Dually derivatized chitosan nanoparticles, and methods of making, and using the same for gene transfer in vivo

Abstract

To provide a nanoparticle comprising dually derivatized chitosan, and methods of making and using the same, for delivering nucleic acids, e.g., gene transfer, in vivo.SOLUTION: A chitosan functionalized with (1) arginine (Arg) and (2) a hydrophilic polyol (HP) at a specific Arg: HP ratio exhibits significantly improved transfection efficiency.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to nanoparticles comprising dually derivatized chitosan and methods of making and using same for delivery of nucleic acids, such as, for example, gene transfer, in vivo. [Background technology]

[0002] Chitosan is a non-toxic cationic copolymer of N-acetyl-D-glucosamine and D-glucosamine. Chitosan has been used as a DNA delivery vehicle to transfect cells because it is a biocompatible, non-toxic polysaccharide that can form complexes with nucleic acids. Due to the complexity and potential toxicity of viral vectors, there has been much interest in using chitosan in the non-viral delivery of nucleic acids.

[0003] Many chitosan / DNA complexes, including those between modified chitosan and nucleic acid, have been investigated in an attempt to identify compositions that are well suited for gene transfer.See, for example, WO2010 / 088565;WO2008 / 082282.The complexes have been found to differ in solubility, tendency to aggregate, stability of the complex, particle size, ability to release DNA, and transfection efficiency, among other properties.

[0004] Thus, there is a need for new compositions and methods for in vivo gene transfer with improved transfection efficiency. The compositions and methods described herein help meet these and other needs. Summary of the Invention

[0005] Provided herein is a surprising discovery that chitosan functionalized with (1) arginine (Arg) and (2) hydrophilic polyol (HP) at a specific Arg:HP ratio exhibits significantly improved transfection efficiency. It has been shown that arginine and gluconic acid act synergistically as functional groups to increase the transfection efficiency of chitosan nanoparticles. See, for example, PCT / CA2013 / 050218. Disclosed herein is a surprising discovery that other molecules can be used in place of gluconic acid as derivatives of chitosan and still exhibit this synergistic effect. Furthermore, an optimized functionalization ratio of the two substituents is provided at which the derivatized chitosan exhibits maximum transfection efficiency. Thus, provided herein is a novel composition that facilitates the delivery of nucleic acids to cells, tissues, and organs, for example, in vivo. In particular, provided herein are doubly derivatized chitosan-based nanoparticles, in which the chitosan is functionalized with the cationic amino acid arginine (Arg) and a hydrophilic polyol (HP) in an optimized ratio of final degree of functionalization of Arg:final degree of functionalization of HP (i.e., Arg:HP).

[0006] In a preferred embodiment, the nanoparticles comprise chitosan conjugated to both a plurality of arginines and a plurality of hydrophilic polyols. See, e.g., Formula I. [ka] In the formula, n is an integer from 1 to 650; α is the degree of functionalization of arginine, β is the degree of functionalization of the hydrophilic polyol, Each R 1 are each independently selected from hydrogen, acetyl, arginine, and a hydrophilic polyol.

[0007] In a preferred embodiment, the nanoparticles comprise chitosan conjugated to arginine. See Formula II. In a preferred embodiment, the arginine serves to increase the solubility of chitosan and / or bind the derivatized chitosan to nucleic acids at high pH. [ka]

[0008] In another embodiment, the nanoparticles include chitosan bound to a hydrophilic polyol, preferably having a carboxyl or aldehyde group for binding to chitosan. In one embodiment, the hydrophilic polyol is selected from the group consisting of a molecule having a carboxyl or aldehyde group for binding to chitosan and a saccharide, and the hydrophilic polyol is not gluconic acid. In one embodiment, the hydrophilic polyol has a carboxyl group. In one embodiment, the hydrophilic polyol having a carboxyl group for binding to chitosan is selected from the group consisting of gluconic acid and threonic acid. In one embodiment, the hydrophilic polyol is gluconic acid, see for example Formula III. In another embodiment, the hydrophilic polyol is threonic acid, see for example Formula IV. In another embodiment, the hydrophilic polyol is a saccharide, which may be a natural saccharide, or a synthetic saccharide, or an acidic form of a saccharide. Non-limiting examples include glyceraldehyde, threose, erythrose, ribose, arabinose, xylose, lyxose, allose, glucose, altrose, mannose, gulose, idose, galactose, and talose. In one embodiment, the hydrophilic polyol is glucose, see e.g., Formula V. In one embodiment, the hydrophilic polyol is threose, see e.g., Formula VI. [ka]

[0009] The doubly derivatized chitosan described herein comprises arginine and a hydrophilic polyol with an optimized final functionalization ratio of Arg:HP. In a preferred embodiment, the doubly derivatized chitosan has a final functionalization ratio, or molar ratio, of Arg:HP that is between about 1:1 and about 10:1. In another embodiment, the final functionalization ratio of Arg:HP is between about 3:1 and about 7:1. In another embodiment, the final functionalization ratio of Arg:HP is about 5:1.

[0010] In particular, chitosan-nucleic acid polyplexes formed with such doubly derivatized chitosans ("DD-chitosans") exhibit higher transfection efficiency than nucleic acid polyplexes formed with unfunctionalized chitosans, singly derivatized chitosans, or doubly derivatized chitosans with a final Arg:HP functionalization ratio between 1:1 and 10:1. Other desirable properties imparted by the use of doubly functionalized chitosans in the polyplexes described herein include improved ability to penetrate mucus barriers, improved polyplex stability at pH greater than 6.5, reduced aggregation at high polyplex concentrations, reduced cytotoxicity, and enhanced intracellular release of nucleic acids. Additionally, in some preferred embodiments, the subject DD-chitosan polyplex compositions may be administered (e.g., systemically) at physiological pH.

[0011] Thus, in one aspect, the present invention provides a DD-chitosan nucleic acid polyplex, which comprises chitosan doubly derivatized with arginine and a hydrophilic polyol.

[0012] In one embodiment, the nucleic acid of the DD-chitosan nucleic acid polyplex is DNA.

[0013] In one embodiment, the nucleic acid of the DD-chitosan nucleic acid polyplex is RNA.

[0014] In one embodiment, the nucleic acid of the DD-chitosan nucleic acid polyplex is an artificial nucleic acid. In a preferred embodiment, the artificial nucleic acid is selected from the group consisting of peptide nucleic acid (PNA), phosphorodiamidate morpholino oligo (PMO), locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA).

[0015] 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. In a preferred embodiment, the therapeutic RNA is selected from the group consisting of antisense RNA, siRNA, small hairpin RNA, microRNA, and enzymatic RNA.

[0016] In one embodiment, the therapeutic nucleic acid is DNA.

[0017] In one embodiment, the therapeutic nucleic acid comprises a nucleic acid sequence that encodes a therapeutic protein.

[0018] In one aspect, the present invention provides a composition comprising a plurality of DD-chitosan nucleic acid polyplexes.

[0019] In one embodiment, the composition has a pH between 3.0 and 8.0, more preferably between 4.0 and 7.0, and most preferably between 4.5 and 6.5.

[0020] In one aspect, the present invention provides a pharmaceutical composition comprising the DD-chitosan nucleic acid polyplex of the present invention. In a preferred embodiment, the DD-chitosan nucleic acid polyplex comprises a therapeutic nucleic acid.

[0021] In one aspect, the present invention provides a method of treating a disease comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition of the present invention.

[0022] In one embodiment, the subject pharmaceutical compositions are administered at physiological pH.

[0023] In one embodiment, the subject pharmaceutical compositions are administered systemically.

[0024] In one embodiment, the subject pharmaceutical composition is administered locally to a target tissue. In a preferred embodiment, the subject pharmaceutical composition is administered to a mucosal tissue. In one embodiment, the mucosal tissue is a gastrointestinal (GI) tissue.

[0025] In one aspect, the present invention provides a vaccine comprising a DD-chitosan nucleic acid polyplex, wherein the nucleic acid encodes an antigen.

[0026] In one aspect, the invention provides a method for vaccinating a patient, the method comprising administering to the patient a vaccine of the invention.

[0027] In one aspect, the present invention provides an immunogenic composition comprising a DD-chitosan nucleic acid polyplex, wherein the nucleic acid encodes an immunogen.

[0028] In one embodiment, the present invention provides a method for initiating or increasing an immune response to a molecule of interest. The method comprises administering to a patient an immunogenic composition of the present invention, in which the nucleic acid encodes an epitope of the molecule of interest. In another embodiment, the present invention provides a method for modulating an immune response, in which the patient comprises administering to a patient a chitosan-nucleic acid polyplex of the present invention, in which the nucleic acid encodes an immunomodulatory cytokine. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 shows the in vitro transfection efficiency (SEAP (ng) / protein (mg); y-axis) of 5 kDa chitosan (x-axis) functionalized with gluconic acid only at a final functionalization degree of 3% or 10%, with arginine only at a final functionalization degree of 10%, or with both gluconic acid and arginine at a final functionalization degree ratio of arginine to gluconic acid of about 1:1 or about 3.3:1. [Diagram 2]Figure 2 shows the transfection efficiency (SEAP (ng) / protein (mg); y-axis) of 5 kDa chitosan (x-axis) functionalized with gluconic acid only for a final functionalization degree of 3% to 10%, with arginine only for a final functionalization degree of 52%, or with both gluconic acid and arginine for a final functionalization degree ratio of arginine to gluconic acid of about 6:1 or about 17:1. [Diagram 3] Transfection efficiency (SEAP (ng) / protein (mg); y axis) of 5 kDa chitosan (x-axis) functionalized with gluconic acid only for a final functionalization degree of 3% to 10%, with arginine only for a final functionalization degree of 26%, or with both gluconic acid and arginine for a final functionalization degree ratio of arginine to gluconic acid of approximately 4:1. [Figure 4] Figure 2 shows the transfection efficiency (SEAP (ng) / protein (mg); y-axis) of 5 kDa chitosan (x-axis) functionalized with threonic acid alone for a final functionalization degree of 3%, or with arginine alone for a final functionalization degree of 29%, or with both threonic acid and arginine for a final functionalization degree ratio of arginine to threonic acid of approximately 10:1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Chitosan is a deacetylated form of chitin, a polymer of N-acetylglucosamine that is the main component of the exoskeleton of crustaceans (e.g., shrimp, crabs, and lobsters). Chitosan is formed from chitin by deacetylation and is thus not a single polymer but a class of molecules with different molecular weights and different degrees of deacetylation. The percentage of deacetylation in commercially available chitosan is generally between 50-100%.

[0031] The chitosan derivatives described herein are produced by functionalizing the resulting free amino groups with positively charged and / or hydrophilic moieties as described herein. The derivatized chitosans described herein have several properties that make them favorable for use as nucleic acid delivery vehicles, including: they efficiently bind and complex with negatively charged nucleic acids, they can be formed into nanoparticles of controllable size, they can be taken up by cells, and they can release nucleic acids intracellularly in a timely manner.

[0032] Chitosans having any degree of deacetylation (DDA) greater than 50% may be functionalized between 1% and 50% and used in the present invention (the percent of functionalization is determined relative to the number of free amino moieties on the chitosan polymer). The degree of deacetylation and the degree of functionalization impart a specific charge density to the functionalized chitosan derivative. The resulting charge density affects solubility, binding and subsequent release of nucleic acids, and interaction with the cell membrane of mammalian cells. Thus, in accordance with the present invention, these properties must be optimized for optimal effectiveness. Exemplary chitosan derivatives are described in Baker et al., 11 / 657,382, filed Jan. 24, 2007, which is incorporated herein by reference. In one embodiment, the doubly derivatized chitosan described herein includes chitosans 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%.

[0033] The chitosan derivatives described herein are soluble at neutral and physiological pH and, for purposes of the present invention, have a range of average molecular weights, including molecular weights ranging from 3 to 110 kDa. The embodiments described herein feature derivatized chitosans of low average molecular weight (less than 25 kDa, e.g., about 5 kDa to about 25 kDa), which can have desirable delivery and transfection properties, and which are small in size and have favorable solubility. Derivatized chitosans of low average molecular weight are generally more soluble than derivatized chitosans of higher molecular weight, and therefore produce nucleic acid / chitosan complexes that more readily release nucleic acid and provide enhanced cellular transfection. Much literature has been devoted to optimizing all of these parameters for chitosan-based delivery systems.

[0034] Chitosan is a molecule having the structure of formula I, where n is any integer, and each R 1 are each independently selected from acetyl, or hydrogen, and R is selected from hydrogen; 1 Those skilled in the art will recognize that a chitosan having an average molecular weight of, for example, 3 kD to 110 kD generally refers to a plurality of chitosan molecules each having a weight average molecular weight of, for example, 3 kD to 110 kD, each of which may have a different chain length (n+2). It is also well recognized that a chitosan referred to as an "n-mer chitosan" does not necessarily include a chitosan molecule of formula I, each of which has a chain length of n+2. Rather, as used herein, an "n-mer chitosan" refers to a plurality of chitosan molecules, each of which may have a different chain length, each of which has an average molecular weight that is substantially the same or equal to a chitosan molecule having a chain length of n. For example, a 24-mer chitosan may include a plurality of chitosan molecules, each of which has a different chain length, e.g., ranging from 7 to 50, but has a weight average molecular weight that is substantially the same or equal to that of a chitosan molecule having a chain length of 24.

[0035] The functionalized chitosan derivatives described herein are doubly derivatized chitosan compounds, such as chitosan-Arg-HP compounds. Generally, chitosan-Arg-HP compounds have the structure of Formula I below: [ka] In the formula, n is an integer from 1 to 650; α is the final degree of functionalization of Arg, β is the final degree of functionalization of HP; Each R 1 are each independently selected from hydrogen, acetyl, Arg, and HP.

[0036] The doubly derivatized chitosan of the present invention may be functionalized with the cationic amino acid, arginine.

[0037] The doubly derivatized chitosans of the present invention may also be functionalized with hydrophilic polyols, which may serve to increase the hydrophilicity of the chitosan (including Arg-chitosan) and / or may provide hydroxyl groups.

[0038] Chitosan functionalized with arginine (Arg) and a hydrophilic polyol (HP) of formula VII: [ka] During the ceremony: R 2 is selected from H, and hydroxyl; R 3 is selected from H, and hydroxyl; There is provided a chitosan derivative nanoparticle comprising said chitosan, wherein X is selected from C2-C6 alkylene optionally substituted with one or more hydroxyl substituents.

[0039] In some embodiments, the chitosan derivative nanoparticles comprise a hydrophilic polyol of formula VII: [ka] During the ceremony: R 2 is selected from H, and hydroxyl; R 3 is selected from H, and hydroxyl; X is selected from C2-C6 alkylene optionally substituted with one or more hydroxyl substituents; However, the hydrophilic polyol is not gluconic acid.

[0040] The term "C2-C6 alkylene" as used herein refers to a straight-chain or branched divalent hydrocarbon radical that may contain one or more carbon-carbon multiple bonds. For the avoidance of doubt, the term "C2-C6 alkylene" as used herein encompasses divalent radicals of alkanes, alkenes, and alkynes.

[0041] The hydrophilic polyol of the present invention may have a carbon backbone of 3, 4, 5, 6, or 7. In one embodiment, the hydrophilic polyol of the present invention having 3 to 7 carbons may have 2, 3, 4, 5, or 6 hydroxyl groups. In one embodiment, the hydrophilic polyol of the present invention having 3 to 7 carbons may have one or more carbon-carbon multiple bonds. In a preferred embodiment, the hydrophilic polyol of the present invention comprises a carboxyl group. In a further preferred embodiment, the hydrophilic polyol of the present invention comprises an aldehyde group. When the hydrophilic polyol of the present invention comprises an aldehyde group, those skilled in the art will recognize that such hydrophilic polyols include both linear structures (aldehyde) and cyclic structures (hemiacetal).

[0042] Non-limiting examples of hydrophilic polyols include gluconic acid, threonic acid, glucose, and threose. See, for example, Formulae III-VI. Examples of other such hydrophilic polyols, which may have carboxyl and / or aldehyde groups, or may be sugars or acid forms thereof, are included in Tables 1-3 below. One of skill in the art will recognize that Tables 1-3 provide non-limiting examples of hydrophilic polyols, and further, that the hydrophilic polyols shown are not limited to the stereochemistry shown.

[0043] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0044] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0045] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0046] In a preferred embodiment, HP may be selected from the group consisting of 2,3-dihydroxylpropanoic acid; 2,3,4,5,6,7-hexahydroxylheptanal; 2,3,4,5,6-pentahydroxylhexanal; 2,3,4,5-tetrahydroxylhexanal; and 2,3-dihydroxylpropanal.

[0047] In a preferred embodiment, the HP may be selected from the group consisting of D-glyceric acid, L-glyceric acid, L-glycero-D-mannoheptose, D-glycero-L-mannoheptose, D-glucose, L-glucose, D-fucose, L-fucose, D-glyceraldehyde, and L-glyceraldehyde.

[0048] In a preferred embodiment, the α:β ratio is between about 1:1 and about 10:1. In a preferred embodiment, the ratio is between about 3:1 and about 7:1. In a most preferred embodiment, the ratio is about 5:1.

[0049] A non-limiting method of the present invention for conjugating chitosan with arginine or hydrophilic polyols in aqueous media is described herein, which uses the well-known water-soluble 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to catalyze the amide formation between the amines on the chitosan backbone and the carboxylic acids on the hydrophilic polyols bearing Boc-protected Arg or carboxyl groups.

[0050] Typically, chitosan in a dilute HCl solution with pH adjusted to the pH of the desired coupling, e.g., 6.0±0.5, more preferably 6.0±0.2, is first coupled to either Arg, e.g., Boc-Arg, or a hydrophilic polyol having a carboxyl group, purified, and then coupled to a second functional group. For example, if chitosan is coupled to Arg first, the chitosan coupled to Arg (Arg-chitosan) may be purified and then coupled to a hydrophilic polyol. Conversely, if chitosan is coupled to a hydrophilic polyol first, the chitosan coupled to the hydrophilic polyol may be purified and then coupled to Arg. Regardless of the order of coupling, Arg and hydrophilic polyol may be coupled to chitosan using well-known methods.

[0051] For example, Arg may be attached to chitosan or chitosan functionalized with polyol (polyol-chitosan) by adding a mixture of Boc-Arg and aqueous NHS solution with an adjusted pH to chitosan in dilute HCl, then adding aqueous EDC solution and allowing coupling to occur at room temperature for 24 hours. The concentration of chitosan amine, pH of reaction, and molar ratios of Arg-COOH to chitosan-amine and EDC:NHS:Arg-COOH may be pre-calculated and satisfied to have a reproducible final functionalization degree of Arg. Boc-Arg-chitosan may be purified before the Boc group deprotection (De-Boc) reaction. The Boc group deprotection (De-Boc) reaction may proceed in HCl medium with controlled HCl concentration and reaction time. The depolymerization of chitosan during the de-Boc reaction may be monitored by measuring the change in viscosity of the reaction solution (almost no depolymerization of chitosan was observed), and the efficiency of the de-Boc reaction may be confirmed by nuclear magnetic resonance (NMR) for de-Boc-Arg-chitosan and Boc-Arg-chitosan. The degree of functionalization may be determined from the C, N elemental analysis of the purified de-Boc-Arg-chitosan.

[0052] Hydrophilic polyols bearing carboxyl groups may be coupled with chitosan or chitosan coupled with Arg (Arg-chitosan) at a reaction pH of 6.0±0.3. At this pH, the carboxylic acid groups of the hydrophilic polyols may be attacked by the unbound amines on the chitosan backbone via a nucleophilic substitution reaction mechanism. Those skilled in the art will recognize that in the case of such coupling of hydrophilic polyols with Arg-chitosan, the nucleophilic substitution reaction may occur predominantly with the amine groups on the chitosan backbone, but a small amount of the hydrophilic polyol may also form covalent bonds with the amine groups of Arg via the same mechanism.

[0053] Natural sugar hydrophilic polyols may be conjugated to chitosan or Arg-conjugated chitosan (Arg-chitosan) using reductive amination followed by reduction with NaCNBH3 or NaBH4.

[0054] Boc-Arg-chitosan, de-Boc-Arg-chitosan, polyol-chitosan, and / or doubly derivatized chitosan may be purified via precipitation, or column processing, or periodic dialysis, or inverse-flow dialysis against water using dialysis tubing of appropriate molecular weight cut-off (MWCO), or through tangential flow filtration (TFF) and diafiltration cartridges.

[0055] Thus, "doubly derivatized chitosan," or "DD-chitosan," also refers to a doubly functionalized chitosan ("doubly functionalized chitosan," or "DF-chitosan"), e.g., a chitosan that is conjugated with both Arg and a hydrophilic polyol, where both Arg and the hydrophilic polyol are covalently attached to the chitosan. Arg may be covalently attached to the chitosan either as a single amino acid or as a polypeptide.

[0056] As used herein, unless otherwise indicated, the terms "peptide" and "polypeptide" are used interchangeably.

[0057] The term "polypeptide" is used in the broadest sense to refer to conventional polypeptides (i.e., short polypeptides containing L- or D-amino acids), as well as peptide equivalents, peptide analogs, and peptidomimetics that retain the desired functional activity. Peptide equivalents may differ from conventional peptides by the replacement of one or more amino acids with similar organic acids, amino acids, etc., or by substitutions or modifications of side chains or functional groups.

[0058] As known to those skilled in the art, peptidomimetics may have one or more peptide bonds replaced by alternative bonds. As known in the art, part or all of the peptide backbone may also be replaced by conformationally restricted, cyclic alkyl, or aryl substituents to restrict the mobility of functional amino acid side chains.

[0059] The polypeptides of the present invention may be produced by recognized methods, including recombinant and synthetic methods well known in the art. Techniques for synthesizing peptides are well known and include those described in Merrifield, J. Amer. Chem. Soc. 85:2149-2456 (1963), Atherton, et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989), and Merrifield, Science 232:341-347 (1986).

[0060] As used herein, a "linear polypeptide" refers to a polypeptide that does not have branching groups covalently attached to the side chains of its constituent amino acids. As used herein, a "branched polypeptide" refers to a polypeptide that includes branching groups covalently attached to the side chains of its constituent amino acids.

[0061] As mentioned above, the final functionalization degree of Arg of the DD-chitosan of the present invention, or the final functionalization degree of polyol, may be determined by elemental analysis. In the following, an exemplary calculation of the final functionalization degree of, for example, arginine and gluconic acid attached to the chitosan backbone is described. For clarity, the following example uses a 20-mer chitosan molecule of 100% DDA.

[0062] Chitosan, 100% DDA, is a homopolymer of D-glucosamine; its repeating unit has 6 C atoms and 1 N atom, with a C / N molar ratio of 6 / 1.

[0063] Due to the depolymerization process, the terminal unit at one end of the polymer is a 2,5-anhydro-D-mannose unit, which has a C / N ratio of 6 / 0.

[0064] Thus, in the example of a 20-mer chitosan, there are 19 units of D-glucosamine and 1 unit of 2,5-anhydro-D-mannose. Thus, the overall C / N ratio is:

number

[0065] This ratio is normalized for the known average integer value of nitrogen to 6 carbons (which is the average number of C atoms per unit):

number

[0066] In coupling of chitosan with, for example, arginine, the arginine moiety has 6 C and 4 N. For a chitosan modified with 20 repeat units of arginine with an average arginine functionalization degree of R%, the following relationship represents the C and N contributions from chitosan and arginine:

number

[0067] The molar ratio of C / N is calculated from the experimentally derived mass ratio of C / N. Therefore, knowing the molar ratio of C / N, the degree of functionalization of R%, i.e., the percentage of amino groups on the chitosan backbone that are linked with arginine groups, can be derived.

[0068] Coupling of gluconic acid with chitosan:

[0069] The gluconic acid moiety has 6 C and 0 N. For a chitosan modified with 20 repeat units of gluconic acid with a degree of functionalization of G%, the following relationship represents the C and N contributions from chitosan and gluconic acid:

number

[0070] The molar ratio of C / N is calculated from the experimentally derived mass ratio of C / N. Therefore, knowing the molar ratio of C / N, the degree of functionalization, G%, i.e., the percentage of amino groups on the chitosan backbone that are linked with gluconic acid moieties, can be derived.

[0071] Coupling of arginine with chitosan followed by coupling of gluconic acid:

[0072] For the coupling of gluconic acid with R-chitosan of 20 repeat units with a predetermined final degree of arginine functionalization (R%), the following relationship represents the C and N contributions from chitosan, arginine, and gluconic acid:

number

[0073] The molar ratio of C / N is calculated from the experimentally derived mass ratio of C / N. Therefore, since R% is predetermined, the degree of functionalization of G% can be derived once the molar ratio of C / N is known.

[0074] As mentioned above, the "final degree of functionalization" of Arg or HP as used herein refers to the percentage of amino groups on the chitosan backbone that are functionalized with Arg or HP, respectively. Thus, the terms "α:β ratio", "final degree of functionalization ratio" (e.g., the ratio of the final degree of functionalization of Arg:final degree of functionalization of HP), etc. may be used interchangeably with the terms "molar ratio" or "number ratio".

[0075] In one embodiment, the doubly derivatized chitosan is not a chitosan derivative nanoparticle, or a chitosan derivative nanoparticle polyplex, as exemplified in PCT application PCT / CA2013 / 050218, particularly in the Examples of PCT / CA2013 / 050218. In one embodiment, the chitosan derivative nanoparticle is not one of the following nanoparticles: a. Chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine at a final functionalization degree of 52% and gluconic acid at a final functionalization degree of 8%. b. chitosan derivative nanoparticles with an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 3%; c. chitosan derivative nanoparticles with an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; d. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 6%; e. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 9%; f. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 40, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; g. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 10, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; h. chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; i. chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine at a final functionalization degree of 26% and gluconic acid at a final functionalization degree of 6%; or j. Chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine to a final degree of functionalization of 52% and gluconic acid to a final degree of functionalization of 8%.

[0076] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with gluconic acid at a final functionalization level of 1%, 2%, 4%, 7%, 8%, 10%, 15%, 20%, 25%, 30% or more. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine at a final functionalization level of about 1% to about 25%. In another embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine at a final functionalization level of about 27% to about 51%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine at a final functionalization level of about 53% to about 70%. In another embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine at a final functionalization level of 26% or not 52%.

[0077] In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated with arginine to a final degree of functionalization of 26% and with gluconic acid to a final degree of functionalization of not 3%.

[0078] In one embodiment, the chitosan derivative nanoparticles comprise chitosan bound to arginine with a final functionalization degree of 26% and bound to gluconic acid with a final functionalization degree that is not 5%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan bound to arginine with a final functionalization degree of 26% and bound to gluconic acid with a final functionalization degree that is not 6%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan bound to arginine with a final functionalization degree of 26% and bound to gluconic acid with a final functionalization degree that is not 9%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan bound to arginine with a final functionalization degree of 26% and bound to gluconic acid with a final functionalization degree that is not 3%, 5%, 6%, or 9%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan bound to arginine with a final functionalization degree of 52% and bound to gluconic acid with a final functionalization degree that is not 8%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to gluconic acid with a final functionalization degree selected from 3%, 5%, 6%, and 9%, and conjugated to arginine with a final functionalization degree other than 26%. In one embodiment, the chitosan derivative nanoparticles comprise chitosan conjugated to gluconic acid with a final functionalization degree of 8%, and conjugated to arginine with a final functionalization degree other than 52%.

[0079] In some embodiments, where appropriate, DD-chitosan includes DD-chitosan derivatives, such as DD-chitosan incorporating additional functionalization, such as DD-chitosan with attached ligands. It will be understood that "derivatives" includes the broad category of chitosan-based polymers that contain covalently modified N-acetyl-D-glucosamine units and / or D-glucosamine units, as well as chitosan-based polymers that incorporate other units or are conjugated with other moieties. Derivatives are often based on modification of the hydroxyl groups or amine groups of glucosamine, such as the modifications made with 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, uronic acid modified chitosan, glycol chitosan, and the like. For further teachings regarding chitosan derivatives, see, e.g., pp. 63-74 of “Non-viral Gene Therapy”, K. Taira, K. Kataoka, T. Niidome (editors), Springer-Verlag Tokyo, 2005, ISBN 4-431-25122-7; Zhu et al., Chinese Science Bulletin, December 2007, vol. 52 (23), pp. 3207-3215; and Varma et al., Carbohydrate Polymers 55 (2004) 77-93.

[0080] A dispersion consists of a particulate material, known as the dispersed phase, distributed in a continuous medium. A "dispersion" of DD-chitosan nucleic acid polyplexes is a composition that includes hydrated DD-chitosan nucleic acid polyplexes, where the polyplexes are distributed in a medium.

[0081] As used herein, a "pre-concentrated" dispersion is a dispersion that has not been subjected to the concentration process to form a concentrated dispersion.

[0082] As used herein, "substantially free" of polyplex sediments means that the composition contains virtually no particles observable upon visual inspection.

[0083] As used herein, physiological pH refers to a pH between 6 and 8.

[0084] "DD-chitosan nucleic acid polyplex," or grammatical equivalents thereof, refers to a complex comprising a plurality of DD-chitosan molecules and a plurality of nucleic acid molecules. In a preferred embodiment, a doubly derivatized chitosan is complexed with said nucleic acid.

[0085] DD-chitosan nucleic acid polyplexes include a nucleic acid component and a DD-chitosan component. Chitosan and DD-chitosan nucleic acid polyplexes can be prepared by any method known in the art. For example, the concentrations of functionalized chitosan and nucleotide feedstocks can be adjusted to accommodate various amine to phosphate ratios (N / P), mixing ratios, and nucleotide concentrations of interest. In some embodiments, particularly in small batches, e.g., batches less than 2 mL, the functionalized chitosan and nucleotide feedstocks can be mixed by slowly dropping the nucleotide feedstock into the functionalized chitosan feedstock while vortexing the container. In other embodiments, the functionalized chitosan and nucleotide feedstocks can be mixed by in-line mixing of two fluid streams. In other embodiments, the resulting polyplex dispersion can be concentrated by TFF. A preferred method for polyplex formation is disclosed in WO 2009 / 039657, which is expressly incorporated herein by reference in its entirety.

[0086] Generally, the nucleic acids of the present invention will contain phosphodiester bonds, although in some cases, nucleic acid analogs may have alternative backbones or other modifications or moieties incorporated for any of a variety of purposes, such as stability and protection. Other contemplated nucleic acid analogs include those with non-ribose backbones. Additionally, naturally occurring nucleic acids, analogs, and mixtures of both may be made. Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids include, but are not limited to, DNA, RNA, and hybrids, and include any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, and the like. Nucleic acids include any form of DNA, any form of RNA, including triplex, double or single stranded, antisense, siRNA, ribozymes, deoxyribozymes, polynucleotides, oligonucleotides, chimeras, microRNA, and derivatives thereof. Nucleic acids include artificial nucleic acids, including, but not limited to, peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligos (PMOs), locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs).

[0087] In one embodiment, the nucleic acid component comprises a therapeutic nucleic acid. The subject DD-chitosan nucleic acid polyplexes allow for the use of any therapeutic nucleic acid known in the art. Therapeutic nucleic acids include therapeutic RNAs, which are RNA molecules capable of exerting a therapeutic effect in mammalian cells. Therapeutic RNAs include, but are not limited to, antisense RNA, siRNA, small hairpin RNA, microRNA, and enzymatic RNA. Therapeutic nucleic acids include, but are not limited to, nucleic acids intended to form triplex molecules, protein-binding nucleic acids, ribozymes, deoxyribozymes, and small nucleotide molecules.

[0088] Many kinds of therapeutic RNA are known in the art.See, for example, Grimm et al., Therapeutic application of RNAi is mRNA targeting finally ready for prime time? J. Clin. Invest., 117:3633-3641, 2007; Aagaard et al., RNAi therapeutics: Principles, prospects and challenges, Adv. Drug Deliv. Rev., 59:75-86, 2007; Dorsett et al., siRNAs: Applications in functional genomics and potential as therapeutics, Nat. Rev. Drug Discov., 3:318-329, 2004.These include double-stranded small interfering RNA (siRNA).

[0089] Therapeutic nucleic acids also include nucleic acids that encode therapeutic proteins, including cytotoxic proteins and prodrugs.

[0090] In a preferred embodiment, the nucleic acid component comprises a therapeutic nucleic acid construct. The therapeutic nucleic acid construct is a nucleic acid construct capable of exerting a therapeutic effect. The therapeutic nucleic acid construct may comprise a nucleic acid encoding a therapeutic protein and a nucleic acid that produces a transcript that is a therapeutic RNA. The therapeutic nucleic acid may be used to provide gene therapy by acting as a replacement or enhancer of a defective gene, or to compensate for the lack of a particular gene product by encoding a therapeutic product. The therapeutic nucleic acid may suppress the expression of an endogenous gene. The therapeutic nucleic acid may code for all or part of a transcript and may function by recombining with DNA already present in the cell, thereby replacing a defective portion of the gene. The therapeutic nucleic acid may code for a portion of a protein and exert an effect by co-suppression of the gene product. In a preferred embodiment, the therapeutic nucleic acid is selected from the therapeutic nucleic acids disclosed in USSN 11 / 694,852, which is expressly incorporated herein by reference.

[0091] In a preferred embodiment, the therapeutic nucleic acid encodes a therapeutic protein selected from the group consisting of hormones, enzymes, cytokines, chemokines, antibodies, mitogens, growth factors, differentiation factors, factors affecting angiogenesis, factors affecting thrombosis, factors affecting blood glucose levels, factors affecting glucose metabolism, factors affecting lipid metabolism, factors affecting blood cholesterol levels, factors affecting blood LDL or HDL levels, factors affecting cell apoptosis, factors affecting food intake, factors affecting energy expenditure, factors affecting appetite, factors affecting nutrient absorption, factors affecting inflammation, and factors affecting bone formation. Particularly preferred are therapeutic nucleic acids encoding insulin, leptin, glucagon antagonists, GLP-1, GLP-2, ghrelin, cholecystokinin, growth hormone, coagulation factors, PYY, erythropoietin, inflammation inhibitors, IL-10, IL-17 antagonists, TNFα antagonists, growth hormone releasing hormone, or parathyroid hormone.

[0092] Expression control region

[0093] In a preferred embodiment, the polyplexes of the invention comprise a therapeutic nucleic acid that is a therapeutic construct that includes an expression control region operably linked to a coding region. The therapeutic construct produces a therapeutic nucleic acid that may be therapeutic in its own right or may encode a therapeutic protein.

[0094] In some embodiments, the expression control region of the therapeutic construct has a constitutive activity. In many preferred embodiments, the expression control region of the therapeutic construct does not have a constitutive activity. This provides dynamic expression of the therapeutic nucleic acid. "Dynamic" expression means expression that changes over time. Dynamic expression may include several periods of low or no expression separated by periods of detectable expression. In many preferred embodiments, the therapeutic nucleic acid is operably linked to a regulatable promoter. This provides regulatable expression of the therapeutic nucleic acid.

[0095] Expression control regions include regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that provide an influence on expression of an operably linked therapeutic nucleic acid.

[0096] The expression control elements included herein can be from bacteria, yeast, plants, or animals (mammalian or non-mammalian). Expression control regions include full-length promoter sequences, such as native promoters and enhancer elements, as well as subsequences or polynucleotide variants that retain all or part of the full-length or native function (e.g., nutritional regulation or cell / tissue specific expression to some degree). As used herein, the term "functional" and grammatical variations thereof, when used in reference to a nucleic acid sequence, subsequence, or fragment, means that the sequence has one or more functions of the native nucleic acid sequence (e.g., the native or unaltered sequence). As used herein, the term "mutation" refers to substitutions, deletions, additions, or other modifications of the sequence (e.g., chemical derivatives, such as modified forms that are resistant to nucleases).

[0097] As used herein, the term "operably linked" refers to a physical juxtaposition of components that is characterized by allowing the components to function in their intended manner. In an example of an expression control element that is operably linked to a nucleic acid, the relationship is such that the control element controls expression of the nucleic acid. Generally, expression control regions that control transcription are aligned near the 5' end (i.e., "upstream") of the nucleic acid to be transcribed. Expression control regions can also be located at the 3' end (i.e., "downstream") of the transcribed sequence, or within the transcript (e.g., in an intron). Expression control elements can be located away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides away from the nucleic acid). An example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5', or 3' of the transcribed sequence, or within the transcribed sequence.

[0098] Some expression control regions confer regulatable expression to an operably linked therapeutic nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a therapeutic nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as inhibitory. Generally, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0099] Numerous regulatable promoters are known in the art. Preferred inducible expression control regions include expression control regions that include inducible promoters stimulated by small molecule compounds. In one embodiment, the expression control region is orally deliverable but responsive to chemicals not normally found in food. Specific examples can be found, for example, in U.S. Patent Nos. 5,989,910; 5,935,934; 6,015,709; and 6,004,941.

[0100] 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 genome of the target cell. In a preferred embodiment, the integration sequence(s) function in combination with an integration means selected from the group consisting of mariner, sleeping beauty, FLP, Cre, ΦC31, R, lambda, and integration means from integrating viruses such as AAV, retroviruses, and lentiviruses.

[0101] In one embodiment, the subject compositions further comprise a non-therapeutic construct in addition to the therapeutic construct, said non-therapeutic construct comprising 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 is preferably selected from the group consisting of mariner, sleeping beauty, FLP, Cre, ΦC31, R, lambda, and integration means from integrating viruses such as AAV, retroviruses, and lentiviruses.

[0102] For further teachings, see WO2008020318, which is expressly incorporated herein by reference in its entirety. In one embodiment, the nucleic acid of the DD-chitosan nucleic acid polyplex is an artificial nucleic acid.

[0103] Preferred artificial nucleic acids include, but are not limited to, peptide nucleic acids (PNAs), phosphorodiamidate morpholino oligos (PMOs), locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs).

[0104] 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, small hairpin RNA, microRNA, and enzymatic RNA.

[0105] In one embodiment, the therapeutic nucleic acid is DNA.

[0106] In one embodiment, the therapeutic nucleic acid comprises a nucleic acid sequence that encodes a therapeutic protein.

[0107] In a preferred embodiment, the doubly derivatized (DD) chitosan nucleic acid polyplex is not a polyplex that comprises a nucleic acid encoding secreted alkaline phosphatase (SEAP) or luciferase complexed to one of the following chitosan derivative nanoparticles: a. Chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine at a final functionalization degree of 52% and gluconic acid at a final functionalization degree of 8%. b. chitosan derivative nanoparticles with an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final functionalization degree of 26% and gluconic acid for a final functionalization degree of 3%; c. chitosan derivative nanoparticles with an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; d. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 6%; e. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 20, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 9%; f. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 40, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; g. chitosan derivative nanoparticles having an amine / phosphate (N / P) ratio of 10, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; h. chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine for a final degree of functionalization of 26% and gluconic acid for a final degree of functionalization of 5%; i. chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine at a final functionalization degree of 26% and gluconic acid at a final functionalization degree of 6%; or j. Chitosan derivative nanoparticles, said nanoparticles comprising 24-mer chitosan conjugated with arginine to a final degree of functionalization of 52% and gluconic acid to a final degree of functionalization of 8%.

[0108] Polyplex

[0109] In preferred embodiments, the polyplexes of the composition comprise chitosan molecules having an average molecular weight before functionalization of less than 110 kDa, more preferably less than 65 kDa, more preferably less than 50 kDa, more preferably less than 40 kDa, and most preferably less than 30 kDa. In some embodiments, the polyplexes of the composition comprise chitosan having an average molecular weight before functionalization of less than 15 kDa, less than 10 kDa, less than 7 kDa, or less than 5 kDa.

[0110] In preferred embodiments, the polyplexes comprise chitosan molecules having, on average, 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 polyplexes comprise chitosan molecules having, on average, less than 95 glucosamine monomer units, less than 65 glucosamine monomer units, less than 45 glucosamine monomer units, or less than 35 glucosamine monomer units.

[0111] In a preferred embodiment, the subject polyplexes have an amine to phosphate (N / P) ratio of 2 to 100, such as 2 to 50, such as 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 5. Preferably, the N / P ratio is inversely proportional to the molecular weight of the chitosan, i.e., the lower the molecular weight of the DD-chitosan, the higher the N / P ratio, and vice versa.

[0112] In preferred embodiments, the subject polyplexes have an average hydrodynamic diameter of less than 1000 nm, more preferably less than 500 nm, and most preferably less than 200 nm.

[0113] In one embodiment, the DD-chitosan nucleic acid polyplexes have an average zeta potential of at least 0 mV at an acidic pH, eg, less than pH 7, and most preferably at a pH of about 4 to 6.

[0114] In one embodiment, the DD-chitosan nucleic acid polyplexes have an average zeta potential at acidic pH between +1 and +60 mV, more preferably between +1 and +40 mV, more preferably between +1 and +30 mV.

[0115] In preferred embodiments, the polyplexes have a low net positive charge, are neutral, or have a net negative charge at physiological pH and a pKa less than 6. Such DD-chitosan nucleic acid polyplexes exhibit reduced cytotoxicity, enhanced intracellular release of nucleic acids.

[0116] The DD-chitosan nucleic acid polyplexes of the composition are preferably homogeneous with respect to polyplex size. Thus, in a preferred embodiment, the composition has a low average polydispersity index ("PDI"). In a particularly preferred embodiment, the dispersion of DD-chitosan nucleic acid polyplexes has a PDI of less than 0.5, more preferably less than 0.4, more preferably less than 0.3, and most preferably less than 0.25.

[0117] The polyplexes of the subject compositions are preferably substantially size stable in the composition. In a preferred embodiment, the compositions of the invention comprise polyplexes whose average diameter increases by less than 100%, more preferably less than 50%, and most preferably less than 25% in 6 hours, more preferably less than 12 hours, more preferably less than 24 hours, and most preferably less than 48 hours at room temperature. In a particularly preferred embodiment, the compositions of the invention comprise polyplexes whose average diameter increases by less than 25% in at least 24 hours, or at least 48 hours, at room temperature.

[0118] The polyplexes of the subject compositions are preferably substantially stable in size under refrigerated conditions, hi a preferred embodiment, the compositions of the invention comprise polyplexes that increase in average diameter by less than 100%, more preferably less than 50%, and most preferably less than 25% over 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours at 2-8°C.

[0119] The polyplexes of the subject compositions are preferably substantially size stable under freeze-thaw conditions. In a preferred embodiment, the compositions of the invention comprise polyplexes that increase in average diameter by less than 100%, more preferably less than 50%, and most preferably less than 25% at room temperature after thawing from freezing at -20°C to -80°C, in 6 hours, more preferably 12 hours, more preferably 24 hours, and most preferably 48 hours.

[0120] In a preferred embodiment, the composition has a nucleic acid concentration of greater than 0.5 mg / ml and is substantially free of precipitated polyplexes. More preferably, the composition has a nucleic acid concentration of at least 0.6 mg / ml, more preferably at least 0.75 mg / ml, more preferably at least 1.0 mg / ml, more preferably at least 1.2 mg / ml, and most preferably at least 1.5 mg / ml and is substantially free of precipitated polyplexes. The composition is hydrated. In a preferred embodiment, the composition is substantially free of uncomplexed nucleic acid.

[0121] In a preferred embodiment, the DD-chitosan nucleic acid polyplex compositions are isotonic. Achieving isotonicity while maintaining polyplex stability is highly desirable in the formulation of pharmaceutical compositions, and these preferred compositions are well suited for pharmaceutical formulations and therapeutic applications.

[0122] Generally, a composition comprising a DD-chitosan nucleic acid polyplex is used to contact a target cell. Such contact generally results in delivery of a nucleic acid for expression by the target cell. Compositions suitable for the DD-chitosan nucleic acid polyplexes described herein are well known in the art and are generally described below.

[0123] In a preferred embodiment, the polyplexes of the composition comprise chitosan molecules having an average molecular weight of less than 50 kDa prior to functionalization, an average of less than 310 glucosamine monomer units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least 0 mV at acidic pH, a PDI of less than 0.5, a nucleic acid concentration of greater than 0.5 mg / ml, and are substantially free of precipitated polyplexes, said polyplexes being size stable and increasing in average diameter by less than 100% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the DD-chitosan nucleic acid polyplex composition is isotonic and substantially size stable under freeze-thaw conditions.

[0124] In a preferred embodiment, the polyplexes of the composition comprise chitosan molecules having an average molecular weight of less than 65 kDa prior to functionalization, an average of less than 400 glucosamine monomer units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +40 mV at acidic pH, a PDI of less than 0.4, a nucleic acid concentration of greater than 0.5 mg / ml, and are substantially free of precipitated polyplexes, said polyplexes being size stable and increasing in average diameter by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the DD-chitosan nucleic acid polyplex composition is isotonic and substantially size stable under freeze-thaw conditions.

[0125] In a preferred embodiment, the polyplexes of the composition comprise chitosan molecules having an average molecular weight of less than 40 kDa prior to functionalization, an average of less than 250 glucosamine monomer units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.3, a nucleic acid concentration of greater than 0.5 mg / ml, and are substantially free of precipitated polyplexes, said polyplexes being size stable and increasing in average diameter by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the DD-chitosan nucleic acid polyplex composition is isotonic and substantially size stable under freeze-thaw conditions.

[0126] In a preferred embodiment, the polyplexes of the composition comprise chitosan molecules having an average molecular weight of less than 30 kDa prior to functionalization, an average of less than 190 glucosamine monomer units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.25, a nucleic acid concentration of greater than 0.5 mg / ml, and are substantially free of precipitating polyplexes, said polyplexes being size stable and increasing in average diameter by less than 25% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the DD-chitosan nucleic acid polyplex composition is isotonic and substantially size stable under freeze-thaw conditions.

[0127] In a preferred embodiment, the polyplexes of the composition comprise chitosan molecules having an average molecular weight of less than 30 kDa prior to functionalization, an average of less than 190 glucosamine monomer units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.25, a nucleic acid concentration of greater than 0.75 mg / ml, and are substantially free of precipitated polyplexes, said polyplexes being size stable and increasing in average diameter by less than 25% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the DD-chitosan nucleic acid polyplex composition is isotonic and substantially size stable under freeze-thaw conditions.

[0128] In a preferred embodiment, the polyplexes of the composition comprise chitosan molecules having an average molecular weight of less than 15 kDa prior to functionalization, an average of less than 95 glucosamine monomer units, an N / P ratio of 2 to 5, an average hydrodynamic diameter of less than 200 nm, an average zeta potential of +1 to +30 mV at acidic pH, a PDI of less than 0.25, a nucleic acid concentration of greater than 1.0 mg / ml, and are substantially free of precipitated polyplexes, said polyplexes being size stable and increasing in average diameter by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours. In a preferred embodiment, the DD-chitosan nucleic acid polyplex composition is isotonic and substantially size stable under freeze-thaw conditions.

[0129] Powder formulation

[0130] The DD-chitosan nucleic acid polyplex composition of the present invention comprises a powder. In a preferred embodiment, the present invention provides a dry powder of the DD-chitosan nucleic acid polyplex composition. In a preferred embodiment, the dry powder of the DD-chitosan nucleic acid polyplex composition is produced through dehydration of the chitosan-nucleic acid polyplex dispersion of the present invention.

[0131] Pharmaceutical preparations

[0132] The present invention also provides "pharmaceutical acceptable" or "physiologically acceptable" formulations comprising the DD-chitosan nucleic acid polyplex compositions of the present invention. Such formulations can be administered in vivo to a subject to perform a therapeutic method.

[0133] As used herein, the terms "pharmaceutical acceptable" and "physiologically acceptable" refer to carriers, diluents, excipients, etc. that may be administered to a subject, preferably without causing undue side effects (e.g., nausea, abdominal pain, headache, etc.) Formulations for such administration include sterile aqueous and non-aqueous solutions, suspensions, and emulsions.

[0134] Pharmaceutical preparations can be made from carriers, diluents, excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, etc., suitable for 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, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases, and preservatives, among other additives, can also be included.

[0135] The excipients may include salts, isotonicity agents, serum proteins, buffers or other pH adjusting agents, antioxidants, viscosity enhancing agents, uncharged polymers, preservatives, or cryoprotectants. The excipients used in the compositions of the present invention may further include isotonicity agents and buffers or other pH adjusting agents. These excipients may be added to achieve a preferred range of pH (about 6.0-8.0) and osmolality (about 50-400 mmol / L). Examples of suitable buffers are acetate, borate, carbonate, citrate, phosphate, and sulfonated organic molecule buffers. Such buffers may be included in the compositions at a concentration of 0.01 to 1.0% (w / v). The isotonicity agent may be selected from any isotonicity agent known in the art, such as, for example, mannitol, dextrose, glucose, and sodium chloride, or other electrolytes. Preferably, the isotonicity agent is glucose or sodium chloride. The isotonicity agent may be used in an amount that provides the composition with an osmolality that is the same as or similar to that of the biological environment into which the isotonicity agent is introduced. The concentration of the isotonicity agent in the composition depends on the nature of the particular isotonicity agent used and may range from about 0.1 to 10%. When glucose is used, the isotonicity agent is preferably used at a concentration of 1 to 5% w / v, particularly 5% w / v. When the isotonicity agent is sodium chloride, it is preferably employed in an amount of up to 1% w / v, particularly 0.9% w / v. The composition of the present invention may further comprise a preservative. Examples of preservatives are polyhexamethylene biguanidine, benzalkonium chloride, stabilized oxychloro complexes (such as those known as Purite®), phenylacetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, and thimerosal. Generally, such preservatives are included at a concentration of about 0.001 to 1.0%. Additionally, the composition of the present invention may contain a cryoprotectant, preferred cryoprotectants being glucose, sucrose, mannitol, lactose, trehalose, sorbitol, colloidal silicon dioxide, dextran preferably having a molecular weight less than 100,000 g / mol, glycerol, and polyethylene glycol having a molecular weight less than 100,000 g / mol, or mixtures thereof.The most preferred cryoprotectants are glucose, trehalose, and polyethylene glycol. Typically, such cryoprotectants are included at a concentration of about 0.01 to 10%.

[0136] Pharmaceutical preparations can be formulated to suit the intended route of administration. For example, for oral administration, the composition can be combined with excipients and used in the form of tablets, troches, capsules, such as gelatin capsules, or coatings, such as enteric coatings (Eudragit® or Sureteric®). Pharmaceutically compatible binders and / or auxiliary materials can be included in the oral preparations. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a 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 stearic acids; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or seasonings.

[0137] The formulation may also include carriers to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including implants, and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone or in combination with a wax may be employed.

[0138] Suppositories and other rectally administrable formulations (e.g., formulations that can be administered by enema) are also contemplated.Further, regarding rectal delivery, see, e.g., Song et al., Mucosal drug delivery: membranes, methodologies, and applications, Crit. Rev. Ther. Drug. Carrier Syst., 21:195-256, 2004; Wearley, Recent progress in protein and peptide delivery by noninvasive routes, Crit. Rev. Ther. Drug. Carrier Syst., 8:331-394, 1991.

[0139] Other pharmaceutical formulations suitable for administration are known in the art and are applicable to 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 Co., Inc., Lancaster, Pa., (1993)).

[0140] Administration

[0141] In one embodiment, the use of DD-chitosan in DD-chitosan nucleic acid polyplexes provides sustained polyplex stability at physiological pH, which provides for effective systemic administration, as well as other forms of administration.

[0142] Any of a number of routes of administration are possible, and the choice of a particular route will depend, in part, on the target tissue. Syringes, endoscopes, cannulas, intubation tubes, catheters, and other items may be used for administration.

[0143] A dose for treating a subject, or an "effective amount," is an amount sufficient to prevent or inhibit the progression or worsening of a disorder, condition, or symptom, but preferably to ameliorate one, some, or all of the symptoms of the condition to a measurable or detectable extent. Thus, in the case of a condition or disorder treatable by expressing a therapeutic nucleic acid in a target tissue, the amount of therapeutic RNA or therapeutic protein produced to ameliorate a treatable condition by the method of the invention will depend on the condition and the desired outcome, and can be readily ascertained by one of skill in the art. The appropriate amount will depend on the condition being treated, the desired therapeutic effect, and the individual subject (e.g., bioavailability in the subject, sex, age, etc.). An effective amount can be ascertained by measuring the relevant physiological effect.

[0144] 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 chitosan-based nanoparticle of the present invention.

[0145] Parenteral Administration

[0146] The compounds of the invention may be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle injectors (including microneedles), needle-free injectors, and infusion techniques.

[0147] Parenteral formulations are commonly aqueous solutions which may contain excipients such as salts, carbohydrates, and buffers, although for some applications, parenteral formulations may be more suitably formulated as sterile nonaqueous solutions or as a dry form for use in combination with a suitable carrier, such as sterile, pyrogen-free water.

[0148] The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0149] The solubility of compounds used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

[0150] Formulations for parenteral administration may be formulated to be immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Thus, the compounds of the present invention may be formulated as solids, semi-solids, or thixotropic liquids for administration as an injection depot that provides modified release of the active compound.

[0151] Oral route

[0152] The subject compositions may be administered orally. Oral administration involves a swallowing motion so that the compound enters the digestive tract. The compositions of the present invention may also be administered directly into the digestive tract.

[0153] Formulations suitable for oral administration include solid formulations such as tablets, capsules, particles or coated particles, liquids, or coated capsules containing powders, lozenges (including liquid-filled), chewables, multiparticulates and nanoparticulates, gels, films, vaginal suppositories, and sprays.

[0154] Liquid formulations include suspensions, solutions, syrups and elixirs. Liquid formulations may be prepared by the reconstitution of a solid.

[0155] Tablet dosage forms generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Generally, disintegrants comprise 1% to 25% by weight of the dosage form, preferably 5% to 20% by weight.

[0156] Binders are generally used to give tablets cohesion. Suitable binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. Tablets also contain diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and calcium hydrogen phosphate dihydrate.

[0157] Tablets may also optionally contain surface-active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants, such as silicon dioxide and talc. If included, the surface-active agents may constitute 0.2% to 5% by weight of the tablet, and the glidants may constitute 0.2% to 1% by weight of the tablet.

[0158] Tablets also generally contain a lubricant such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of sodium lauryl sulfate and magnesium stearate. Lubricants generally comprise from 0.25% to 10% by weight of the tablet, preferably from 0.5% to 3%.

[0159] Other possible ingredients include antioxidants, colorants, flavorants, preservatives, and taste-masking agents.

[0160] Tablet blends may be compressed directly or by roller to form tablets. Alternatively, tablet blends, or portions of blends, may be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers, may be coated or uncoated; it may be encapsulated.

[0161] Tablet formulation is discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

[0162] Consumable oral films for human or veterinary use are generally water-soluble or water-swellable pliable thin film dosage forms that may be fast dissolving or mucoadhesive and generally include film-forming polymers, binders, solvents, humectants, plasticizers, stabilizers or emulsifiers, viscosity control agents, and solvents. Some formulation components perform more than one function.

[0163] Multiparticulate beads comprising the compositions of the present invention are also included in the present invention.

[0164] Other possible ingredients include antioxidants, colorants, flavors and flavor enhancers, preservatives, saliva stimulants, cooling agents, co-solvents (including oils), emollients, weighting agents, anti-foaming agents, surfactants, and flavor masking agents.

[0165] Films according to the invention are generally prepared by evaporative drying of thin aqueous films coated onto a peelable support or paper. This may be done in a drying oven or tunnel, usually a combined coater dryer, or by freeze-drying or vacuuming.

[0166] Solid formulations for oral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0167] Other suitable delivery technologies are known, such as high energy dispersions and permeable coated particles.

[0168] Topical administration

[0169] The compounds of the invention may also be administered topically, i.e., dermally or transdermally, to the skin or mucosa. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions.

[0170] Other means of local administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (eg Powderject™, Bioject™, etc.) injection.

[0171] Formulations for topical administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0172] Inhaled / Intranasal

[0173] The compounds of the invention may generally be administered intranasally or by inhalation in the form of a dry powder (either alone, admixed in a dry mix with, for example, lactose, or as mixed component particles) from a dry powder inhaler, or as an aerosol spray from a pressurized container, pump, spray, atomizer, or nebulizer, with or without the use of a suitable propellant.

[0174] Capsules, effervescents, 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.

[0175] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0176] Rectal / vaginal administration

[0177] 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 alternatives may be used as appropriate.

[0178] Formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release.

[0179] Ocular / Aural Administration

[0180] The compounds of the present invention may also be administered directly to the eye or ear, generally in the form of drops.Other formulations suitable for administration to the eye and ear include ointments, biodegradable (e.g., absorbent gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and microparticle systems.The formulations may also be delivered by iontophoresis.

[0181] Formulations for ocular / aural administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0182] Usage

[0183] In one embodiment, the DD-chitosan nucleic acid polyplex compositions of the invention may be used for therapeutic treatments, sometimes referred to herein as therapeutic compositions.

[0184] Therapeutic proteins of the invention are produced by polyplexes of the invention that include therapeutic nucleic acids, as discussed below. Uses of the subject proteins described below relate to uses of the subject polyplexes that affect the use of such proteins.

[0185] Therapeutic proteins contemplated for use in the present invention have a wide variety of activities and are used in the treatment of a wide variety of disorders. The following description of the activities of Therapeutic proteins and indications treatable with Therapeutic proteins of the present invention is exemplary and not intended to be exhaustive. The term "subject" refers to animals, with mammals being preferred, and humans being particularly preferred.

[0186] A partial list of therapeutic proteins and target diseases is provided in Table 4.

[0187] [Table 4]

[0188] In another embodiment, the therapeutic compositions of the present invention comprise a therapeutic nucleic acid, e.g., a therapeutic RNA, that does not code for a therapeutic protein. For example, by selecting a therapeutic RNA that targets a gene involved in the mechanism of a disease and / or an undesired cellular or physiological condition, the subject compositions may be used in the treatment of a wide range of diseases and conditions. The subject compositions have properties such that the therapeutic RNA used is not limited in terms of the range of target selection. Thus, the subject compositions are used in any disease or condition that contains a compatible target.

[0189] Preferred tissues, diseases and conditions include, by way of example and not by way of limitation, the following: [Table 5-1] [Table 5-2]

[0190] Hyperglycemia and weight

[0191] Therapeutic proteins include insulin and insulin analogs. Diabetes is a debilitating metabolic disease caused by the absence (type 1) or insufficient (type 2) production of insulin from the beta cells of the pancreas (Unger, RH et al., Williams Textbook of Endocrinology Saunders, Philadelphia (1998)). Beta cells are specialized endocrine cells that produce and store insulin for release after a meal (Rhodes, et. al. J. Cell Biol. 105:145 (1987)), and insulin is a hormone that promotes the transport of glucose from the blood to tissues that need it. Patients with diabetes must frequently monitor their blood glucose levels, and many require multiple insulin injections each day to stay alive. However, these patients rarely achieve ideal glucose levels with insulin injections (Turner, RC et al. JAMA 281:2005 (1999)). Furthermore, sustained elevation of insulin levels can lead to adverse side effects such as hypoglycemic shock and reduced sensitivity of the body's response to insulin. Thus, diabetic patients also develop long-term complications such as cardiovascular disease, kidney disease, blindness, nerve damage, and impaired wound healing (UK Prospective Diabetes Study (UKPDS) Group, Lancet 352, 837 (1998)).

[0192] Disorders treatable by the methods of the invention include hyperglycemic conditions, such as insulin-dependent (type 1) or non-insulin-dependent (type 2) diabetes, and physiological conditions or disorders associated with or resulting from hyperglycemic conditions. Thus, hyperglycemic conditions treatable by the methods of the invention also include histopathological changes associated with chronic or acute hyperglycemia (e.g., diabetes). Specific examples include pancreatic degeneration (beta cell destruction), renal tubular calcification, liver degeneration, eye damage (diabetic retinopathy), diabetic foot, ulcers in mucous membranes such as the mouth and gums, excessive bleeding, delayed blood clotting and wound healing, and increased risk of coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity.

[0193] The subject compositions are useful for reducing glucose, improving glucose tolerance, treating hyperglycemic conditions (e.g., diabetes), or treating physiological disorders associated with or resulting from hyperglycemic conditions. Such disorders include, for example, diabetic (autonomic) neuropathy, nephropathy (kidney damage), skin infections and other skin disorders, slow or delayed healing of injuries or wounds (e.g., resulting in diabetic carbuncles), eye damage (retinopathy, cataracts) that can lead to blindness, diabetic foot, and accelerated periodontitis. Such disorders also include increased risk of developing coronary heart disease, stroke, peripheral vascular disease, dyslipidemia, hypertension, and obesity.

[0194] As used herein, the term "hyperglycemic" or "hyperglycemia" refers to an abnormally high level of glucose present in the blood of a subject, either temporary or chronic, when used in relation to a condition of a subject. The condition can be caused by a delay in glucose metabolism or glucose absorption, such that the subject shows a state of glucose intolerance or high glucose levels not commonly seen in normal subjects (e.g., in glucose intolerant subdiabetic subjects at risk of developing diabetes, or in diabetic subjects). Fasting plasma glucose (FPG) levels are less than about 110 mg / dl in normal blood sugar, between about 110 mg / dl and 126 mg / dl in impaired glucose metabolism, and greater than about 126 mg / dl in diabetes.

[0195] Disorders treatable by producing proteins in the mucosal tissue of the gastrointestinal tract also include obesity, or undesirable body weight. Leptin, cholecystokinin, PYY, and GLP-1 reduce hunger, increase energy expenditure, cause weight loss, or provide normal glucose homeostasis. Thus, in various embodiments, the method of the invention for treating obesity, or undesirable body weight, or hyperglycemia includes the use of a therapeutic nucleic acid encoding leptin, cholecystokinin, PYY, or GLP-1. In another embodiment, a therapeutic RNA targeting ghrelin is used. Ghrelin increases appetite and hunger. Thus, in various embodiments, the method of the invention for treating obesity, or undesirable body weight, or hyperglycemia includes the use of a therapeutic RNA targeting ghrelin to reduce the expression of ghrelin. Treatable disorders also include disorders commonly associated with obesity, such as abnormally high serum / plasma LDL, VLDL, triglycerides, cholesterol, plaque formation leading to narrowing or blockage of blood vessels, hypertension / stroke, and increased risk of coronary heart disease.

[0196] As used herein, the term "obese" or "obesity" refers to a subject who weighs at least 30% more than an age- and sex-matched normal subject. "Undesirable weight" refers to a subject who weighs 1%-29% more than a matched normal subject, and to a subject who is normal with respect to weight but who wishes to lose weight or prevent weight gain.

[0197] In one embodiment, the therapeutic protein of the present invention is a glucagon antagonist. Glucagon is a peptide hormone produced by the beta cells of pancreatic islets and is the main regulator of glucose metabolism (Unger RH & Orci LN Eng. J. Med. 304:1518(1981); Unger RH Diabetes 25:136 (1976)). Like insulin, blood glucose concentration mediates the secretion of glucagon. However, in contrast to insulin, glucagon is secreted in response to a decrease in blood glucose. Thus, circulating concentrations of glucagon are highest during fasting and lowest during feeding. Glucagon levels increase to prevent insulin from promoting glucose storage 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-induced diabetic rats, within 15 minutes of an intravenous bolus (0.75 μg / g body weight) of this glucagon antagonist, blood glucose levels were reduced by 37% (Van Tine BA et. al. Endocrinology 137:3316 (1996)). In another embodiment, the present invention provides a method for treating diabetes, or hyperglycemia, comprising using a therapeutic RNA to reduce the level of glucagon production from the pancreas.

[0198] In another embodiment, the therapeutic protein of the present invention useful for treating hyperglycemic conditions or undesirable weight gain (e.g., obesity) is glucagon-like peptide-1 (GLP-1). GLP-1 is a hormone released from L-cells in the intestine during a meal and stimulates beta cells in the pancreas to increase insulin secretion. GLP-1 has other 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 concentrations, increases beta cell mass, stimulates insulin biosynthesis and secretion in a glucose-dependent manner, and GLP-1 may increase tissue sensitivity to insulin (Kieffer TJ, Habener JF Endocrin. Rev. 20:876 (2000)). Thus, regulated release of GLP-1 in the gastrointestinal tract concomitant with a meal may provide a therapeutic effect on hyperglycemic conditions or undesirable weight gain. GLP-1 analogs that are resistant to dipeptidyl peptidase IV (DPP IV) provide a longer duration of action and improved therapeutic value. Thus, GLP-1 analogs are preferred therapeutic polypeptides. In another embodiment, the present invention provides a method for treating diabetes or hypertension, comprising using a therapeutic RNA to reduce the level of DPP IV.

[0199] In another embodiment, the therapeutic protein of the present invention useful for treating hyperglycemic conditions is an antagonist to the hormone resistin. Resistin is an adipocyte-derived factor whose expression is elevated in diet-induced and hereditary forms of obesity. Neutralization of circulating resistin improves blood glucose and insulin action in obese mice. Conversely, administration of resistin to normal mice impairs glucose tolerance and insulin action (Steppan CM et. al. Nature 409:307 (2001)). Thus, production of a protein that antagonizes the biological action of resistin in the gastrointestinal tract may provide an effective treatment for insulin resistance and hyperglycemic conditions associated with obesity. In another embodiment, the present invention provides a method for treating diabetes, hypertension, comprising using a therapeutic RNA to reduce the expression level of resistin in adipose tissue.

[0200] In another embodiment, a therapeutic polypeptide of the invention useful for treating hyperglycemic conditions or undesirable body weight (e.g., obesity) is leptin. Leptin is produced primarily by adipocytes, but also in small amounts in the stomach in a meal-dependent manner. Leptin relays information about adipocyte metabolism and body weight to appetite centers in the brain, which signal to decrease food intake (promoting satiety) and increase the body's energy expenditure.

[0201] In another embodiment, a therapeutic polypeptide of the invention useful for treating hyperglycemic conditions 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 a proteolytic cleavage product of Acrp30 consisting of the globular head domain to mice results in significant weight loss (Fruebis J. et al. Proc. Natl Acad. Sci USA 98:2005 (2001)).

[0202] In another embodiment, a therapeutic polypeptide of the present invention useful for treating hyperglycemic conditions or undesirable body weight (e.g., obesity) is cholecystokinin (CCK). CCK is a gastrointestinal peptide secreted from the intestine in response to certain nutrients in the gastrointestinal tract. The release of CCK is proportional to the amount of food consumed and is believed to signal the brain to end eating (Schwartz MW et. al. Nature 404:661-71(2000)). Thus, elevated CCK may reduce food intake and promote weight loss or weight stabilization (i.e., prevent or inhibit weight gain).

[0203] Regarding PYY, see, e.g., le Roux et al., Proc Nutr Soc. 2005 May; 64(2):213-6.

[0204] Immune system disorders

[0205] In one embodiment, the therapeutic composition of the present invention has immunomodulatory activity. For example, the therapeutic polypeptide of the present invention may be useful for treating immune system deficiencies or disorders by activating or inhibiting the proliferation, differentiation, or motility (chemotaxis) of immune cells. Immune cells are developed through the hematopoietic process, which produces myeloid cells (platelets, red blood cells, neutrophils, and macrophages) and lymphoid cells (B lymphocytes and T lymphocytes) from pluripotent stem cells. The causes of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorder, acquired (e.g., by chemotherapy or toxins), or infectious.

[0206] The therapeutic compositions of the present invention may be useful for treating hematopoietic cell deficiencies or disorders. For example, the therapeutic polypeptides of the present invention can be used to increase the differentiation or proliferation of hematopoietic cells, including pluripotent stem cells, for the purpose of treating those disorders associated with a decrease in a particular (or many) type of hematopoietic cell. Examples of immune deficiency 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, lymphopenia, phagocytosis and bactericidal dysfunction, severe combined immunodeficiency (SCID), Wiskott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria.

[0207] The therapeutic composition of the present invention may be useful for treating autoimmune disorders.Many autoimmune disorders are caused by the inappropriate recognition of self as foreign by immune cells.This inappropriate recognition leads to an immune response that leads to the destruction of host tissue.Therefore, administration of the therapeutic composition of the present invention that inhibits immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, may be an effective treatment for preventing autoimmune disorders.

[0208] Examples of autoimmune disorders treatable 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, ophthalmia, bullous pemphigoid, pemphigus, polyendocrinopathy, purpura, Reiter's disease, stiff man syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barré syndrome, insulin-dependent diabetes mellitus, Crohn's disease, ulcerative colitis, and autoimmune inflammatory eye disease.

[0209] Similarly, allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems may be treated with the therapeutic compositions of the present invention. Additionally, these molecules may be used to treat anaphylaxis, hypersensitivity to antigenic molecules, or blood type incompatibility.

[0210] 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 due to the destruction of transplanted tissue through an immune response by host immune cells. Similarly, an immune response is also involved in GVHD, where foreign transplanted immune cells destroy host tissue. Administration of the therapeutic compositions of the present invention to inhibit immune responses, particularly T-cell proliferation, differentiation, or chemotaxis, may be an effective treatment to prevent organ rejection or GVHD.

[0211] Similarly, the therapeutic compositions of the present invention may be used to alleviate inflammation. For example, therapeutic polypeptides may inhibit the proliferation and differentiation of cells involved in inflammatory responses. These molecules may be used to treat chronic and acute inflammatory conditions, including inflammation associated with infections (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 due to overproduction of cytokines (e.g., TNF or IL-1). In one embodiment, therapeutic RNAs targeting TNFα are used in the subject compositions to treat inflammation. In another preferred embodiment, therapeutic RNAs targeting IL-1 are used in the subject compositions to treat inflammation. siRNA therapeutic RNAs are particularly preferred. Inflammatory disorders of interest for treatment in the present invention include, but are not limited to, chronic obstructive pulmonary disease (COPD), interstitial cystitis, and inflammatory bowel disease.

[0212] Coagulation disorders

[0213] In some embodiments, the therapeutic compositions of the present invention may be used to regulate hemostatic activity (stopping bleeding) or thrombolytic activity (thrombus formation). For example, by increasing hemostatic or thrombolytic activity, the therapeutic compositions of the present invention may be used to treat blood clotting disorders (e.g., afibrinogenemia, factor deficiency), platelet abnormalities (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, the therapeutic compositions of the present invention, which may increase hemostatic or thrombolytic activity, may be used to inhibit or dissolve clots. These therapeutic compositions may be important in the treatment of heart attacks (infarctions), strokes, or scarring. In one embodiment, the therapeutic polypeptides of the present invention are clotting factors (e.g., factors VIII, IX, or X) useful for the treatment of hemophilia or other clotting disorders.

[0214] Hyperproliferative disorders

[0215] In one embodiment, the therapeutic composition of the invention is capable of modulating cell proliferation. Such therapeutic polypeptides may be used to treat hyperproliferative disorders, including tumors.

[0216] Examples of hyperproliferative disorders that may be treated by the therapeutic compositions of the present invention include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, breast, and genitourinary tract.

[0217] Similarly, other hyperproliferative disorders may also be treated with the therapeutic compositions of the present invention, including, but not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom's macroglobulinemia, Gaucher's disease, histiocytosis, and any other hyperproliferative disease other than tumors located in the organ systems listed above.

[0218] Delivery to the circulatory system provides access for therapeutic proteins to a wide variety of tissues. Alternatively, the therapeutic compositions of the invention may stimulate the proliferation of other cells that may suppress hyperproliferative disorders.

[0219] For example, hyperproliferative disorders can be treated by increasing immune response, particularly by improving the quality of the antigen of the hyperproliferative disorder, or by expanding, differentiating or mobilizing T cells. This immune response can be increased by either enhancing existing immune response or initiating new immune response. Alternatively, reducing immune response can be a method of treating hyperproliferative disorders, such as using chemotherapeutic agents.

[0220] infectious disease

[0221] In one embodiment, the therapeutic composition of the present invention may be used to treat infectious diseases. For example, infectious diseases may be treated by increasing immune response, particularly increasing proliferation and differentiation of B cells and / or T cells. This immune response may be increased either by enhancing an existing immune response or by initiating a new immune response. Alternatively, the therapeutic composition of the present invention may directly inhibit infectious agents without necessarily inducing an immune response.

[0222] Viruses are an example of infectious agents that can cause diseases or symptoms that can be treated by the therapeutic composition 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, shingles, etc.), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (e.g., smallpox, smallpox, etc.), Reoviridae (e.g., rotavirus), Retroviridae (HTLV-I, HTLV-II, lentivirus), and Togaviridae (e.g., rubivirus). Viruses within these families can cause a variety of diseases or conditions, 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, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's sarcoma, warts), and viremia. The therapeutic compositions of the present invention can be used to treat any of these conditions or diseases.

[0223] Similarly, bacterial or fungal agents that may cause diseases or conditions that may 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, Nocardia), Aspergillus, Bacillaceae (e.g., Bacillus anthracis, Clostridium), Bacteroidetes, Blastomyces, Bordetella, Borrelia, Brucella, Candida, Campylobacter, and the like. Bacteria such as Streptococcus, Coccidioides, Cryptococcus, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionella, Leptospira, Listeria, Mycoplasmales, Neisseriaceae (e.g., Acinetobacter, Neisseria gonorrhoeae, Neisseria meningitidis), Pasteurellaceae infections (e.g., Actinobacillus, Haemophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcus. These bacterial or fungal families may cause the following diseases or conditions, including but not limited to bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS-related infections), paronychia, prosthetic-related infections, Reiter's disease, respiratory tract infections, such as whooping cough or sinusitis, sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid, 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 (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. The therapeutic compositions of the present invention may be used to treat any of these conditions or diseases.

[0224] Additionally, parasitic agents causing diseases or conditions that may be treated by the therapeutic compositions of the present invention include, but are not limited to, the following families: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, diamoebiasis, dourine, ectoparasitosis, giardiasis, helminthiasis, leishmaniasis, theileriosis, toxoplasmosis, trypanosomiasis, and trichomonas. These parasites may cause a variety of diseases or conditions, including, but not limited to, scabies, scrub typhus, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver diseases, lung diseases, opportunistic infections (e.g., AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The therapeutic compositions of the present invention may be used to treat any of these conditions or diseases.

[0225] reproduction

[0226] The therapeutic compositions of the invention can be used to differentiate, proliferate, and attract cells to promote tissue regeneration (see Science 276:59-87 (1997)). Tissue regeneration can be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), aging, disease (e.g., osteoporosis, osteoarthritis, periodontal disease, liver failure), surgery, including cosmetic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

[0227] The therapeutic compositions of the present invention may promote regeneration of various tissues, including, but not limited to, organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal, or cardiac muscle), blood vessels (including vascular endothelium), nerves, hematopoietic organs, and skeletal tissues (bone, cartilage, tendons, and ligaments). Preferably, regeneration occurs with little or no scarring. Regeneration may also include angiogenesis.

[0228] Additionally, the therapeutic compositions of the present invention may increase regeneration of tissues that are difficult to heal. For example, increased tendon / ligament regeneration will speed up recovery time after injury. The therapeutic compositions of the present invention may also be used prophylactically to avoid injury. Specific diseases that may be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Further examples of tissue regeneration of non-healing wounds include bedsores, ulcers associated with vascular insufficiency, surgical wounds, and traumatic wounds.

[0229] Similarly, nerve tissue and brain tissue can also be regenerated by using the therapeutic compositions 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 disorders (e.g., spinal cord injury, head trauma, cerebrovascular disease, and stalk). In particular, peripheral neuropathy, peripheral neuropathies (e.g., resulting from chemotherapy or other medical therapies), focal neuropathy, and all diseases related to central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can be treated using the therapeutic compositions of the present invention. In relation to CNS disorders, numerous means are known in the art to facilitate therapeutic access to brain tissue, including methods of disrupting the blood-brain barrier and methods of combining therapeutic agents with moieties that provide transport into the CNS. In one embodiment, the therapeutic nucleic acid is designed to encode a fusion protein, the fusion protein comprising a transport moiety and a therapeutic protein. Alternatively, the subject compositions may be delivered directly to the CNS.

[0230] chemotaxis

[0231] In one embodiment, the therapeutic composition of the invention may modulate chemotaxis. For example, in one embodiment, the therapeutic polypeptide of the invention has chemotactic activity. Chemotactic molecules attract or recruit cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial cells, and / or endothelial cells) to a particular site in the body, such as a site of inflammation, infection, or hyperproliferation. The recruited cells can then fight off and / or heal a particular injury or disorder.

[0232] For example, the therapeutic polypeptides of the present invention may increase the chemotactic activity of certain cells. These chemotactic molecules may then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotactic molecules may be used to treat tissue wounds and other trauma by attracting immune cells to the location of injury. The chemotactic molecules of the present invention can also attract fibroblasts, which may be used to treat wounds.

[0233] It is also contemplated that the therapeutic compositions of the present invention may 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 chemotactic inhibitors.

[0234] Particularly preferred for use are pre-therapeutic proteins that are activated in the vicinity of the target tissue.

[0235] 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 beta, etc.) for treating growth disorders or wasting syndromes; and polypeptides for providing passive immunity or protection in a subject against foreign antigens or pathogens (e.g., Helicobacter pylori), or for providing treatment of cancer, arthritis, or cardiovascular disease. (e.g., human or humanized) antibodies for providing a therapeutic agent; cytokines, interferons (e.g., interferons (IFN), IFN-α2b, IFN-2α, IFN-αN1, IFN-β1b, IFN-γ), interleukins (e.g., IL-1 through IL-10), tumor necrosis factors (TNF-α, TNF-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), gonadotropins, chemotactic factors, 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, tyrosine phosphate phosphate (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 (e.g., FAbs) thereof (e.g., Orthoclone OKT-e (anti-CD3), GPIIb / IIa monoclonal antibodies). Therapeutic RNAs targeting nucleic acids encoding such factors are also contemplated.

[0236] vaccine

[0237] In one embodiment, the present invention provides a method for vaccinating a patient, said method comprising administering a composition of the present invention capable of producing a desired epitope. In a preferred embodiment, said composition comprises a therapeutic nucleic acid construct capable of expressing a protein comprising said epitope.

[0238] Cosmetic Use

[0239] In one embodiment, the present invention provides a DD-chitosan nucleic acid polyplex for cosmetic use. The cosmetic product of interest comprises a DD-chitosan nucleic acid polyplex in a formulation suitable for cosmetic use. EXAMPLES

[0240] Example 1: Formation of doubly derivatized chitosan and DNA polyplexes

[0241] Chitosan was doubly derivatized with arginine and gluconic acid or with arginine and threonic acid (DD-chitosan) according to well-known methods. DD-chitosan was polyplexed with DNA vectors encoding either secreted alkaline phosphatase (SEAP) or luciferase siRNA.

[0242] Example 2: Transfection with DNA polyplexes in vitro

[0243] Generally, in vitro transfection of 293T cells with DD-chitosan nucleic acid polyplex formulations was carried out in two steps: cell preparation followed by transfection.

[0244] Example 3: Maintenance of cell lines

[0245] The 293T cell line was kindly provided by the laboratory of Dr. Timothy Kieffer at the University of British Columbia (Vancouver, Canada) and was prepared as follows: human kidney cells were transformed with SV40 T antigen, grown in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin / streptomycin, and maintained at less than 80% confluency.

[0246] Example 4: Preparation of cells for transfection

[0247] Cells were prepared for transfection as follows: The day before transfection, 293T cells were plated in 6-well tissue culture plates (4.5x10 cells) in 3 mL of complete medium (high glucose DMEM + 10% FBS + penicillin / streptomycin). 5 On the day of transfection, cells were washed once with phosphate-buffered saline (PBS), trypsinized with 0.5 mL of 0.05% trypsin, added 0.5 mL of complete medium, and cell numbers were determined for two selected wells by counting 10 μL using a hemocytometer. When cells were approximately 50% confluent (approximately 7x10 5 If the cells were too sparse or too confluent, then transfection was not allowed to proceed.

[0248] Example 5: Transfection of cells

[0249] Transfections were performed as follows: First, the medium was removed from each well, 1 mL of Opti-mem (pH 7.4) was added to each well, swirled gently, and then removed. (Six wells were washed simultaneously to prevent cells from being dislodged). Next, an additional 1 mL of Opti-mem (pH 7.4) was added to each well, taking care not to dislodge the cells. Polyplex samples were then added to each well (2 μg DNA of interest), swirled, and incubated at 37°C for 2 hours. After incubation, the medium was removed and replaced with 2 mL of complete medium, and incubated again at 37°C. At the required time point, the supernatant was removed and stored at -20°C for subsequent SEAP assays.

[0250] Example 6: SEAP Protein Assay

[0251] The SEAP assay was performed using the SEAP Chemiluminescent Assay kit. All reagents for the assay were equilibrated at 25°C for 30 minutes before use. Placental alkaline phosphatase was dissolved at 1 mg / mL in 1X dilution buffer from the kit supplemented with 0.1% bovine serum albumin and 50% glycerol, and then assay standards were prepared by 10-fold serial dilution in DMEM to 0.01 pg / uL. Standards and thawed samples were then diluted 1 / 4 in dilution buffer, heat inactivated at 65°C for 30 minutes, incubated on ice for 2 minutes, centrifuged (16100x rcf for 2 minutes at room temperature), and the supernatant was transferred to a new tube. After equilibration at 25°C for 5 minutes, 50 uL of samples and standards were added to each well of the Microlite-1 plate, respectively. Inactivation buffer (50 uL) was then added to each well, mixed by gently pipetting up and down to avoid creating bubbles, and incubated for 5 min. Substrate / enhancer reagent was prepared with a 1:19 substrate to enhancer ratio and incubated for 5 min. Substrate / enhancer was then added to each well and incubated for 20 min, then the plate was read on a luminometer (Lmax11384, Molecular Devices) with a 1 sec integration time.

[0252] Example 7: Results

[0253] Figures 1-3 show the transfection efficiency of chitosan derivatized with different percentages of arginine or gluconic acid, and of chitosan doubly derivatized with both gluconic acid and arginine. Figures 1-3 show the synergistic effect when chitosan is doubly derivatized with both arginine and gluconic acid. Although the highest synergy is seen with a final functionalization degree of gluconic acid of about 3%, synergistic effects can be seen when chitosan is doubly functionalized with arginine with a final functionalization degree of 10% and gluconic acid with a final functionalization degree ranging from 3% to 10% (Figure 1). Although the highest synergistic effects are seen with a final functionalization degree of gluconic acid of about 8%, synergistic effects can also be seen when chitosan is doubly functionalized with arginine with a final functionalization degree of 52% and gluconic acid with a final functionalization degree ranging from 3% to 8% (Figure 2). Also, chitosan doubly functionalized with arginine and gluconic acid with a final degree of functionalization of 26% and 6% showed the greatest synergy in transfection efficiency (Figure 3). Finally, synergy can also be seen when chitosan is doubly derivatized with arginine and an alternative moiety such as threonic acid instead of gluconic acid. Synergy is observed when chitosan is doubly functionalized with arginine with a final degree of functionalization of 29% and threonic acid with a final degree of functionalization from 3% (Figure 4).

[0254] Example 8:

[0255] Chitosan is doubly derivatized with arginine and a HP selected from the group consisting of 2,3-dihydroxylpropanoic acid; 2,3,4,5,6,7-hexahydroxylheptanal; 2,3,4,5,6-pentahydroxylhexanal; 2,3,4,5-tetrahydroxylhexanal; and 2,3-dihydroxylpropanal (DD-chitosan) and analyzed according to Examples 1 to 7. DD-chitosan is polyplexed with DNA vectors encoding either secreted alkaline phosphatase (SEAP) or luciferase siRNA.

[0256] All citations are expressly incorporated herein by reference in their entirety.

[0257] All patents and patent publications related to this specification are hereby incorporated by reference.

[0258] Certain modifications and improvements will occur to those skilled in the art upon reading the above description. Such modifications and improvements have been excluded from this specification for purposes of brevity and readability, but it should be understood that they properly lie within the scope of the following claims.

Claims

1. Chitosan functionalized with arginine (Arg) and a hydrophilic polyol (HP) of formula VII: 【Chemistry 1】 During the ceremony: R 2 is selected from H, and hydroxyl; R 3 is selected from H, and hydroxyl; X is C optionally substituted with one or more hydroxyl substituents 2 -C 6 Chitosan derivative nanoparticles comprising a chitosan selected from alkylenes.

2. A hydrophilic polyol of formula VII: 【Chemistry 2】 During the ceremony: R 2 is selected from H, and hydroxyl; R 3 is selected from H, and hydroxyl; X is C optionally substituted with one or more hydroxyl substituents 2 -C 6 a hydrophilic polyol selected from the group consisting of alkylenes; The chitosan derivative nanoparticles of claim 1 , with the proviso that the hydrophilic polyol is not gluconic acid.

3. The chitosan derivative nanoparticles according to claim 2 , wherein the hydrophilic polyol has a carboxyl group.

4. The chitosan derivative nanoparticles of claim 3 , wherein the hydrophilic polyol is threonic acid.

5. The chitosan derivative nanoparticles of claim 2, wherein the hydrophilic polyol is a saccharide selected from the group consisting of glyceraldehyde, threose, erythrose, ribose, arabinose, xylose, lyxose, allose, glucose, altrose, mannose, gulose, idose, galactose, and talose.

6. The hydrophilic polyol is 2,3-dihydroxylpropanoic acid; 2,3,4,5,6,7-Hexahydroxylheptanal; 3. The chitosan derivative nanoparticles of claim 2, which are selected from the group consisting of 2,3,4,5,6-pentahydroxylhexanal; 2,3,4,5-tetrahydroxylhexanal; and 2,3-dihydroxylpropanal.

7. 11. The chitosan derivative nanoparticle of any of the preceding claims, comprising chitosan-nucleic acid polyplexes comprising chitosan molecules having an average molecular weight before functionalization of less than 65 kDa, an average of less than 400 glucosamine monomer units, an N / P ratio of 2 to 20, an average hydrodynamic diameter of less than 500 nm, an average zeta potential of at least 0 mV at acidic pH, a PDI of less than 0.5, and a nucleic acid concentration of more than 0.5 mg / ml, wherein the chitosan-nucleic acid polyplexes are substantially free of precipitated polyplexes, and the chitosan-nucleic acid polyplexes are stable in size and increase in average diameter by less than 100% for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours at room temperature.

8. The chitosan derivative nanoparticle of claim 7, wherein the average diameter of the chitosan-nucleic acid polyplexes increases by less than 50% at room temperature for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours.

9. 2. The chitosan derivative nanoparticle of any of the preceding claims, wherein said nanoparticles have a ratio of final degree of Arg functionalization:final degree of HP functionalization of between about 1:1 and about 10:

1.

10. 10. The chitosan derivative nanoparticles of claim 9, wherein the ratio of the final degree of functionalization of Arg to the final degree of functionalization of HP is between about 3:1 and about 7:

1.

11. The chitosan derivative nanoparticles of claim 10, wherein the ratio of the final degree of functionalization of Arg to the final degree of functionalization of HP is about 5:

1.

12. 12. A composition comprising the nanoparticles of any one of claims 1 to 11, wherein the chitosan forms a complex with a nucleic acid to form a doubly derivatized (DD) chitosan-nucleic acid polyplex.

13. The composition of claim 12 , wherein the nucleic acid is DNA or RNA.

14. The composition of claim 12 or 13, wherein the amine to phosphate ratio of the DD-chitosan nucleic acid polyplex is between 2 and 100.

15. The composition of claim 14, wherein the amine to phosphate ratio of the DD-chitosan nucleic acid polyplex is between 2 and 50.

16. The composition of claim 15, wherein the amine to phosphate ratio of the DD-chitosan nucleic acid polyplex is between 2 and 30.

17. The composition of claim 16, wherein the amine to phosphate ratio of the DD-chitosan nucleic acid polyplex is between 2 and 15.

18. 18. A method of delivering a nucleic acid molecule to a cell, comprising contacting the cell with a composition according to any one of claims 12 to 17.

19. The method of claim 18 , wherein the cell is in vivo.

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