was tested according to the ratios in the table below. After 2 hours, the following physicochemical properties were determined: free DNA, pH, diameter, and PDI.

[0497]

[0498] 11.2 Results

[0499]

[0500]

[0501] Free DNA determination of FaSSIF-polyplex samples. Figure 5 As shown, no free DNA was observed.

[0502] Lane descriptions are provided below.

[0503]

[0504] 12. Preparation of PEGylated Polyplexes and In Vitro Transfection

[0505] 12.1 In vitro transfection reagents

[0506]

[0507] 12.2 In vitro transfection consumables

[0508] Material Catalog Number Poly-L-Lysine 96-well Clear Bottom TC Plate 356516 96-well PP, round bottom 3365 15mL tube 430790 50mL tube 430828 Axygen MaxyClear Snaplock Microtubes, 1.5mL 14222155

[0509] In vitro transfection equipment

[0510] describe Manufacturer Centrifuge, Allegra 6R Beckman 12-channel pipette, P 50-1200 Biohit Sartorius 12-channel pipette, P 30-300 Biohit Sartorius 12-channel pipette, P 10-100 Biohit Sartorius Vi-Cell Automatic Cell Viability Analyzer Beckman Incubator, Model 3110 ThermoFisher Centrifuge, Model 5415D Eppendorf MSD Plate Reader Meso Scale Diagnostics Ltd. <![CDATA[Cirascan TM Imager]]> Quanterix / Aushon Titer Plate Shaker, Model 00122 Labline Instruments

[0511] 12.4 Procedure

[0512] Test Items

[0513] As described herein, aliquots of N:P 7 polyplexes (CMC-INT06-024C, DD-X-DNA (pVax-opt-hIL10) polyplexes, 5% trehalose, 0.25 mg / mL) were PEGylated using PEG-PGA (mPEG5K-b-PLE10 in 5% trehalose) to achieve the target N:P:A ratios in the table below. The resulting PEGylated polyplexes were tested for particle diameter, PDI, zeta potential before and after freeze / thaw. Thawed samples were tested for in vitro transfection.

[0514]

[0515]

[0516] 12.5 In vitro transfection procedure

[0517] Cell preparation

[0518] HEK293T cells (ATCC) at passage 19 were passaged and centrifuged to remove trypsin and then resuspended twice in DMEM supplemented with 10% FBS. Cell viability and counting were performed in a Vi-Cell automated cell viability analyzer to verify cell count and viability. Cells were diluted to 1.25 × 10 in DMEM supplemented with 10% FBS. 5 The cells / mL concentration was determined and dispensed into 96-well clear bottom TC plates (200 uL, 25,000 cells per well). The plates were incubated at room temperature for 15-20 minutes and then incubated overnight at 37°C / 5% CO2 before use for transfection.

[0519] Dilution of polyplexes

[0520] Unless otherwise specified, test samples are diluted to 0.125mg / mL DNA, target concentration. Control samples (CMC-INT06-024 and CMC-JAN01-010) are provided with 0.25mg / mL DNA, diluted to 0.14mg / mL in water. All test and control samples are then serially diluted in water, 1 / 1.35, diluted 10 times in total. These serial dilutions are further diluted 17.7 times in Opti-MEM, and 35.6uL of this dilution polyplex is added to each well in duplicate (the final dosage range of the control sample from 282ng polyplex to 19ng polyplex, and the test sample from 251ng polyplex to 17ng polyplex).

[0521] Cell transfection

[0522] Transfection was performed as follows. First, the culture medium was removed from each HEK293T well, and then 0.1 mL Opti-MEM (pH 7.4) was added and then removed. The polyplex sample diluted in Opti-MEM (see the previous section) was added to each well and incubated at 37°C for 3 hours. After incubation, the culture medium was removed and replaced with 0.2 mL complete medium and incubated again at 37°C. After 48 hours, the supernatant was removed and immediately used for MSD determination. The remaining cells were lysed to determine the total protein.

[0523] Total protein quantification

[0524] Using DC TM Protein assay uses BSA for standard curve to determine total protein in each well. Once the supernatant is removed for ELISA, the remaining cell layer is lysed in lysis buffer at 4°C for 10 minutes. Before transferring to a v-bottom 96-well plate, the lysate is pipetted several times (while minimizing bubble formation). The lysate is then clarified by centrifugation at 1000g for 5 minutes at 4°C.

[0525] Preparation of protein standard curve

[0526] Protein Assay Standard II (bovine serum albumin) stock solution was prepared at 5 mg / ml in milli-Q water and stored at 4° C. A standard curve was prepared by making a 1:2 serial dilution of the protein standard in lysis buffer (the same buffer used to prepare the samples).

[0527] Lowry / DCTM Protein Assay

[0528] Working reagent A' was prepared by adding 20 μl of reagent S to each 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 was added to each well. 200 μl of reagent B was then added to each well. The plate was shaken for 5 seconds and incubated at room temperature for 15 minutes to develop color. The absorbance was measured at 750 nm on a SpectraMAX plate reader. The absorbance of the standards was plotted against the concentration of the standards. SpectraMax software curve fitting analysis was used, and the four-parameter algorithm provided the best curve fit for the standard curve. The software also inferred the protein concentration of the sample from the protein standard curve.

[0529] Quantification of hIL-10 protein by MSD assay

[0530] The supernatant was centrifuged at 1000 × g for 10 minutes and then diluted appropriately. All reagents used for the assay were equilibrated to room temperature before use. Standards for the assay were prepared by reconstituting hIL-10 in diluent 2 to a specified concentration, incubating at room temperature for 15 minutes, and then serially diluting 4 times with diluent 2 to prepare a standard curve for 7 doses. 50 μL of standard was added in duplicate to 2 columns 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 300 rpm for 2 hours at room temperature. The plate was washed and then 25 μL of the diluted detection antibody was added. Incubated at 300 rpm for 2 hours at room temperature. The plate was washed and then 150 μL of 2X read buffer T was added. Barcode reading was used using Mesoscale (automatic assignment of VPLEX single points). The absorbance of the standard was plotted against the concentration of the standard, and the amount of hIL-10 in the sample was derived using Prism GraphPad software with a four-parameter algorithm. Once the amount of hIL-10 in each well was determined, it was compared to the amount of hIL-10 from DCs. TM Protein assays were normalized to total protein (ie, ng hIL-10 / mg total protein). Dose response curves were prepared in GraphPad Prism by plotting ng hIL-10 / mg total protein versus the amount of transfected DNA (ng). EC50 and predicted maximum amount of hIL-10 per mg total protein were determined.

[0531] 12.6 Results

[0532] All particles were stable and showed no aggregation. The zeta potential of all samples decreased to -3 mV after PEGylation. The polyplexes showed the same properties after freeze-thaw. The in vitro transfection results of unPEGylated and PEGylated samples were similar ( Figure 6 ).

[0533]

[0534] 13. PEGylation of polystyrene beads

[0535] References: CMC-INT06-EXP-039 and CMC-INT06-EXP-055

[0536] 13.1 Procedure

[0537] PEGylation of orange polystyrene beads

[0538] In general: Transfer 1 mL of amine-modified polystyrene beads to a 7 mL glass vial with a magnetic stir bar. Add 5 uL 1M HCl. In a 1.5 mL eppendorf tube, dissolve 70 mg PEG NHS and 8 mg EDC-HCl in 1 mL water until a clear solution forms. Slowly add 0.7 mL of this solution to the polystyrene suspension. Stir the solution overnight at room temperature while protecting from light. Purify and concentrate the PEGylated orange particles as described below. Use particle size and zeta potential measurements to confirm the PEGylation of the PS particles.

[0539] PEGylation of green polystyrene beads

[0540] In general: Transfer 2 mL of amine-modified green polystyrene beads to a 7 mL glass bottle with a magnetic stir bar. Add 10 uL of 1M HCl. In a 1.5 mL eppendorf tube, dissolve 100 mg of PEG-NHS and 5 mg of EDC-HCl in 1 mL of water until a clear solution forms. Slowly add 0.9 mL of this solution to the polystyrene suspension. Stir the solution overnight at room temperature while protecting from light. Purify and concentrate the PEGylated green particles as described below. Use particle size and zeta potential measurements to confirm the PEGylation of the PS particles.

[0541] PEGylation of blue polystyrene beads

[0542] In general: 1 mL of amine-modified blue polystyrene beads was transferred to a 7 mL glass bottle with a magnetic stir bar. 2 mL of water was added. In a 1.5 mL eppendorf tube, 270 mg of PEG-NHS and 10.34 mg of EDC-HCl were dissolved in 1 mL of water until a clear solution was formed. 0.9 mL of this solution was slowly added to the polystyrene suspension. The solution was stirred overnight at room temperature while protected from light. The PEGylated blue particles were purified and concentrated as described below. Particle size and zeta potential measurements were used to confirm the PEGylation of the PS particles.

[0543] Purification and concentration of PEGylated polystyrene

[0544] Purify and concentrate the PEGylated PS particles as follows. Divide 300uL of the reaction mixture into an Amicon Ultra filter. Centrifuge at 5000rpm for 10 minutes. Remove the liquid at the bottom and add 300uL of water to the top of the filter (the beads remain on the filter). Repeat centrifugation and washing four times to wash the beads. The beads from different filters are pooled on a single filter. Add 300uL of water and centrifuge again. The green and orange beads are suspended in up to about 1.5mL of water.

[0545] The hydrodynamic diameter of the polystyrene particles was measured by mixing 10 uL of the suspension in 390 uL of water. The zeta potential of the polystyrene particles was measured by mixing 20 uL of the particle suspension in 780 uL of 10 mM NaCl.

[0546] 13.2 Results

[0547] At the end of the reaction, a significant decrease in the zeta potential indicates that PEGylation has occurred.

[0548]

[0549] 14. Preparation of RITC-DDX+DNA Polyplex and PEGylated RITC-DD-X+DNA Polyplex

[0550] Reference: Provided below

[0551] 14.1 RITC-DD-X reagent

[0552]

[0553]

[0554] 14.2RITC-DD-X Equipment

[0555] describe Manufacturer NMR, Avance II 500MHz, probe BBI Bruker

[0556] 14.3 Procedure

[0557] RITC labeled DD-X

[0558] Based on the method derived from Carbohydrate Polymers 72 (2008) 616-624, RITC was conjugated to DD-X. Briefly, DD-X stock solution (batch JAN03-009) was dissolved in water to reach a concentration of 40mM and pH 5.8. The DD-X solution was mixed with an equal volume of DMSO and stirred at room temperature for 1 hour, then bubbling with nitrogen for 15 minutes. In a separate container, RITC was dissolved in DMSO (target 3.7mg / mL) and added dropwise to the DD-X / DMSO mixture (RITC / DMSO:DD-X / DMSO 1:8, v / v). The resulting mixture was then stirred at room temperature for 65 hours. RITC-labeled DD-X was purified by water dialysis and then freeze-dried. 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. The conjugation of RITC and DD-X was confirmed by H-NMR spectroscopy: RITC-DD-X was dissolved in (8 mg / mL) D2O+DCl / D2O to a pH of about 2.5.

[0559] RITC-DD-X+DNA polyplex

[0560] A 50 / 50 blend of RITC-DD-X (as described above) and unlabeled DD-X was prepared and then used to prepare N:P 20 polyplexes (0.1 mg / mL DNA) with GWiz-GFP plasmid DNA in 5% trehalose according to the drip method procedure described herein.

[0561] RITC-DD-X+DNA polyplex

[0562] A blend of 33 / 67 RITC-DD-X (as described above) and unlabeled DD-X was prepared and then used to prepare N:P 7 polyplexes (0.25 mg / mL DNA) with pVax-opt-hIL10 plasmid DNA in 5% trehalose according to the in-line mixing procedure described herein.

[0563] PEGylated RITC-DD-X+DNA polyplex

[0564] An aliquot of N:P 7 polyplex was PEGylated using PEG-PGA as described herein. The resulting PEGylated polyplex was tested for particle size, PDI, and zeta potential. The resulting formulations other than N:P 7 polyplex were produced.

[0565]

[0566] 14.4 Results

[0567] Particle size, PDI and zeta potential results

[0568] Sample name N:P:A Ratio Diameter(nm) PDI Zeta potential (mV) CMC-INT06-070 7:1:0 125.2±0.3 0.17±0.01 32.23±1.44 CMC-INT06-075A 7:1:1.75 144.93±1.19 0.19±0.01 -3.04±0.43 CMC-INT06-075B 7:1:2.5 142.67±2.5 0.17±0 -3.06±0.36 CMC-INT06-075C 7:1:3.2 150.04±0.7 0.17±0.02 -3.33±0.1 CMC-INT06-075D 7:1:1.75 Visible aggregation Visible aggregation Visible aggregation CMC-INT06-075E 7:1:2.5 Visible aggregation Visible aggregation Visible aggregation CMC-INT06-075-F 7:1:3.2 Visible aggregation Visible aggregation Visible aggregation CMC-INT06-075G 7:1:1.75 142.10±1.04 0.17±0.0 -3.01±0.55 CMC-INT06-075H 7:1:2.5 146.5±1.21 0.17±0.02 -2.73±0.58 CMC-INT06-075I 7:1:3.2 149.77±1.83 0.17±0.01 -3.14±0.68

[0569] Multimeric complexes CMC-INT06-075 D, E and F aggregate.

[0570] 15. Assessment of the aggregation of different PS beads within type III mucin and quantification of their diffusion through 1 and 3 μm transwells

[0571] Reference: CMC-INT06-EXP-042

[0572] 15.1 Mucin diffusion materials

[0573]

[0574] 15.2 Mucus diffusion equipment

[0575] describe Manufacturer Plate reader, EnVision 2105 PerkinElmer Analytical Balance Mettler Toledo Incubator, Model 3110 ThermoFisher Microscope, EVOS FL Auto Thermofisher

[0576] 15.3 Procedure

[0577] Preparation of type III mucin suspension

[0578] The Type III mucin suspension is prepared by dispensing the Type III mucin into water to achieve a target concentration of 5% w / w to 0.1% w / w as desired.

[0579] Aggregation in mucin testing

[0580] The test sample (RITC-labeled polyplex, amine-modified PS beads, or PEGylated PS beads) was combined with the type III mucin suspension in microtubes at various concentrations to achieve the final target concentration of the test sample and mucin. The sample-mucin samples were protected from light and mixed at 30 rpm at room temperature. At 30 and 60 minutes, the mixture was aliquoted onto an Ibidi u-slide and observed under an EVOS microscope for visible aggregation.

[0581] Assessment of diffusion across mucin-loaded transwells

[0582] Sample preparation

[0583] The test sample (RITC-labeled polyplex, amine-modified PS beads or PEGylated PS beads) was combined with the type III mucin suspension in a microtube to achieve a final target concentration of the test sample and a final mucin concentration of 0.4%. The samples were protected from light and mixed at 40 rpm for 1 hour before loading into the transwell insert.

[0584] Transwell preparation and sample diffusion

[0585] The bottom of the transwell was filled with 0.6 mL of 0.4% type III mucin. HTS transwell inserts (with 1 um or 3 um PET membrane) were loaded with 0.1 mL of premixed sample + mucin. The control was simply not loaded with 0.4% mucin. The reference (simulating 100% diffusion) was the premixed sample + mucin (0.7 mL) without the transwell insert. After incubation at 37°C for 20 hours, diffusion of the test sample into the bottom well was evaluated by a fluorescent plate reader (Envision).

[0586] 15.4 Results

[0587] Aggregation in mucin testing

[0588] like Figure 7 As shown, both RITC-labeled polyplexes and amine-modified PS beads showed aggregation in mucin after 1 hour of incubation. PEGylated PS beads showed no visible aggregation.

[0589] Assessment of diffusion across mucin-loaded transwells

[0590] like Figure 8 As shown, the PEGylated PS beads had the highest diffusion level across the 3um or 1um PET membrane after 20 hours of incubation in 0.4% mucin compared to the amine-modified PS beads and RITC-labeled polyplexes. This is due to less aggregation of the PEGylated PS beads in mucin.

[0591] 16. Effect of Transwell Repeated PEGylated Polyplex Preparations on Diffusion Across Type III Mucin and Pluronics F127

[0592] 16.1 Materials

[0593]

[0594] 16.2 Equipment

[0595] describe Manufacturer Plate reader, EnVision 2105 PerkinElmer Analytical Balance Mettler Toledo Incubator, Model 3110 ThermoFisher Microscope, EVOS FL Auto Thermofisher

[0596] 16.3 Procedure

[0597] 4% Pluronics F127 solution preparation

[0598] Pluronics F127 was dissolved in water to reach 4% w / w and filtered at 0.22 um.

[0599] Preparation of type III mucin suspension

[0600] Type III mucin suspensions were prepared as described herein.

[0601] Aggregation in mucin testing

[0602] The test samples (PEGylated RITC-DD-X + DNA polyplexes, RITC-labeled polyplexes, amine-modified PS beads, or PEGylated PS beads) were combined with the type III mucin suspension in microtubes at various concentrations to achieve the final target concentration of the test sample and mucin. The sample-mucin samples were protected from light and mixed at 30 rpm at room temperature. At 30 and 60 minutes, the mixtures were aliquoted onto Ibidi u-slides and observed under an EVOS microscope for visible aggregation.

[0603] Assessment of diffusion across mucin-loaded transwells

[0604] Sample preparation

[0605] The test samples (PEGylated RITC-DD-X + DNA polyplex, RITC labeled polyplex) were combined with type III mucin suspensions in microtubes with or without Pluronics F127 solution to achieve the final target concentration of the test sample and a final mucin concentration of 0.5%. Controls (amine-modified PS beads, PEGylated PS beads) were combined with mucin only to a final mucin concentration of 0.5%. Samples were protected from light and mixed at 40 rpm for 1 hour before loading into transwell inserts.

[0606] Transwell preparation and sample diffusion

[0607] The bottom of the transwell was filled with 0.6 mL of 0.5% type III mucin. The HTS transwell insert with 3um PET membrane was loaded with 0.1 mL of premixed sample + mucin. The reference (simulating 100% diffusion) was premixed sample + mucin (0.7 mL) without transwell insert. After incubation at 37°C for 20 hours, diffusion of the test sample into the bottom well was evaluated by a fluorescent plate reader (Envision).

[0608] 16.4 Results

[0609] PEGylation of PS beads

[0610] PEGylation of amine-modified PS beads increased mucin diffusion by nearly 10-fold ( Fig. 9 ). Fluorescence microscopy showed that PEGylated PS beads did not aggregate in mucin, whereas non-PEGylated PS beads severely aggregated under the same conditions.

[0611] Diffusion of PEGylated polyplexes in mucins

[0612] PEGylation of the polyplexes (N:P 7) increased mucin diffusion from 32% to 50-52%, which is approximately 60% higher than the diffusion of the unPEGylated polyplexes ( Fig. 9 ). The N:P:A ratios tested behaved similarly on mucin spreading. Fluorescence microscopy revealed no significant morphological differences between PEGylated and non-PEGylated polyplexes.

[0613] Effects of Pluronics F127 on mucin diffusion

[0614] The presence of Pluronics F127 increased mucin diffusion (10-15%) in both non-PEGylated and PEGylated polyplexes ( Fig.10 ).

[0615] 17. Lyophilization of PEGylated polyplexes prepared in 2.5% or 5% trehalose at c125 at respective NPA ratios

[0616] Reference: CMC-INT06-EXP-103

[0617] 17.1 Freeze-dried materials

[0618]

[0619]

[0620] 17.2 Freeze Drying Equipment

[0621] project supplier Lyostar II Freeze Dryer FTS Stainless steel tray SP scientific Oven, Model 0F-02 Jeio ​​Tech Autoclave N / A

[0622] 17.3 Procedure

[0623] As previously described, DD-X-DNA (pVax-PD-L1-Fc) polyplexes were produced at an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL DNA with 5% trehalose. An aliquot of the polyplex was then PEGylated (mPEG5K-b-PLE10) as previously described. The resulting formulations, except for the N:P 7 polyplex, were produced and filled into 2 mL glass vials (1 mL and 0.1 mL fill volumes were tested).

[0624]

[0625] Lyophilize the samples using the following controlled freeze-drying cycle:

[0626] Freezing: from +20°C to -40°C (at 1°C / min), then at -40°C for 120 minutes.

[0627] Primary drying: 3800 min at -32°C and 63 mTorr

[0628] Secondary drying: P = 63 mTorr, shelf temperature -32°C to 30°C (0.2°C / min), then at 30°C for 360 minutes.

[0629] At the end of the secondary drying, the vials were purged with nitrogen, stoppered, and allowed to equilibrate to room temperature. Short-term stability was performed at room temperature (ambient humidity), in a desiccator at room temperature, and at 4°C. At selected time points, 1 mL samples were rehydrated with water and incubated for 15 minutes before analysis. Samples were analyzed for particle size, PDI, zeta potential, and free DNA as described herein.

[0630] 17.4 Results

[0631] The lyophilized cake showed no signs of collapse or cracking. The 0.1 mL sample was circular due to insufficient volume to form a cake. No free DNA was observed in the rehydrated sample.

[0632] Fig.13 The stability of freeze-dried PEGylated polyplexes is shown for up to 4 weeks at room temperature and 4° C. Samples were rehydrated to the volume prior to freeze-drying.

[0633] 18. Screening of potential lyophilized excipients for in-pipeline compounding of PEG-PP by freeze-thaw method

[0634] 18.1 Excipient Screening Materials

[0635] Material Catalog Number Mannitol M4125-100G Kollidon 12PF(PVP 2kDa) 50348141 PEG 4kDa 95904-250G-F

[0636] 18.2 Procedure

[0637] Preparation of mannitol solution

[0638] Typically, mannitol is dissolved in water at a concentration of 0.125 g / mL. The resulting solution is filtered using a 0.2 um filter. If necessary, the mannitol solution is diluted to the desired concentration required for subsequent formulations.

[0639] Preparation of Kollidon 12 (PVP 2) solution

[0640] Typically, PVP 2 is dissolved in water at a concentration of 0.05 g / mL. The resulting solution is filtered using a 0.2 um filter. If necessary, the PVP 2 solution is diluted to the desired concentration required for the subsequent formulation.

[0641] Preparation of PEG 4kDa solution

[0642] Typically, PEG 4kDa is dissolved in water at a concentration of 0.05 g / mL. The resulting solution is filtered using a 0.2 um filter. If necessary, the PEG 4kDa solution is diluted to the desired concentration required for subsequent formulations.

[0643] Preparation of formulations

[0644] As previously described, DD-X-DNA (pVax-PD-L1-Fc) polyplexes were generated at an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL DNA without other excipients. An aliquot of the polyplex was then PEGylated (mPEG5K-b-PLE10) as previously described.

[0645]

[0646] Aliquots of unPEGylated polyplex (CMC-INT06-112A) or PEGylated polyplex (CMC-INT06-112B) were mixed with water or various concentrations of different excipients to achieve the following final target excipient concentrations and a final DNA concentration of 0.1 mg / mL.

[0647]

[0648]

[0649] The resulting formulation was aliquoted and: filled into cryovials and frozen at -80°C; or lyophilized as described herein. Thawed samples were analyzed for particle size, PDI, zeta potential, pH, supercoiled DNA % and free DNA as described herein. Lyophilized samples were assigned individual sample IDs as provided below.

[0650]

[0651]

[0652] Lyophilized samples were rehydrated with water to the original concentration before drying and analyzed for particle size, PDI, zeta potential, pH, % supercoiled DNA, and free DNA as described herein.

[0653] 18.3 Results

[0654] The non-PEGylated polyplex (CMC-INT06-112 A1) in water alone aggregated severely after freeze-thaw. Therefore, no further testing was performed on this sample.

[0655] The PEGylated polyplex preparations in water or any of the excipients or concentrations tested were translucent and other physicochemical properties were unchanged from those before freeze-thaw.

[0656]

[0657]

[0658] Appearance of freeze-dried cake

[0659] The lyophilized cake of the non-PEGylated polyplex (CMC-INT06-116 A1) in water alone had an inconsistent appearance and collapsed. The lyophilized cakes of the PEGylated polyplex formulations (CMC-INT06-116 B1 to CMC-INT06-116B11) in water or any excipient or tested concentration were uniform and had no cracking and slightly shrunken.

[0660] Physicochemical properties of freeze-dried samples after rehydration

[0661] The unpegylated polyplex (CMC-INT06-116 A1) in water alone aggregated severely after rehydration. Therefore, no further testing was performed on this sample. The pegylated polyplex formulations in water or any excipient or tested concentration were translucent and other physicochemical properties did not change compared to before freeze-thaw.

[0662]

[0663]

[0664] 18.4 Conclusion

[0665] In the absence of excipients, PEGylated polyplexes using PGA(1.3k)-PEG(5k) at a N:P:A ratio of 7:1:17.5 prevented aggregation of the polyplexes after freeze-thaw or lyophilization. Trehalose, mannitol, PEG4kDa or PVP 2kDa were not required to prevent aggregation of the PEGylated polyplexes after freeze-thaw or lyophilization, and they did not cause aggregation at the concentrations tested.

[0666] Lyophilization of PEGylated polyplexes concentrated and diluted in 19.5% trehalose

[0667] Reference: CMC-INT06-EXP-110 and CMC-INT06-128

[0668] 19.1 Procedure

[0669] As previously described, DD-X-DNA (pVax-PD-L1-Fc) polyplexes were produced at an N:P ratio of 7:1 and a DNA concentration of 0.25 mg / mL DNA with or without 5% trehalose. Aliquots of the polyplexes were then PEGylated (mPEG5K-b-PLE10) as previously described and then concentrated using a TFF process. The resulting formulations, except for the N:P 7 polyplex, were produced and filled into 2 or 10 mL glass vials (0.3 mL, 1 mL, and 2 mL fill volumes were tested).

[0670]

[0671] Lyophilize the samples using the following controlled freeze-drying cycle:

[0672] Freezing: from +20°C to -40°C (at 1°C / min), then at -40°C for 120 minutes.

[0673] Primary drying: 3800 min at -32°C and 63 mTorr

[0674] Secondary drying: P = 63 mTorr, shelf temperature -30°C to 30°C (0.2°C / min), then at 30°C for 6 hours.

[0675] At the end of the secondary drying, the vials were purged with nitrogen, stoppered, and allowed to equilibrate to room temperature. The samples were rehydrated with water to achieve different multiples of concentration: 1X or 10X). The samples were analyzed for particle size, PDI as described herein.

[0676] 19.2 Results

[0677] The freeze-dried sample in the 1 mL fill volume had a firm, slightly shrunken and cracked cake appearance. The freeze-dried samples rehydrated to 1 mg / mL and 10 mg / mL had a translucent appearance with no visible aggregates. The freeze-dried sample rehydrated to 25 mg / mL formed a viscous gel ( Fig.12 ). The freeze-dried sample rehydrated to 50 mg / mL formed a paste because the volume of water was insufficient to form a suspension.

[0678] Freeze-dried samples rehydrated to 10 mg / mL and 25 mg / mL were tested for nanoparticle size, PDI, and zeta potential, with no apparent aggregation at 4 °C for at least 48 h under either condition ( Fig.12 ).

[0679] 20. Freeze drying of PEG-PP (various NPA ratios) prepared in 4.5% sucrose or water

[0680] Reference: CMC-INT06-129

[0681] 20.1 Procedure

[0682] As previously described, DD-X-DNA (pVax-PD-L1-Fc) polyplexes were generated at a 7:1 N:P ratio and a DNA concentration of 0.125 mg / mL DNA without other excipients. Aliquots of the polyplexes were then PEGylated (mPEG5K-b-PLE10) as previously described. Additional test samples CMC-INT06-124A and CMC-INT06-124B have been described herein.

[0683]

[0684] The resulting formulation was filled into 2 mL glass vials (0.3 mL and 1 mL fill volumes were tested) and lyophilized using the following controlled freeze drying cycle:

[0685] Freezing: from 20°C to -40°C (at 1°C / min), then at -40°C for 120 minutes.

[0686] Primary drying: 3660 minutes at -35°C and 63 mTorr

[0687] Secondary drying: P = 63 mTorr, shelf temperature -35°C to 30°C (0.2°C / min), then at 30°C for 360 minutes.

[0688] At the end of the secondary drying, the vials were purged with nitrogen, stoppered, and allowed to equilibrate to room temperature and stored at 4°C until use.

[0689] For excipient-free samples (CMC-INT06-129 A, CMC-INT06-129 B, CMC-INT06-129 C), short-term stability was performed at 4°C. 4.5% sucrose samples (CMC-INT06-129 D and CMC-INT06-129E) were tested at time 0. At selected time points, the samples were rehydrated with water to reach the concentration before lyophilization. The particle size, PDI and zeta potential of the rehydrated samples were analyzed as described herein.

[0690] 20.2 Results

[0691] All formulations were successfully lyophilized. Formulations without lyoprotectants showed retraction / radial shrinkage; those with lower NPA ratios (i.e., NPA = 7:1:7) and lower fill volumes (0.3 mL) were more susceptible to static electricity (upward movement of the cake, lateral movement, cracking, etc.). Formulations containing 4.5% sucrose showed only limited retraction and had a smooth surface.

[0692] Description of freeze-dried samples

[0693]

[0694] Appearance, particle size, PDI and zeta potential of rehydrated samples at time 0.

[0695]

[0696]

[0697] The stability of the lyophilized samples (particle size, PDI and zeta potential of the rehydrated samples) for up to 4 weeks at 4 °C in the absence of excipients was Fig.14 The results showed no change in particle size and zeta potential, and limited or no increase in PDI.

[0698] The following table provides an overview of current findings:

[0699]

[0700] Embodiment 2:

[0701] Unless otherwise described, the general methods, compositions, and procedures were as described in Example 1 above.

[0702] 80 μL of reversibly PEGylated dual derivatized chitosan polyplex (DDX) was administered to the mouse bladder at 250 μg / mL, where PEG contained a polyglutamic acid tail and the polyplex contained an NPA ratio of 7:1:7 (medium PEGylation) or 7:1:17.5 (high PEGylation). As a control, 80 μL of unPEGylated polyplex at a NP ratio of 7:1 was administered to the mouse bladder. The administered formulation was incubated in the bladder for 1 hour, and the bladder contents were then collected for analysis. Fig.15 As shown, incubation in the bladder resulted in severe aggregation of the non-PEGylated polyplexes. In contrast, neither the highly PEGylated polyplexes nor the moderately PEGylated polyplexes showed detectable aggregation after incubation in the bladder.

[0703] Embodiment 3:

[0704] The in vivo gene transfer efficiency of reversibly PEGylated and unPEGylated DDX particles was compared. UnPEGylated DDX particles at NP7, NP15, and NP20 and PEGylated DDX at NPA 7:1:3.5 (low PEGylation) and NPA 7:1:17.5 (high PEGylation) were tested. As described in Examples 1 and 2, reversibly PEGylated DDX particles were coated with PEG-polyglutamate. Each DDX formulation was tested at two different concentrations (125 μg / mL and 1,000 μg / mL). The formulations were injected by intracolonic instillation (ICI) in three 150 μL doses. After 24 hours, colon samples were collected and analyzed for transgene mRNA expression. As Fig.16 As shown, the PEGylated DDX particles achieved significantly improved gene delivery (>10-fold) as detected by an increase in copy number expression. The % of samples with detectable gene expression was significantly increased in the PEGylated DDX formulations.

[0705] In addition to mRNA expression, expression of transgene encoded protein was also evaluated. Unpegylated DDX particles at NP15 (Group 2) and NP20 (Group 3) and pegylated DDX at NPA7:1:3.5 (Group 7) and NPA 7:1:17.5 (Groups 5 and 6) were tested. Reversibly pegylated DDX particles were coated with PEG-polyglutamate as described in Examples 1 and 2. Each DDX formulation was tested at 125 μg / mL. Formulations were injected by intracolonic instillation (ICI) in three 150 μL doses. After 24 hours, cell samples were collected and analyzed for transgene protein expression using a Mesoscale Discovery immunoassay. As Fig.17 As shown, expression of the transgene-encoded protein was significantly increased in the colon of mice treated with PEGylated DDX particles relative to non-PEGylated DDX particles.

[0706] The protein expression experiment was repeated with unpegylated DDX particles at a 7:1 NP ratio and pegylated DDX particles at a 7:1:17.5 NPA ratio, each at 1000 μg / mL. The formulations were administered using a 3×150 μL ICI procedure; colon sections were harvested 24 hours after administration, and protein lysates were used to quantify human PD-L1-Fc protein using a Mesoscale Discovery immunoassay. Fig.18 As shown, expression of the transgene-encoded protein was significantly increased in the colon of mice treated with PEGylated DDX particles relative to non-PEGylated DDX particles.

[0707] Embodiment 4:

[0708] The general method outlined in the previous examples was used to produce PEGylated DDX nanoparticles and their physicochemical parameters were determined. Double derivatized polyplexes with a final functionalization degree of 25% cation (arginine (R)) and 10% polyol (glucose (G)) were prepared at NP20:1 (unPEGylated) and NPA10:1:5 (PEGylated) ratios. Lyophilization protectants (5% trehalose or 1% mannose) were selected based on the improved lyophilization protection provided for the corresponding unPEGylated and PEGylated polyplexes. The preparations were frozen or lyophilized and then melted or rehydrated when applicable. The supercoiled DNA %, appearance, particle size, PDI, zeta potential and pH of the test samples were tested.

[0709] like Fig.19 As shown, at t=0, the supercoiled DNA% is significantly lower for the non-PEG lyophilized than the equivalent PEGylated formulation. The supercoiled DNA% is indicative of the amount of nucleic acid delivery material and should preferably be >80% and more preferably >90%.

[0710] Polyplexes were prepared with a 10:1:5 NPA (PEGylation) ratio. For these polyplexes, the final degree of functionalization of cation / polyol was 28% R and 10% G, and the DNA concentration was 125 μg / mL. The lyoprotectant 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. Fig. 20 As shown, lyophilized PEGylated DDX formulations were stable for up to 12 weeks when stored at 4°C or room temperature (RT).

[0711] Polyplexes were prepared at a ratio of 10:1:5 for NPA (PEGylated) and 20:1 for NP (unPEGylated). For these polyplexes, the final degree of functionalization of the cation / polyol was 28% R and 10% G, and the DNA concentration was 1000 μg / mL. Lyoprotectants (5% trehalose or 1% mannitol) were selected based on the improved lyophilization protection provided to the corresponding unPEGylated and PEGylated polyplexes. 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). The samples were tested for particle size and PDI. Fig.21 As shown, the lyophilized PEGylated formulation rehydrates stably up to 10 mg / mL, while the non-PEGylated formulation can be stably rehydrated to 2 mg / mL. Although the 2 mg / mL rehydration concentration provides a useful gene delivery formulation, the unexpectedly significant increase in the achievable stable DNA concentration of the PEGylated formulation (10 mg / mL) has significant benefits in terms of the dose volume required to achieve a therapeutic or clinically relevant effect.

[0712] Polyplexes were prepared at a ratio of 10:1:5 for NPA (PEGylated) and 10:1 for NP (unPEGylated). For these polyplexes, the final degree of functionalization of cation / polyol was 28% R and 10% G. DNA concentrations of 125 μg / mL containing 1% mannitol (PEGylated polyplexes) or 5% trehalose (unPEGylated polyplexes) were applied to mouse bladders, incubated for 1 hour, and bladder contents were collected for analysis. The visual appearance and nanoparticle size of the samples were examined by dynamic light scattering. Fig. 22 As shown, the PEGylated polyplexes showed no detectable aggregation, while the non-PEGylated polyplexes showed severe aggregation, as shown by the increase in size and polydispersity index (PDI) in the left image and the appearance of white clots in the collected urine sample in the right image.

[0713] Polyplexes were prepared at a ratio of 10:1:5 for NPA (PEGylated) and 20:1 for NP (unPEGylated). For these polyplexes, the final degree of functionalization of cations / polyols was 28% R and 10% G. Formulations containing 1% mannitol (PEGylated polyplexes) or 5% trehalose (unPEGylated polyplexes) were analyzed 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 passed through 0.2 μm pore filters (13 mm diameter, 1 cm 2Surface area) filtration: PES (polyethersulfone), PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene) and Nylon. The samples were tested for nanoparticle size, zeta potential, conductivity, pH, cation (arginine) and polyol (glucose) content, and DNA content. Fig.23 As shown, the non-PEGylated polyplex preparations exhibited a significantly greater decrease in DNA concentration after filtration (15-32%) compared to the PEGylated polyplex preparations (<10%). These results suggest that the non-PEGylated polyplexes aggregate under the assay conditions.

[0714] To confirm Fig.23 Based on the results shown in, the production of PEGylated and non-PEGylated DDX chitosan-DNA polyplexes was scaled up and the filtration rates of the formulations were tested at larger volumes. For these polyplexes, the final degree of functionalization of the cation / polyol was 28% R and 10% G. The sterile filtration suitability of formulations containing 1% mannitol (PEGylated polyplexes) or 5% trehalose (non-PEGylated polyplexes) was analyzed. 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 passed through a 0.8 / 0.2 μm pore PES filter stack (25 mm diameter, 2.8 cm 2 Filtration was performed using a constant pressure Vmax filtration setting at 30 psig (e.g. Fig.24 Schematic representation). The filtration rate was determined as the mass of filtrate collected per surface area. The samples before and after filtration were tested for nanoparticle size, zeta potential, conductivity, pH, cation / polyol content, and DNA content.

[0715] like Fig.24 As shown, the non-PEGylated polyplex preparations showed a significantly greater decrease in DNA concentration after filtration (19%) compared to the PEGylated polyplex preparations (<10%). 2 The non-PEGylated polyplex preparations blocked the filter device when the maximum concentration provided the membrane surface area. In contrast, the PEGylated polyplex preparations blocked the filter device at concentrations up to 5.35 g polyplex / cm 2 All tested polyplex concentrations did not clog the filter, indicating that the surface area was higher than 5.35 g / cm 2 The PEGylated polyplex preparations were still filterable.

[0716] by Fig.25Polyplexes were prepared with the NPA (PEGylated) or NP (unPEGylated) ratios shown in and a DNA concentration of 0.125 mg / mL (c125). The cation / polyol number ratios were 25% R and 10% G. The lyoprotectant was 1% mannitol (1% Man). The preparations were either freeze-thawed (FT) or freeze-dried and rehydrated (FD). The samples were tested for supercoiled DNA %, nanoparticle size, and zeta potential. Fig.25 As shown, the non-PEGylated DDX formulations precipitated after freeze / thaw and lyophilization / rehydration. It was not possible to measure supercoiled DNA% on 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 formulation indicated a certain degree of aggregation after freeze / thaw, but significantly less than the non-PEGylated material. After lyophilization / rehydration, all PEGylated DDX formulations tested (NPA 10:1:5 to 10:1:1) did not show any detectable aggregation.

[0717] A PEGylated DDX polyplex was prepared and delivered to the small intestine of dogs by direct instillation. Fig.26 As shown, mRNA copy number was detected 24 hours after delivery, demonstrating gene delivery in dogs.

[0718] The PEGylated polyplexes were mixed with acetate buffer at various ratios to simulate a low pH environment, and the pH of the resulting solution was determined. The zeta potential of the particles was also monitored. When the pH was below the pKa of the anionic anchoring region of the polymer coating (approximately 4.25), the zeta potential increased dramatically, indicating release of the polymer coating. Fig. 27 .

[0719] Embodiment 5:

[0720] Reversibly PEGylated polyplexes containing dual derivatized chitosan (Arg:gluconic acid) were prepared with a N:P:A ratio of 7:1:X and polyglutamic acid anchors of different sizes, wherein X is 3.5, 9, or 14.5. PLE5 refers to a polyplex comprising a PEG-polyglutamic acid (PEG-PLE) polymer, wherein the length of the polyglutamic acid anchor is 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 resulting PEGylated polyplexes are shown in the table below.

[0721]

[0722]

[0723] The resulting PEGylated polyplexes were subjected to particle size measurement and no significant variation in size was found among the prepared polyplexes. Fig.28 Dispersing the polyplexes in water and measuring the pH indicated that both the larger PLE length and the decreased N:A ratio contributed to the increase in the pH of the aqueous dispersion. Fig.29 Likewise, zeta potential measurements showed that longer PLE anchor regions and decreased N:A ratios both resulted in decreased zeta potential. Fig.30 (Left) In addition, the inventors observed that the zeta potential of the PLE25 multiplex plateaued when the N:P:A ratio was adjusted from 7:1:X, X=9 to 7:1:X, X=14.5, indicating that maximal PEGylation was achieved at lower amounts of polymer. Fig.30 (right).

[0724] The PEGylated polyplexes were assayed in a polyaspartic acid (PAA) competition assay, where PAA was mixed in an aqueous buffer containing various concentrations of the PEGylated polyplexes and the accessibility of nucleic acids was observed. Nucleic acid accessibility was measured by a picogreen assay. Accessible nucleic acids bind to picogreen and the increase in fluorescent signal caused by binding is detected. Completely released and / or fully accessible nucleic acids provide the maximum fluorescent signal. The EC50 of the PAA concentration required to achieve half-maximal signal indicates how easily the particle composition is disrupted by PAA contact, which is a measure of the stability of the test polyplex. The results show that the stability of the PLE25 PEGylated polyplex is slightly lower than that of the other polyplexes. Figure 31-32 .

[0725] PEGylated polyplexes were prepared as c125 (125 μg / mL DNA) reaction mixtures and then mixed with FaSSIF-V2 at a 1:2 volume ratio (FaSSIF in excess). DLS measurements were performed at 37°C at time zero (t0) and 30 minutes (t=30) after mixing with FaSSIF-V2 to measure particle size and polydispersity ( Fig.33 ), and also measured the zeta potential and pH of the sample ( Fig.34 Under the conditions tested, the longer the polyanion chain length (e.g., PLE25 was the most stable and PLE5 was the least stable) and the higher the amino functionalization of the chitosan (e.g., N:P:A 14.5 was the most stable and N:P:A 3.5 was the least stable), the more stable the polyplex was.

[0726] Embodiment 6:

[0727] The effect of polyanionic species and molecular weight (MW) on the PEGylated polyplexes was examined. PLE (polyglutamic acid), PLD (polyaspartic acid, PAA) and HA (hyaluronic acid) were studied. A 4.51% trehalose solution was used as a storage stabilizer. A PEG polyanion working solution was prepared.

[0728] PEG-HA 25 does not dissolve in the trehalose diluent and becomes a gel-like substance. The gel does not dissolve after dilution 100 times. PEG-HA 25 was omitted from further analysis. The resulting c250 solution was mixed with the PEG polyanion at a 1:1 v / v ratio by adding the PEG polyanion solution dropwise to the c250 solution while vortexing. After adding the PEG polyanion, continue vortexing for 10 seconds. The following PEGylated polyplex was produced.

[0729]

[0730] The above compositions were incubated at ambient temperature for 1 hour before further analysis.

[0731] In addition, the solution of the double derivatized chitosan nucleic acid polyplex was diluted from 1000 μg / mL nucleic acid concentration (c1000) to 125 μg / mL (c125) nucleic acid concentration in 4.51% trehalose solution. After freeze-thaw (F / T), all samples were tested for appearance, pH, size, zeta potential, and free DNA content. The results are shown in the following table and Figure 35-36 middle.

[0732]

[0733]

[0734]

[0735]

[0736] The polyplex samples were analyzed by agarose gel electrophoresis to detect uncomplexed nucleic acids. Fig.37 In addition to the 7:1:30 N:P:A polyplex PEGylated with PEG-PLD50 ( Fig.37 , bottom rows 6-8), all samples tested showed no detectable uncomplexed nucleic acids.

[0737] The results show the following. For PEGylation, PEG-PLE10, PEG-PLD10 and PEG-PLD50 are compatible with DDX polyplexes at a 7:1 NP ratio. No precipitation was observed in any c125 PEGylated polyplex solution after F / T. PEG-PLE10 and PEG-PLD10 behaved similarly when mixed with polyplexes. The size of the polyplex increased from 140nm to 160nm after PEGylation. And for NPA ratios of 7:1:3.5, 7:1:5, 7:1:9, 7:1:15 and 7:1:30, the size was stable at 160nm. The zeta potential decreased with increasing PEG polyanion ratios, but the decrease slowed down after exceeding 7:1:9, indicating that PEGylation may reach saturation. For PEG-PLD50, after PEGylation, at NPA ratios up to 7:1:15, the polyplex size was comparable to the polyplex before PEGylation (7:1:0). At NPA7:1:30, the polyplex size increased to 440nm after PEGylation. The zeta potential of the PEG-PLD50 PEGylated polyplex decreased with increasing PEG-PLD50 ratios. Under PEG-PLD50, for 7:1:30, free DNA was observed, while no free DNA was seen for the other formulations, indicating that the nucleic acids in the polyplex of 7:1:30 PEG-PLD50 were loosely wrapped and / or that PLD-50 disrupted the binding of the derivatized chitosan to the nucleic acids.

[0738] Embodiment 6:

[0739] The effects of non-covalent reversible PEGylation were compared with covalent PEGylation. Fig.38 As shown, both non-covalent PEGylated polyplexes and covalent PEGylated polyplexes with titratable polyanion anchoring regions are expected to have a zeta potential close to neutral under pH conditions that maintain a negative charge on the polyanion anchoring region. After the polyanion anchoring region is titrated to neutral or positive charge at low pH (e.g., pH 2), the zeta potential of the non-covalent PEGylated polyplex should increase detectably. In contrast, covalently attached PEG cannot be released and the zeta potential should not change much.

[0740] Two different covalent PEGylation strategies were investigated, e.g. Fig.39 In the first, PEG-PLE was cross-linked with doubly derivatized (DDX) chitosan to form a cross-linked PEG-chitosan stock, and then DNA was added to form a polyplex (PEG cross-linked to chitosan). Fig.39 , middle row). In the second, a reversibly PEGylated polyplex was formed and EDC / NHS was added to crosslink the PEG-PA polymer to the polyplex (PEG crosslinks to the polyplex). ( Fig.39 , bottom). After adjusting the pH from 6 to 2, both covalently PEGylated polyplexes showed minimal changes in zeta potential. In contrast, the zeta potential of the reversibly PEGylated polyplex increased significantly. The stability of the polyplexes was also tested by attack with free polyaspartic acid (PAA). Nucleic acids in the polyplexes were released from the reversibly PEGylated polyplexes by PAA attack, and PEG was cross-linked to the chitosan polyplexes. In contrast, cross-linking after polyplex formation provided polyplexes that did not release nucleic acids after PAA attack under the conditions tested.

[0741] The transfection efficiency of the reverse PEGylated polyplexes at N:P:A7:1:X, where X is 9, 3 or 1, was determined and compared with the unPEGylated polyplexes (7:1:0). The results showed that the transfection efficiency was not reduced by reverse PEGylation. Fig.40 .

[0742] Compare the transfection efficiency of reversibly PEGylated and covalently PEGylated polyplexes. Fig.41 As shown, the reversibly PEGylated polyplexes maintained high transfection efficiency, whereas both types of covalently PEGylated polyplexes showed poor transfection efficiency.

[0743] Embodiment 7:

[0744] The transfection efficiency of reversibly PEGylated polyplexes formed by mixing nucleic acid, chitosan and PEG-polyanionic polymer in one step (one step) was compared with that formed by mixing preformed chitosan DNA polyplexes with PEG-polyanionic polymer (two steps). mRNA expression was detected in colon tissue 24 hours after intracolonic delivery of one-step or two-step PEGylated polyplexes. mRNA and protein were detected in bladder tissue 24 hours after intravesical delivery of one-step or two-step PEGylated polyplexes. Figure 42-43 As shown, the results indicate that the one-step polyplex and the two-step polyplex exhibited similar transfection efficiency under the conditions tested. Without wishing to be bound by theory, it is hypothesized that the generally smaller size of the one-step polyplex may exhibit superior diffusion properties in highly viscous mucosa.

[0745] ****

[0746] Equivalent solutions

[0747] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety and for all purposes, and the extent of their reference is as if each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. The above disclosure may cover a plurality of different inventions with independent utility. Although each of these inventions is disclosed in its preferred form, the specific embodiments thereof disclosed and described herein should not be considered restrictive, as many variations are possible. The subject matter of the present invention includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions and / or properties disclosed herein. The following claims specifically point out certain combinations and sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and sub-combinations of features, functions, elements and / or properties may be claimed in this application, an application claiming priority to this application, or a related application. Such claims, whether for different inventions or for the same invention, and wider, narrower, equal or different in scope than the original claims, are also considered to be included in the subject matter of the invention disclosed herein.

[0748] This application also includes the following specific implementation schemes:

[0749] 1. A composition comprising:

[0750] (a) a complex comprising chitosan derivative nanoparticles and at least one nucleic acid molecule, wherein the chitosan derivative nanoparticles comprise amino-functionalized chitosan, wherein the at least one nucleic acid molecule is non-covalently bound to the chitosan derivative nanoparticles at an amino to phosphorus (N:P) molar ratio greater than 3:1, thereby forming a derivatized chitosan nucleic acid complex having a positive charge; and

[0751] (b) a plurality of linear block copolymers non-covalently bound to the chitosan derivative nanoparticles, wherein the linear block copolymers comprise at least one polyanion (PA) anchor region and at least one polyethylene glycol (PEG) tail region, wherein the PEG-PA molecules are non-covalently bound to the chitosan derivative nanoparticles, and

[0752] wherein the composition comprises a molar ratio of amino groups to anions (N:A) greater than about 1:100 and less than about 10:1.

[0753] 2. A composition according to embodiment 1, wherein the linear block copolymer is a diblock copolymer comprising a PA anchor region and a PEG tail region.

[0754] 3. A composition according to embodiment 1, wherein the linear block copolymer is a triblock copolymer comprising a central PA anchor region flanked by two PEG tail regions, or alternatively, a central PEG tail region flanked by two PA anchor regions.

[0755] 4. The composition according to embodiment 1, wherein the PA tail region comprises a polypeptide, wherein the polypeptide is negatively charged.

[0756] 5. The composition according to embodiment 1, wherein the PA tail region comprises a carbohydrate, wherein the carbohydrate is negatively charged.

[0757] 6. A composition according to embodiment 5, wherein the carbohydrate comprises multiple carboxylate, phosphate and / or sulfate moieties.

[0758] 7. A composition according to embodiment 6, wherein the carbohydrate is a glycosaminoglycan.

[0759] 8. A composition according to embodiment 1, wherein the PEG-PA molecule comprises:

[0760] (a) PEG-polyglutamic acid (PEG-PGA) molecule;

[0761] (b) PEG-polyaspartic acid (PEG-PAA) molecules; or

[0762] (c) PEG-hyaluronic acid (PEG-HA) molecules,

[0763] or a combination of one, two or all of (a) to (c).

[0764] 9. A composition according to any one of embodiments 1 to 6, wherein the PEG portion of the PEG-PA molecule comprises 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, more preferably about 3,000 Da to about 5,000 Da, and most preferably about 5,000 Da.

[0765] 10. The composition according to any one of embodiments 1 to 9, wherein the PA portion of the PEG-PA molecule comprises 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, more preferably about 1,500 Da.

[0766] 11. The composition according to any one of embodiments 1 to 9, wherein the PA portion of the PEG-PA molecule comprises a linear polypeptide, for example comprising about 5 to about 25 acidic amino acids.

[0767] 12. The composition of any one of embodiments 1 to 11, wherein 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, more preferably greater than about 5:1 and less than about 30:1, more preferably greater than about 5:1 and less than about 20:1, more preferably greater than about 5:1 and less than about 10:1, and most preferably about 7:1.

[0768] 13. The composition of any one of embodiments 1 to 12, wherein 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, more preferably greater than about 1:25 and less than about 6: 1, more preferably greater than about 1:10 and less than about 6: 1, more preferably greater than about 1:5 and less than about 6: 1.

[0769] 14. A composition according to any one of embodiments 1 to 11, wherein the N:P molar ratio is from about 1:8 to about 30:1 (e.g., to about 20:1, 15:1, 10:1, 8:1, or 7:1), and wherein the P:A molar ratio is from about 1:50 to about 1:5, more preferably, wherein 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, more preferably from about 1:2.5 to about 1, more preferably, wherein the N:P:A ratio is about 7:1:7, about 7:1:12, or about 7:1:17.

[0770] 15. The composition according to any one of embodiments 1 to 14, wherein the chitosan derivative nanoparticles comprise or are functionalized with a polyol of formula II:

[0771]

[0772] in

[0773] (a)R 2 Selected from: H and hydroxyl;

[0774] (b)R 3 is selected from: H and hydroxyl; and

[0775] (c) X is selected from C2-C6 alkylene optionally substituted with one or more hydroxy substituents.

[0776] 16. The composition according to any one of embodiments 1 to 14, wherein the chitosan derivative nanoparticles comprise a polyol of formula III:

[0777]

[0778] in:

[0779] -----Y is =O or -H2;

[0780] R 2 Selected from: H and hydroxyl;

[0781] R 3 Selected from: H and hydroxyl;

[0782] X is selected from: C2-C6 alkylene optionally substituted with one or more hydroxy substituents; and

[0783] represents the bond between the polyol and the derivatized chitosan.

[0784] 17. The composition according to any one of embodiments 1 to 16, wherein the amino-functionalized chitosan is arginine-, lysine- or ornithine-functionalized, preferably arginine-functionalized.

[0785] 18. The composition according to any one of embodiments 1 to 17, wherein the composition:

[0786] (a) stable in simulated intestinal fluid in the fasting state for at least 24 hours; or

[0787] (b) Stable when dispersed in mammalian urine at 37°C for at least 1 hour.

[0788] 19. The composition according to any one of embodiments 1 to 18, wherein the composition further comprises a surfactant, an excipient and / or a storage stabilizer.

[0789] 20. The composition according to embodiment 19, wherein the composition comprises the storage stabilizer, preferably, wherein the storage stabilizer is a monosaccharide, a disaccharide, a polysaccharide or a reduced alcohol thereof, more preferably, wherein the storage stabilizer is selected from trehalose and mannitol.

[0790] 21. The composition according to embodiment 19 or 20, wherein the composition comprises the surfactant, preferably, wherein the surfactant comprises a poloxamer, more preferably, wherein the poloxamer is poloxamer 407.

[0791] 22. The composition of any one of embodiments 1 to 21, wherein the at least one nucleic acid comprises RNA.

[0792] 23. The composition of any one of embodiments 1 to 21, wherein the at least one nucleic acid comprises DNA.

[0793] 24. The composition according to any one of embodiments 1 to 23, wherein the composition is stable or at least stable for 48 hours or 1 week at 4°C in an aqueous dispersion comprising the composition dispersed in purified water.

[0794] 25. The composition according to embodiment 24, wherein the composition is stable in the aqueous dispersion after freezing / thawing and / or drying / rehydration, preferably, wherein the drying comprises spray drying, freeze drying, spray freeze drying, evaporation or supercritical drying, more preferably, wherein the drying comprises freeze drying or spray drying.

[0795] 26. The composition according to embodiment 24 or 25, wherein the composition exhibits a polydispersity index of less than 0.2, for example after at least 48 hours or 1 week at 4°C in the aqueous dispersion.

[0796] 27. A method of making any one of the compositions of embodiments 1 to 26, the method comprising:

[0797] (a) providing a complex comprising the chitosan derivative nanoparticle and the at least one nucleic acid, wherein the chitosan derivative nanoparticle comprises amino-functionalized chitosan; and

[0798] (b) mixing the complex with a solution comprising PEG-PA molecules, thereby forming a reaction mixture comprising the composition.

[0799] 28. A method as described in specific embodiment 27, wherein the complex of (a) is provided at a nucleotide concentration of 0.01 mg / mL to 25 mg / mL, more preferably 0.05 to 10 mg / mL, more preferably 0.10 to 5 mg / mL, more preferably 0.10 to 2 mg / mL.

[0800] 29. A method as described in specific embodiment 27 or 28, wherein (a) and (b) are mixed in a (v / v) ratio of 1:10 to 10:1, preferably 1:5 to 5:1, more preferably 1:2 to 2:1, and more preferably in a ratio of about 1:1.

[0801] 30. A method as described in specific embodiments 27, 28 or 29, wherein the method further comprises concentrating the reaction mixture, preferably, the concentration comprises ultrafiltration, solvent sublimation and / or solvent evaporation, more preferably, the ultrafiltration comprises tangential flow filtration.

[0802] 31. The method of embodiment 27, wherein the amino-functionalized chitosan comprises or is functionalized with a hydrophilic polyol.

[0803] 32. A method of making any one of the compositions of embodiments 1 to 26, the method comprising mixing amino-functionalized chitosan, at least one nucleic acid and a PEG-PA molecule simultaneously or sequentially to form a reaction mixture comprising the composition.

[0804] 33. The method of embodiment 32, 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 combination.

[0805] 34. A method of transfecting a cell with a nucleic acid, the method comprising contacting the cell with a composition according to any one of Embodiments 1 to 22 or a composition produced by any of the methods as described in Embodiments 23 to 33.

[0806] 35. The method of embodiment 34, wherein the cells are cells constituting or derived from mucosal tissue.

[0807] 36. The method of embodiment 35, wherein the mucosal tissue is lung tissue, nasal tissue, eye tissue, vaginal tissue, bladder tissue, or gastrointestinal tissue.

[0808] 37. The method of embodiment 35, wherein the cell is an intestinal cell of a subject, and the contacting comprises administering the composition to the subject orally or rectally.

[0809] 38. The method of embodiment 35, wherein the cell is a bladder cell and the contacting comprises administering the composition intravesically to the subject.

[0810] 39. The method of any one of embodiments 27 to 33, wherein the method provides reduced mucoadhesion compared to particles not containing a polymer component comprising PEG-PA.

Claims

1. A composition comprising: (a) a complex comprising chitosan derivative nanoparticles and at least one nucleic acid molecule, wherein the chitosan derivative nanoparticles comprise amino-functionalized chitosan, wherein the at least one nucleic acid molecule is non-covalently bound to the chitosan derivative nanoparticles at an amino to phosphorus (N:P) molar ratio greater than 3:1, thereby forming a derivatized chitosan nucleic acid complex having a positive charge; and (b) a plurality of linear block copolymers non-covalently bound to the chitosan derivative nanoparticles, wherein the linear block copolymers comprise at least one polyanion (PA) anchor region and at least one polyethylene glycol (PEG) tail region, wherein the PEG-PA molecules are non-covalently bound to the chitosan derivative nanoparticles, and wherein the composition comprises a molar ratio of amino groups to anions (N:A) greater than about 1:100 and less than about 10:

1.

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

3. The composition of claim 1, wherein the linear block copolymer is a triblock copolymer comprising a central PA anchor region flanked by two PEG tail regions, or alternatively, a central PEG tail region flanked by two PA anchor regions.

4. The composition of claim 1, wherein the PA tail region comprises a polypeptide, wherein the polypeptide is negatively charged.

5. The composition of claim 1, wherein the PA tail comprises a carbohydrate, wherein the carbohydrate is negatively charged.

6. The composition of claim 5, wherein the carbohydrate comprises a plurality of carboxylate, phosphate and / or sulfate moieties.

7. The composition of claim 6, wherein the carbohydrate is a glycosaminoglycan.

8. The composition of claim 1, wherein the PEG-PA molecule comprises: (a) PEG-polyglutamic acid (PEG-PGA) molecule; (b) PEG-polyaspartic acid (PEG-PAA) molecules; or (c) PEG-hyaluronic acid (PEG-HA) molecules, or a combination of one, two or all of (a) to (c).

9. The composition of any one of claims 1 to 6, wherein the PEG portion of the PEG-PA molecule comprises 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, more preferably about 3,000 Da to about 5,000 Da, and most preferably about 5,000 Da.

10. The composition of any one of claims 1 to 9, wherein the PA portion of the PEG-PA molecule comprises 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, more preferably about 1,500 Da.

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