Field of the Invention

[0001 ] The present invention relates generally to gene therapy, and more particularly, to an immunomodulatory composition for gene delivery.

Background of the Invention

[0002] Hemophilia A is an inherited bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII) that affects 1 :5000 males. Current treatment relies on regular infusions of FVIII protein, either plasma-derived or recombinant. Albeit effective, this treatment is invasive and expensive (>$ 100,000 per patient annually). A challenge for hemophilia management is the development of neutralizing antibodies (inhibitors) to infused FVIII that occur in 30% of hemophiliacs, which decrease treatment efficacy. Thus, an alternative treatment is desirable.

[0003] Hemophilia B is a monogenetic bleeding disorder attributed to a deficiency of functional factor IX that inflicts 1 in 25,000 males. Current treatment involves protein replacement therapy with recombinant or plasma-derived FIX which is invasive, expensive and inaccessible to hemophiliacs in developing nations. Inhibitor formation to FIX products, occurs in ~3% of patients, is associated with severe allergic anaphylactic reactions and increased morbidity. Hemophilia B is classified by the level of normal plasma FIX activity as severe (<1 %), moderate (1 - 5%) or mild (5-50). This broad therapeutic window is beneficial for the application of gene therapy strategies. Although the therapeutic threshold is 25-50 fold greater than hemophilia A treatment.

[0004] Hemophilia is a suitable model for gene therapy, as restoration of modest FVIII activity can provide phenotypic correction. Current gene therapy strategies, effective in expressing high levels of functional FVIII and FIX in murine and canine models of hemophilia, have shown limited efficacy in clinical trials. These challenges have sparked interest in novel non-viral gene therapy strategies.

[0005] Oral administration of DNA or gene tablet has been considered as a non-invasive approach to gene therapy with enhanced patient compliance. The gastrointestinal (GI) tract is able to absorb plasmid DNA and subsequently secrete the transgene product into blood. To increase uptake efficiency, strategies are being devised to protect and chaperone DNA through the gut.

[0006] Chitosan, a cationic polymer, forms nanoparticles through electrostatic interactions with anionic DNA to facilitate delivery in the gut. These nanoparticles protect entrapped DNA from digestion, increase transcellular and paracellular transport across mucosal epithelium, mediate uptake by the Peyer's patches and have been used for the delivery of therapeutic genes. For example, oral administration of chitosan hFVIII-DNA nanoparticles has been reported by Bowman et al. (J Control Release. 2008). The authors observed evidence of hFVIII in plasma using the thrombin generation assay, and a peak of 2-4% of FVIII activity. However, anti- FVIII antibodies were detected via ELISA assay, indicating the potential for FVIII inhibitor formation which would prevent re-administration of FVIII to rescue declining levels.

[0007] It would be desirable, thus, to provide a method of gene delivery that overcomes one or more of the disadvantages of the prior art.

Summary of the Invention

[0008] A novel tolerogenic formulation is provided in which a particle- forming polysaccharide is coupled to a nucleic acid encoding a therapeutic protein.

[0009] Thus, in one aspect of the invention, a tolerogenic formulation is provided comprising a particle-forming polysaccharide coupled to a nucleic acid that encodes a therapeutic protein.

[0010] In another aspect of the invention, a method of treating a disease or condition resulting from a protein deficiency is provided. The method comprises administering to a mammal a tolerogenic nanoparticle formulation that expresses a target therapeutic protein.

[001 1] These and other aspects of the invention will become apparent in the detailed description that follows by reference to the following figures. Brief Description of the Figures

[0012] Figure 1 graphically illustrates transfection efficiency of chitosan nanoparticles. A) Transfection of cells with nanoparticles containing 5 and 10 g luciferase plasmid in comparison to lipofectamine-DNA complex and naked DNA. Relative light units (RLU) normalized by protein content. B) Cells transfected with cFVIII plasmid and assayed with cFVIII ELISA 24 h post transfection. Bars represent meaniSD;

[0013] Figure 2 graphically illustrates plasma FVIII activity following oral administration at days 1 (n=l l), 4 (n=3), 8 (n=3) and 16 (n=6) post treatment. Bars represent mean±SEM. **p<0.01, compared with untreated hemophilic mice;

[0014] Figure 3 graphically illustrates in vivo cFVIII activity following oral administration of chitosan nanoparticles in hemophilic mice. Bars represent mean±SEM. ***p<0.001 , compared with untreated hemophilic mice.

[0015] Figure 4 graphically illustrates the results of a tail clip assay following oral nanoparticle administration. A) Tail bleeding times presented as a fraction of mice still bleeding as a function of time B) Blood loss determined by measuring hemoglobin absorbance (575nm) of blood collected following tail transection;

[0016] Figure 5 graphically illustrates Plasma cFVIII activity following repeated weekly oral administration. Bars represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001 , compared with untreated hemophilic mice;

[0017] Figure 6 graphically illustrates cFVIII antigen detection in mouse plasma following repeated weekly oral administration. Bars represent mean±SEM. *p<0.05, **p<0.01 , ***p<0.001, compared with untreated hemophilic mice;

[0018] Figure 7 graphically illustrates antibody response to repeated weekly oral administration of chitosan nanoparticles;

[0019] Figure 8 is a schematic illustrating different immunomodulation experiments (A,B,C,D); [0020] Figure 9 graphically illustrates the detection of neutralizing

(inhibitors) and non-neutralizing human FVIII antibody in treated mice using anti- human FVIII ELISA performed on plasma samples;

[0021] Figure 10 graphically illustrates in vitro expression of various hFVIII plasmids in HEK 293 cells;

[0022] Figure 11 graphically illustrates the detection of neutralizing

(inhibitors) and non-neutralizing human FVIII antibody in treated mice using anti- human FVIII ELISA performed on plasma samples;

[0023] Figure 12 graphically illustrates immune tolerance to chitosan nanoparticles in naive hemophilia A mice. (A, D, H) Naive hemophilia A mice fed chitosan nanoparticles containing 100 g human FVIII DNA once a week for a total of eight weeks. (B, E, H) Mice fed nanoparticles during the first eight weeks followed by weekly immunizations with 0.75 U rhFVIII on weeks 9, 10 11 and 12. (C, F, H) Hemophilia A mice fed chitosan nanoparticles once a week for a total of twelve weeks and immunized with rhFVIII on weeks 9 10, 11 and 12, and detection of FVIII antibodies (G);

[0024] Figure 13 graphically illustrates oral tolerance to chitosan nanoparticles in hemophilia A mice with pre-existing hFVIII antibodies. (A, D) Hemophilia A mice given immunized weekly i.p. injections of 0.75 U rhFVIII for a total of four weeks. (B, E) Mice were given four weekly rhFVIII injections followed by weekly oral nanoparticle administration on weeks 5-16. (C, F) Hemophilia A mice immunized weekly with rhFVIII for a total of 16 weeks and fed nanoparticles on weeks 5-16;

[0025] Figure 14 graphically illustrates the results of anti-human FVIII

ELISA performed on plasma samples to detect both neutralizing (inhibitors) and non- neutralizing human FVIII antibodies and illustrates adoptive transfer of splenocytes. (A, C, E) Control mice received splenocytes from naive mice. (B, D, E) Mice received splenocytes from nanoparticle-fed donor mice. Bars represent mean±SEM. ***p<0.001, compared with control; [0026] Figure 15 graphically illustrates plasma hFIX activity following oral administration in hemophilia B mice. Bars represent mean±SEM. ***p<0.001, *p<0.05 compared with untreated hemophilic mice; and

[0027] Figure 16 graphically illustrates the effect of salts (A) and charge ratio

(B) on nanoparticle transfection efficiency.

Detailed Description of the Invention

[0028] A novel tolerogenic nanoparticle formulation is provided. The nanoparticle formulation comprises a particle-forming polysaccharide coupled to a nucleic acid encoding a therapeutic peptide or protein.

[0029] As used herein, the terms "therapeutic protein" is meant to encompass full-length proteins as well as peptide portions thereof.

[0030] The term "tolerogenic" is used herein to refer to a formulation that induces immune tolerance in a host to a therapeutic protein or peptide portion thereof (i.e. antigen) expressed by the formulation, such that on subsequent exposure to the therapeutic protein/peptide antigen, the host is essentially non-responsive, e.g. exhibits minimal, if any, cellular and/or humoral immune responses to the therapeutic protein/peptide antigen. Immune responses include the formation of neutralizing antibodies (therapeutic protein inhibitor) that may prevent subsequent administration of the therapeutic protein/peptide.

[0031] The formulation comprises particle-forming cationic polysaccharides which are capable of coupling to a protein-encoding nucleic acid or gene to form nanoparticles. Examples of suitable polysaccharides include polysaccharides which are biocompatible, biodegradable and which exhibit low toxicity, such as chitosan, chitin, cellulose and biocompatible modified forms of these polysaccharides such as derivatives exhibiting different degrees of deacetylation, different chain lengths, or derivatives incorporating ligands on branch chains. As one of skill in the art will appreciate, the selected polysaccharide may comprise mixtures of one or more different polysaccharides and their derivatives in order to optimize the formulation. For example, the formulation may comprise a mixture of fully deacetylated polysaccharide, e.g. chitosan, along with a fully acetylated polysaccharide, e.g. chitin, to yield a polysaccharide component having a suitable charge density for coupling with nucleic acid. In addition, the selected polysaccharide may be utilized in non- ionized form, or as a salt, including for example, chloride or glutamate salts. For the purposes of the present invention, polysaccharides having a molecular weight of less than about 400 kDa, preferably less than 300 kDa, and more preferably having a molecular weight in the range of about 10-250 kDa. In one embodiment, chitosan is employed in the formulation. Chitosan is composed of β-(1, 4)-linked D-glucosamine and N-acetyl-D-glucosamine.

[0032] Nucleic acid suitable for inclusion in the present nanoparticles encodes a target therapeutic protein. As one of skill in the art will appreciate, the nucleic acid is not limited with respect to the target protein it encodes, and thus, may encode a wide range of target proteins. Examples of suitable therapeutic proteins that may be encoded by nucleic acid incorporated in the present nanoparticles include coagulation factor VIII (FVIII), functional factor IX (FIX), insulin and functionally equivalent variants thereof, including variants that incorporate one or more amino acid deletions, additions or substitutions. Nucleic acid in linear or cyclic form may be utilized, as well as RNA and DNA. DNA is the preferred nucleic acid for use in the formulation.

[0033] The nanoparticles of the present formulation comprise polysaccharide coupled to nucleic acid through an electrostatic interaction between positively charged polysaccharide amine groups and the negatively charged phosphate nucleic acid backbone. The charge ratio of amine groups to phosphate groups is referred to as the N:P ratio. This electrostatic interaction advantageously induces condensation of nucleic acid which protects against DNase digestion, enhances cell attachment and increases intracellular delivery. Thus, the appropriate polysaccharide for inclusion in the nanoparticle formulation will be selected based on the resulting charge ratio with the nucleic acid encoding the target therapeutic protein. Generally, the N:P ratio within the formulation is in the range of about 1 :1 to 10:1, preferably at least about 2:1, and more preferably at least about 3 : 1 or 4: 1.

[0034] The nanoparticles are formed by incubating the selected polysaccharide, e.g. chitosan, and nucleic acid in amounts that result in a charge ratio between the positively charged polysaccharide to negatively charged nucleic acid ranging from 1 :1 to 10:1. The polysaccharide and dissolved nucleic acid are admixed in a suitable solvent and at an appropriate temperature to form nanoparticles.

[0035] The nanoparticle formulation may be combined with one or more pharmaceutically acceptable adjuvants. The expression "pharmaceutically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable. Examples of pharmaceutically acceptable adjuvants include diluents, excipients and the like which do not interfere with the expression of the nucleic acid in the formulation. Reference may be made to "Remington's: The Science and Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, 2005, for guidance on drug formulations generally. The selection of adjuvant depends on the intended mode of administration of the composition. In one embodiment of the invention, the compounds are formulated for oral administration. Compositions for oral administration via tablet, capsule or suspension are prepared using adjuvants including sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; powdered tragancanth; malt; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, chloroquine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tableting agents, anti-oxidants, preservatives, colouring agents and flavouring agents may also be present. Preservatives including anti-microbial agents may also be added to the composition.

[0036] Chitosan nanoparticle formulations may include additives that enhance efficacy, and in particular, enhance the expression of the nucleic acid. For example, inclusion of electrolytes such as sodium sulphate, sodium acetate or tris in an amount sufficient to yield an ionic strength of the formulation in the range of about 25-500 mM, such as 25- 300 mM, and preferably in the range of about 25 -100 mM.

[0037] The present nanoparticles may be modified to enhance efficacy. For example, the nanoparticles may be modified to incorporate ligands that facilitate tissue-specific expression, for example, ligands such as vasointestinal peptide and pituitary adenylate cyclase-activating peptide. [0038] As one of skill in the art will appreciate, to achieve higher expression of a target therapeutic protein, codon optimized genes and/or high expression gene variants and hybrids may be utilized. For example, increased expression of FVIII may be achieved by using B-domain deleted variants. FVIII expression may be further enhanced by utilizing codon optimized sequences.

[0039] The present nanoparticles in accordance with another aspect of the invention may be used in a method to treat a range of diseases or pathological conditions which require protein therapy, including monogenetic disorders or diseases that require reconstitution of a protein, or disorders that may be treated by introduction of a protein. Examples of diseases that may be treated include diseases such as Hemophilia A, Hemophilia B, Hemophilia C, Acquired hemophilia, Hemophilia B leyden, muscular dystrophy, cystic fibrosis, thalassaemia, sickle cell anaemia, Pompe disease, mucopolysaccharidosis, diabetes, alphal -antitrypsin disease, adenosin deaminase deficiency, familial hypercholesterolaemia, Gaucher disease, Fanconi anemia, chronic granulomatous disease, Canavan disease, Tay sachs disease, Fragile X syndrome, Huntington's disease, Alzheimer's, myotonic dystrophy, Marfan syndrome, inflammatory bowel disease, cancer, von Willebrand disease and HIV/AIDS.

[0040] Thus, the present nanoparticle formulation may be useful to treat a disease or condition in a mammal that requires protein therapy in which administration of the formulation to the mammal functions to achieve a therapeutic plasma level of the therapeutic protein it expresses. Alternatively, the nanoparticle formulation may be useful to induce in a mammal an immune tolerance to the therapeutic protein. In this case, the method will generally be utilized in combination with another therapy used to introduce the therapeutic protein to the mammal. As used herein, the term "mammal" is meant to encompass both human and non-human mammals.

[0041] As used herein, the term "treat" or "treatment" is used herein with respect to the present nanoparticle formulation to refer to the use of the formulation to ameliorate or heal a target disease or condition, or to the use of the formulation to facilitate the use of distinct separate therapy to ameliorate or heal a target disease or condition. In facilitating a separate therapy to treat a disease, the nanoparticle formulation may function to alleviate, reduce or prevent undesirable side effects of the separate therapy e.g. an adverse immune response.

[0042] The method comprises administering to a mammal a nanoparticle formulation that expresses a target therapeutic protein, e.g. a deficient protein or a protein that will otherwise treat the disease/disorder, in a therapeutically effective amount, e.g. an amount sufficient to alleviate at least one or more symptoms of the disease. The nanoparticle formulation is administered orally. The formulation, thus, may be in the form of a tablet, capsule, powder, liquid, suspension, or any other form suitable for oral administration.

[0043] Suitable dosages of the formulation will vary with the target therapeutic protein being administered, and the disease/condition being treated. In addition, dosages will depend on whether the nanoparticle formulation is being administered to induce tolerance to the therapeutic protein, or whether it is being administered to achieve therapeutic plasma levels of the therapeutic protein.

[0044] In one embodiment, a method of treating hemophilia A is provided.

The method comprises administration to a mammal of a chitosan nanoparticle encoding FVIII. In another embodiment, a method of treating hemophilia B is provided. The method comprises administration to a mammal of a chitosan nanoparticle encoding FIX. In each case, the nanoparticle formulation may be administered at a dose in the range of about 0.5-500 mg/kg. Frequency of administration may be daily or weekly.

[0045] Thus, the present tolerogenic nanoparticle formulation may be advantageously used to treat target diseases in which protein therapy is required. The formulation provides prophylactic treatment of the disease and is effective to induce immune tolerance, e.g. suppress antibody/inhibitor development to negligible levels for an extended period of time, e.g. for at least 4 weeks, and preferably for at least 8 weeks. It is also effective to result in levels of expression of a therapeutic protein of at least about 5% of the normal endogenous level (i.e. the level of the target protein in a healthy mammal), and preferably results in levels of expression of the protein of at least about 10% of the normal endogenous level. This treatment method may be used on its own, or in combination with conventional/alternative treatments for treatment purposes or to induce immune tolerance. The present nanoparticle formulation, thus, provides a means to utilize proteins for therapeutic purposes that may not otherwise be immunologically tolerated, e.g. exogenous proteins such as proteins modified from their native, endogenous form and proteins from other species.

[0046] Embodiments of the invention are described by reference to the following specific example which is not to be construed as limiting.

Example 1

MATERIALS & METHODS Plasmids

[0047] Plasmid pBK-CMV-cFVIIIcDNABDD, containing the canine FVIII cDNA (cFVIII), was kindly provided by Dr. D. Lillicrap (Queen's University, Kingston, Ontario, Canada). Plasmids pCDNAeGFP (Invitrogen, Burlington, ON, Canada) and pGL3Luc (Promega Corporation, Madison, WI) were prepared using MaxiPrep (Qiagen, Mississauga, Ontario, Canada).

Chitosan/DNA nanoparticle formulation

[0048] Chitosan DNA nanoparticles were formed by complex coacervation method as described previously (Roy et al. Nat Med. 1999; 5: 387-91). Chitosan (CL 213; degree of deacetylation >84%; Novamatrix, Drammen, Norway), 0.02% in 25 mM sodium acetate-acetic acid and plasmid DNA dissolved in 50 mM Na ₂ S0 ₄ was heated to 55°C. The chitosan and DNA solutions were mixed and vortexed for 20 s. A total of 4 distinct formulations of nanoparticles were prepared: 0.02% chitosan with 100 μ§/νηΙ, (10 μg) DNA; 0.02% chitosan with 300 μg/mL (30 μg) DNA; 0.04% chitosan with 100 μg/mL (10 μg) DNA; 0.04% chitosan with 300 μ¾/ηιΙ, (30 g) DNA.

Transmission Electron Microscopy (TEM)

[0049] A Formvar coated Cu ⁺⁺ grid was covered with a 10 μί solution of nanoparticles and left for approximately 1 min. Grids were air-dried, then viewed in a Jem-1200EX Transmission Electron Microscope (JOEL, Tokyo, Japan) operating at 80 KV. Particles were viewed at 6 K, 7.5 K, 10 K, or 15 K times magnification. In vitro transfection of HEK 293 cells

[0050] HEK 293 cells were cultured in 12-well plates with a-MEM, 10% fetal bovine serum, 1% Pen/Strep. Cells were transfected with 5 or 10 μg eGFP, Luciferase or cFVIII plasmid formulated in lipofectamine, chitosan nanoparticles or as naked plasmid and examined 24 hours post-transfection. Expression of eGFP was determined by fluorescent microscope (Leica Microsystems, Germany); Luciferase assay (Promega, USA) was detected on a microplate luminometer (Applied Biosystems, USA) and normalized by protein content determined by Bradford Assay (Pierce, USA); FVIII secretion was assayed by canine FVIII ELISA (Affinity Biologicals, Canada).

Nuclease and proton protection assay

[0051] Chitosan nanoparticles (0.02% chitosan and 100 μg/ml cFVIII plasmid

DNA) were incubated with either 0.25 U or 0.5 U of DNase I (Fermentas, Burlington, Canada) for 15 min at 37°C. DNase was inactivated by incubation at 80°C for 10 min in the presence of EDTA. Plasmid DNA was released from chitosan nanoparticles following incubation for 4 h in the presence of 0.06 U chitosanase enzyme (Sigma, Oakville, Canada). To determine protection against proton digestion, HC1 was used to decrease the nanoparticle solution pH to approximately 1-2 for 1 h and then neutralized with NaOH. Entrapped DNA was released with chitosanase and DNA migration was examined using 1% agarose gel electrophoresis.

Animal Procedures

[0052] Hemophilia A mice (C57BL/6 ^FVII ) were fed chitosan nanoparticles containing either 150 g on three biconsecutive days (50 g on days -4, -2 and 0) or 100 μg cFVIII DNA via gavage using a 20G needle (Popper & Sons, New Hyde Park, NY). Blood samples were collected via orbital plexus in citrate. All experiments were conducted in adherence to the Animal Ethics Guidelines of McMaster University.

FVIII activated partial thromboplastin time (aPTT)

[0053] Plasma clotting time was determined by aPTT in an automated coagulometer (General Diagnostics Coag-A-Mate). Samples and canine reference standards were mixed with equal parts human FVIII deficient plasma, veronal buffer and aPTT reagent (Organon Tecknika Corp, Durham NC, USA) for 3 min at 37°C. Subsequently, 25 mM CaCl ₂ was added and the time to clot formation was measured. Normal canine plasma was used as standard. The aPTT detection limit was 2.4 mU canine FVIII activity. cFVIII antigen and antibody ELISA

[0054] Plasma cFVIII antigen levels were detected using a cFVIII ELISA kit

(Affinity Biologicals, Hamilton, Ontario, Canada), using normal canine plasma as standard. The ELISA detection limit was 3.6 mU canine FVIII antigen.

[0055] A modified ELISA was developed to detect antibodies to canine FVIII expression in mice. Briefly, wells were coated first with goat anti-canine FVIII capture antibody (Affinity Biologicals, Hamilton, Ontario, Canada), followed by normal canine plasma and finally mouse plasma samples. Bound mouse anti-cFVIII antibodies were detected with anti-mouse IgG antibodies (Promega, USA). Positive control plasma obtained from mice immunized with genetically-modified cells- secreting cFVIII (inhibitor titer of 3.4 BU) was used. The antibody ELISA had a detection limit of OD 0.37.

Bethesda assay

[0056] Plasma samples were examined for cFVIII inhibitors using a modified

Bethesda assay (Verbruggen et al. Thromb Haemost. 1 95; 73: 247-51). Briefly, samples were mixed with equal volumes of buffered normal plasma pH 7.4 and incubated for 2 h at 37°C. Bethesda Units were determined using aPTT assay by comparing test samples with human FVIII deficient control plasma activities.

Tail clip assay

[0057] Phenotypic correction in hemophilia A mice following nanoparticle administration was determined by tail clip assay. Hemophilia A mice were treated with nanoparticles (100 μg) and examined on days 1 (n=10), 4 (n=13) or 7 (n=10) post-treatment. Tails were transected at a 2-mm diameter and immediately immersed in normal saline at 37°C. Bleeding was followed visually for 15 minutes. Blood loss was quantified by hemoglobin content that accumulated in the saline solution. Red blood cells were resuspended in lysis buffer (NH4CI 8 g/L; KHCO3 lg/L; EDTA 0.037 g/L) and absorbance was measured at 575 nm. Controls include naive hemophilia A mice (n=6) and normal BALB/c mice (n=5).

Statistics

[0058] Data analysis performed with GraphPad Prism 5 statistical software package. A value of p<0.05(*) was considered significant while p<0.01(**) and p<0.001(***) were considered highly significant.

RESULTS

Formulation and characterization of chitosan nanoparticles

[0059] Chitosan nanoparticles are formed through electrostatic interactions between the positively charged amine groups on chitosan and negatively charged phosphate backbone of DNA. This chitosan-DNA interaction influences the size, morphology and transfection ability of resulting nanoparticles. Thus, four nanoparticle formulations were compared by Transmission Electron Microscopy (TEM). Nanoparticles containing 0.02 % chitosan and 100 μg/mL plasmid DNA appeared to form a combination of rod-like and globular particles of approximately 200 to 400 nm. Chitosan at 0.04 % and 100 μg mL DNA, lead to an increased average particle size of approximately 500 nm in diameter which appears as dense irregular aggregates. In contrast, 0.02% chitosan and 300 [ig/mL DNA resulted in a wider range of particle sizes, from less than 50 nm to nearly 1 μτη in diameter. Finally, 0.04% chitosan and 300 μg/mL DNA lead to a nanoparticle size of several microns. The nanoparticle formulation of 0.02% chitosan and 100 μ§/πιΙ, DNA yielded the smallest particle sizes without producing large aggregates and was subsequently used in this study.

Nanoparticle stability

[0060] The ability of chitosan nanoparticles to protect plasmid DNA from degradation by DNase I and acidic pH (pH 1-2) was determined. Chitosan nanoparticles were subjected to DNase I to mimic enzymatic digestion in the ileum. DNA entrapped in nanoparticles did not migrate in agarose gel electrophoresis. Following chitosanase digestion, intact DNA was evident, although some degradation was observed. In contrast, unformulated plasmid was completely degraded by DNase I .

[0061] Nanoparticles were subsequently subjected to conditions that mimic the low pH environment (pH 1-2) in the stomach. DNA formulated with chitosan remained intact in a low pH environment. In contrast, low pH degraded unformulated DNA.

Transfection efficiency of nanoparticles

[0062] HEK 293 cells were transfected with nanoparticles containing pCDNAeGFP, pGL3Luc or pBK-CMV-cFVIIIc DNA. At 24 h post-transfection, cells transfected with unformulated eGFP plasmid (^g) yielded undetectable eGFP expression. In contrast, >90% of the cells transfected with Lipofectamine expressed eGFP. Nanoparticle transfection also produced eGFP expression in >90% of cells, although with significantly lower intensity, suggesting a reduced eGFP expression. A lower nanoparticle dose ^g total DNA) also transfected cells (data not shown).

[0063] The transfection efficiency of nanoparticles was further investigated with luciferase, a quantifiable reporter gene. Transfection with 10 μg of unformulated luciferase plasmid led to undetectable transgene expression (Figure 1A). Transfection with nanoparticles containing 5 μg of plasmid DNA resulted in an average of 7.3x10 ⁸ RLU/mg, while transfection with 10 μg resulted in an average of 10.6x10 ⁸ RLU/mg. Moreover, Lipofectamine drastically enhanced transfection efficiency (Figure 1 A).

[0064] Finally, transfection with nanoparticles containing 10 μg or or 5 g of canine FVIII plasmid resulted in cFVIII secretion of 1 10 mU/mL/24 h and 79 mU/mL/24 h, respectively (Figure IB).

Oral administration of cFVIII DNA in hemophilic mice

[0065] Encouraged by the in vitro findings, the biodistribution of chitosan nanoparticles and FVIII expression were elucidated in mice. A group of hemophilia A mice were fed chitosan nanoparticles containing a total of 150 μg of cFVIII plasmid DNA on days -4, -2 and 0. A control group received chitosan solution with no DNA following the same regimen. Selected mice (n=2) were sacrificed two days following the final administration, at which time samples of intestine, liver and kidney were harvested and DNA from tissues extracted. Using primers specific to cFVIII that span the B-domain junction, a DNA fragment of the expected 152bp from duodenum, jejunum and ileum of treated mice was successfully amplified. Furthermore, faint amplification bands were obtained from the liver but not the kidney of treated mice. Conversely, the 152 bp cFVIII fragment was not amplified from DNA extracted from tissues of untreated FVIII deficient mice. Positive amplification was observed in control DNA from G8 murine myoblasts transfected with the cFVIII plasmid while no amplification was detected from untransfected cells.

[0066] The change in plasma FVIII activity in treated mice following oral administration was also determined. There was a statistically significant decrease in aPTT (p<0.01) on day one over pre-treatment values (Figure 2), with an average cFVIII activity of 130 mU in treated mice (n=18). However, FVIII activity was temporary and regressed back to baseline by day 4 post-treatment, suggesting a transient FVIII expression. In contrast, control mice (n=7) receiving chitosan solution only without plasmid DNA, did not experience a statistically significant increase in FVIII activity (p=0.69).

[0067] Further, oral administration of chitosan nanoparticles protected hemophilic mice from bleeding. A group of FVIII-deficient mice (n=5) was administered a single dose of nanoparticles containing a total of 150 μg cFVIII plasmid DNA on three biconsecutive days. A second group of hemophilic mice (n=4) received an equal amount of unformulated cFVIII plasmid DNA. On day 2 after treatment, a tail clip assay was performed to assess clot formation. Phenotypic correction was assessed as cessation of bleeding within 15 minutes of trauma. Interestingly, 3 of 5 treated mice stopped bleeding and stable clots were observed. In contrast, all the mice receiving unformulated cFVIII plasmid bled beyond 15 min. FVIII kinetics in hemophilic mice following FVIII DNA ingestion

[0068] The kinetics of circulating FVIII was evaluated. The change in plasma

FVIII activity following oral DNA administration was monitored over a four-week period. Hemophilia A mice (n=30) were fed a single dose of chitosan nanoparticles containing 100 μg cFVIII DNA. Blood samples were collected two weeks pre- treatment, and for groups of mice (n=6) on days 0.5, 1 , 2, 3, 4, 7, 14, 21 , and 28 post- treatment. Untreated hemophilia A mice and wild type mice were used as controls. Circulating FVIII on day one showed a statistically significant increase (p<0.001) over pre-treatment values, which reflects an average FVIII activity of 77 mU in treated mice (Figure 3). However, FVIII activity was temporary; it regressed back to baseline by day 2 post-treatment and remained undetectable for the entire experiment (4 weeks).

[0069] The oral administration of a single dose of chitosan nanoparticles protected hemophilic mice against trauma. A group of FVIII-deficient mice (n=33) was administered nanoparticles containing 100 μg cFVIII plasmid DNA. A tail clip assay was performed on days 1, 4 or 7 post-treatment to determine tail bleeding times (Figure 4A). Wild type mice (n=5) stopped bleeding within 15 minutes while naive Hemophilia A mice (n=6) bled beyond 15 minutes. In contrast, on day 1 post- treatment, 50% of mice bled for less than 15 minutes. The transient nature of phenotypic correction was demonstrated by excessive bleeding of treated mice on days 4 and 7 post-treatment. This trend was further confirmed by measuring blood loss as reflected by hemoglobin concentration of blood collected following tail transaction (Figure 4B). The blood loss in wild type mice was minimal while excessive in naive hemophilia A mice. In mice treated with nanoparticles blood loss was reduced one day after feeding but not on days 4 or 7 post treatment (Figure 4B).

Repeated oral administration of FVIII DNA in hemophilic mice

[0070] The feasibility of re-administering nanoparticles was then determined.

A group of hemophilia A mice (n=7) were fed nanoparticles containing 100 μg of cFVIII DNA weekly for a total of four weeks. Untreated hemophilic mice were used as controls. The plasma FVIII activity in treated mice was on average 100 mU on day 1 (Figure 5) and remained elevated at statistically significantly levels for all four weeks of treatment. A week after the last feed, FVIII activity decreased markedly, although some residual activity remained .

[0071] Complementing these results, canine FVIII antigen levels increased to statistically significant levels on day 1 (p<0.001 ) to >100 mU (Figure 6), and remained detectable throughout four weeks (Figure 6). Canine FVIII antigen was undetectable on week 5, confirming the transient nature of cFVIII expression.

[0072] A major challenge for hemophilia management is the presence of inhibitors, which were not found in any of the treated mice throughout the experiment, as determined by a Bethesda Assay (data not shown). Additionally, a specific ELISA protocol was developed for the detection of anti-cFVIII antibodies. Interestingly, antibodies to cFVIII were undetectable in any of the treated mice during the experiment (Figure 7).

Chitosan/FVIII DNA nanoparticles modulates the immune response

[0073] Oral administration of chitosan nanoparticles containing DNA coding for FVIII modulates the immune response to FVIII that afflicts a certain proportion of hemophiliacs as a result of the therapeutic regimen of regular FVIII infusions. In this regard, the induction of immune tolerance was examined following orally administered chitosan/human FVIII-DNA nanoparticles in a hemophilia A mouse model of the human disease. A standard immunization protocol based on the regular injections of human FVIII for 4 weeks (mimicking the human therapeutic regimen) was utilized, after which mice develop high anti-FVIII antibody titer. Four groups of hemophilia mice were treated as shown in Fig. 8A-D: Two groups (n=14) were fed chitosan nanoparticles containing 100 μg of DNA coding for human FVIII, once per week, for 4 weeks. After 4 weeks, one of the groups (n=6) did not receive any further treatments (Figure 8A), while the other group (n=8) received the standard immunization regimen for a further 4 weeks (8B). The other two groups (n=l l) of mice received the standard immunization regimen for 4 weeks, after which one of the groups (n=6) received no further treatment (Figure 8C), while the other group (n=5) was fed chitosan nanoparticles containing 100 μg of DNA coding for human FVIII, once per week, for 4 weeks (Figure 8D). Mice were bled at regular intervals, and anti- FVIII antibodies measured by ELISA (Figure 9). [0074] The results show that mice that were fed FVIII DNA nanoparticles did not elicit an antibody response when immunized with human FVIII, indicating the immunomodulatory effect of chitosan FVIII-DNA nanoparticles.

Discussion

[0075] The stability and transfection potential of nanoparticles was investigated in this study. Formulated nanoparticles protected DNA from degradation by DNase and from low pH environments. Similarly, nanoparticles were effective at transfecting HEK 293 cells in vitro, as shown using several marker genes.

[0076] Oral administration of chitosan nanoparticles containing cFVIII plasmid provided limited biodistribution beyond the intestinal tissue. Nevertheless, this led to a partial and transient increase in FVIII plasma activity from treated hemophilia mice, with an average of >100 mU cFVIII activity. This level of activity would convert severe or moderate hemophilia to a mild phenotype. The protection against trauma of mice following the tail clip assay also indicates correction of the hemophilic bleeding phenotype.

[0077] Repeated administration of nanoparticles resulted in the restoration of transient FVIII activity. The presence of FVIII was confirmed with the associated detection of cFVIII antigen following re-administration. However, this transgene expression was absent a week following final administration. These results indicate the potential of re-administration of nanoparticles to provide sustainable FVIII levels for the treatment of hemophilia. Interestingly, while therapeutic levels were achieved in this study, inhibitors were undetectable for at least 5 weeks. The sustainable activity of plasma FVIII further indicates the absence of neutralizing antibodies. Moreover, non-neutralizing antibodies to the relatively large FVIII transgene were also undetectable.

[0078] Determination of immune responses to FVIII following oral nanoparticle administration did not elicit an antibody response. Further, subsequent immunization with recombinant human FVIII protein did not produce antibodies. This is in contrast to the high antibody titers produced in naive mice following immunization. These results indicate that the present orally administered nanoparticles induce immune tolerance in hemophilic mice. This immunomodulatory effect is important in the treatment of hemophilia, since treatment of neutralizing antibodies to FVIII is the greatest clinical and economic challenge in the management of hemophilia.

[0079] Together these data indicate that administration of chitosan nanoparticles is an effective treatment for hemophilia A without inducing immune response complications. Furthermore, the present invention can be used to immunomodulate hemophiliacs.

Example 2 - Chitosan/DNA nanoparticle formulation induces prophylactic tolerance

[0080] A human FVIII (hFVIII) plasmid was selected to replace the canine

FVIII used in Example 1 to enable challenge with injected recombinant human FVIII (rhFVIII). The transgene expression of a number of human FVIII plasmids were examined in vitro (Figure 10). HEK 293 cells were transfected with lipofectamine containing 0.5 pmol of pcDNA/BDDFVIII, MFGBDDhFVIII, pcDNA-IVS-hFVIII, pSP64-hFVIII, pBIISK-BDDhFVIII or pSBT/mCAGFVIII. Media containing no plasmid or transfection agent was used as control. Expression of hFVIII was highest for pcDNA-IVS-hFVIII, which contains the human IVS2 intronic sequence and is used in the remainder of this study.

[0081] An optimized formulation of chitosan nanoparticles was used to compensate for the lower activity of human FVIII relative to canine FVIII. The formation of chitosan nanoparticles is dependent on the electrostatic interactions between the positively charge amine groups on chitosan and the negatively charged phosphate backbone of DNA. The formulation of Examplel consisted of chitosan chloride complex with DNA at an N:P charge ratio of 4:1 incorporating 50 mM sodium sulphate and 25 mM sodium acetate-acetic acid. The optimized formulation used in the following studies consisted of chitosan chloride salt 213 (CL213) at an N:P ratio of 3:1 with 50 mM sodium sulphate only. This optimized formulation was shown to increase transfection 4-fold (Fig.10).

Immune response modulation following oral chitosan/hFVIII administration

[0082] Immune response modulation following orally administered chitosan- hFVIII nanoparticles was tested. Hemophilia A mice (n=14) were fed nanoparticles containing 100 g hFVIII DNA once weekly for a total of four weeks (Figure 11A- B). A subgroup of these mice (n=8) were subsequently immunized with 0.75 U rhFVIII once a week for a total of four weeks, which represent weeks 5, 6, 7 and 8 post-treatment (Figure 1 IB). Blood plasma samples were examined for both neutralizing (inhibitors) and non-neutralizing antibodies by anti-hFVIII EL1SA. hFVIII antibodies were undetectable following oral administration of chitosan nanoparticles (Figure 11 A, 1 IE). Furthermore, subsequent immunization with rhFVIII did not produce hFVIII antibodies that were statistically significant from controls (Figure 1 IB, HE). This is in contrast to immunization of na'ive hemophilia A mice, which produced high antibody titres (Figure 1 1C,11E). Taken together, these results suggest that pre-treatment with orally administered nanoparticles provides protection against antibody formation following rhFVIII challenge.

[0083] The modulation of pre-existing antibodies following oral nanoparticle administration was also examined. Hemophilia A mice (n=l 1) were immunized with weekly i.p. injections with 0.75 U rhFVIII for a total of four weeks (Figure 11C, 1 ID). A subgroup of mice (n=5) were subsequently fed chitosan nanoparticles containing 100 μg hFVIII DNA on weeks 5, 6, 7 and 8 (Figure 11D). Immunized mice produced high antibody titres that persist for up to 8 weeks (Figure HE). Treatment with nanoparticles did not significantly reduce these antibody titres; although, the average antibody titre was lower (Figure 1 ID, 1 IE).

Oral nanoparticle administration induce long-term tolerance to hFVIII

[0084] The prophylactic tolerance induced in na'ive mice was further examined over a 16-week period. A group of hemophilia A mice (n=5) were fed chitosan nanoparticles containing 100μg hFVIII DNA weekly for a total of eight weeks. The repeated oral administration of chitosan nanoparticles did not produce detectable antibody or inhibitor levels as determined by anti-hFVIII ELISA (Figure 12A, 12G) and Bethesda assay (Figure 12D, 12H), respectively. A second group of mice (n=7) were also fed chitosan nanoparticles for a total of eight weeks and subsequently immunized with 0.75 U rhFVIII on weeks 9, 10, 11 and 12. The majority of immunized treated mice did not develop detectable antibodies over the 16- week study period (Figure 12B, 12G), which correlated with inhibitor titres that were not significant from controls (Figure 12E, 12H). This indicates that oral nanoparticle administration induces tolerance to rhFVIII challenge. Finally, a third group of hemophilia A mice (n=8) were fed weekly for a total of twelve weeks concurrently with rhFVIII immunizations on weeks 9, 10, 11 and 12. Low level antibody titres were detected on week 12 which reduced to background by week 16 (Figure 12C, 12G). Nevertheless, inhibitor titres were not significantly different from controls (Figure 12F, 12H). These results indicate that pre-treatment with nanoparticles can induce oral tolerance to suppress antibody titres and prevent inhibitor development to FVIII challenge.

Immune response modulation in the presence of pre-existing FVIII inhibitors

[0085] Next it was determined whether or not repeated administration of nanoparticles can reduce antibody (neutralizing and non-neutralizing antibodies) and inhibitor (neutralizing antibodies) titres in hemophilia A mice with pre-existing antibodies. A group of hemophilia A mice (n=7) were immunized i.p. with 0.75 U rhFVIII over a four week period followed by oral administration of nanoparticles on weeks 5, 6, 7, 8, 9, 10, 11 and 12. Antibody (Figure 13B, 13G) and inhibitor (Figure 13E, 13H) titres peaked on week 4 and declined steadily on weeks 8, 12 and 16. Indeed there is a significant decrease in the antibody response by weeks 12 and 16. This indicates that repeated administration of nanoparticles contribute to the reduction of antibody and inhibitor titres. However, this reduction may be explained by the natural decline of antibody and inhibitor titres shown in a control group of hemophilia A mice (n=5) immunized for four weeks (Figure 13A, 13G and Figure 13D, 13H). A comparison of these two groups did not reveal any significant differences over the 16 week study period (Figure 13G, 13H). This data indicate that oral nanoparticle administration may not suppress antibody and inhibitor titres in hemophilia A mice with pre-existing antibodies.

[0086] It was also determined whether or not repeated nanoparticle administration could limit the increase in antibody and inhibitor titres in hemophilia A mice with pre-existing antibodies while receiving continuous immunizations. A group of hemophilia A mice (n=8) were immunized weekly with 0.75 U rhFVIII for sixteen weeks and were fed nanoparticle containing 100 μg hFVIII from weeks 5 to 16. The antibody titres increased on weeks 8, 12 and 16 (Figure 13C, 13G) while inhibitor levels reached a plateau by week 8 (Figure 13F, 13H). This data suggest that oral administrations of nanoparticles are unable to suppress the antibody response to repeated immunizations with the presence of pre-existing antibodies. The inhibitor titre was not suppressed on week 8, however the plateau reached on weeks 8, 12 and 16 may be attributed to repeated nanoparticle administration.

Adoptive transfer of splenocytes from tolerized mice into naive recipient mice

[0087] To determine if oral administration of chitosan nanoparticles result in the production of regulatory T cells that mediate tolerance, splenocytes from tolerized mice were tested for their ability to transfer tolerance to naive recipient mice. Hemophilia A mice donor mice (n=5) were fed nanoparticles containing 100 g hFVIII DNA weekly for four weeks (Figure 14B). Splenocytes were isolated and injected intravenously into naive hemophilia A mice (n=5). Recipient mice were challenged i.p. with 0.75 U rhFVIII on days 1, 7, 14 and 21 following splenocyte transfer (Figure 14B). Splenocyte transfer protected naive recipient mice from high titre antibody development (Figure 14D, 14E) compared with controls (Figure 14A, 14C, 14E). The apparent one-week delay in the development of antibodies in tolerized mice may be attributed to the passive immunity provided by splenocyte transfer against immunization on day 1 but not on days 7, 14 and 21. Taken together, the suppression of hFVIII antibody titres suggests that T regulatory cells mediate suppression and may be involved the development of tolerance; however, alternative mechanisms leading to tolerance known to artisans are also contemplated.

Discussion

[0088] Example 1 shows that sustainable canine FVIII expression and phenotypic correction in hemophilic mice is achieved following oral gene therapy. Of particular interest, both antibody and inhibitor development was not detected despite plasma FVIII levels of > 100 mU/mL. Consistent with these findings, human FVIII- specific antibodies were not produced to nanoparticle treatment containing human FVIII DNA.

[0089] It is shown that prior oral administration of chitosan nanoparticles containing human FVIII DNA in hemophilia A mice suppressed systemic antibody formation following challenge with recombinant FVIII protein compared with immunized controls. Inhibitor formation is believed to be T helper dependant. FVIII expression in the gut-associated lymphoid tissue may recruit CD4 ⁺ Foxp3 ⁺ regulatory T cells to facilitate peripheral tolerance mechanisms that may involve immune suppressive cytokines IL-10 and TGF-β. This systemic immune tolerance could be transferred to naive mice. The transient FVIII-specific tolerance observed during transfer may be attributed to the immunization protocol used, which consisted of four weekly i.p. injections of rhFVIII without adjuvant and low engraftment of regulatory T cells.

[0090] Prophylactic mucosal tolerance to human FVIII was long-lasting.

Tolerized mice did not produce systemic FVIII-specific antibody response for at least 8 weeks following immunization protocols. Low level antibody response in a subset of tolerized mice may be attributed to batch variation or protein sequence differences between mucosal presented FVIII transgene and recombinant FVIII used for immunization.

[0091] In contrast, the oral administration of nanoparticles had limited immunomodulatoy effect against pre-existing inhibitors. The continuous decline in antibody titres over 16 weeks with repeated nanoparticle administration emulated the trend observed in the control group following discontinued immunization. This suggests that nanoparticle treatment did not contribute to the regression of antibody titres in mice with pre-existing antibodies. Although, mucosal tolerance is believed to suppress antigen-presenting cell activation and may not affect clearance of long-lived plasma cells and circulating antibodies.

[0092] Furthermore, mucosal expression of hFVIII did not illicit a prime- boost response characteristic of DNA vaccines. In this study, prior mucosal gene therapy suppressed development of antibodies to immunization while FVIII gene expression did not boost antibody titres in mice with pre-existing antibodies.

[0093] Clinically, it is unlikely to suspend protein replacement therapy during tolerance induction protocols. To better recapitulate clinical practice, nanoparticles were administered contemporaneously with weekly rhFVIII immunization. However, concurrent nanoparticle treatment did not suppress continuous immune response activation for 12 weeks, although inhibitor titres appeared to plateau. [0094] Inhibitor development is a significant challenge to clinical management of hemophilia. Oral gene therapy may provide long-term prophylactic tolerance in paediatric patients without need of immunosuppressive agents or risk of insertional mutagenesis.

Example 3 - Chitosan nanoparticle-mediated FIX gene delivery to treat hemophilia B FIX transgene expression in hemophilia B mice

[0095] The transgene expression kinetics of FIX delivery in hemophilia B mice was examined. Hemophilia B mice (n=16) were fed chitosan nanoparticles containing 100 μg hFIX. Blood plasma samples were collected on days 1 , 2, 3, 4 and 14 post-treatment. Untreated hemophilic mice were used as controls. The hFIX activity reached peak expression on day 1 post-treatment and regressed back to baseline by day 3 post-treatment (Figure 15). The detection of hFIX activity on day 2 post treatment may be attributed to the longer half-life circulating FIX than hFVIII. Nevertheless, the in vivo activity observed was low.

Discussion

[0096] Oral administration of chitosan nanoparticles containing human FIX

DNA provided detectable FIX transgene expression. This transient expression provided FIX plasma activity in treated mice with average values of >14mU and >9.5 mU FIX activity at 24 and 48 hours post treatment, respectively. This level of FIX activity is sufficient to convert a severe hemophilia disorder to a moderate phenotype. This level of expression is significant considering the 25-50 fold higher physiological levels of FIX compared with FVIII ^ /mL vs. 100-200ng/mL).

[0097] Thus, therapeutically relevant expression of FIX can be achieved by oral gene therapy using chitosan nanoparticles. These findings show that oral gene therapy is a feasible treatment for hemophilia B.

Example 4 - Chitosan DNA nanoparticle formulations

[0098] Chitosan nanoparticle formulations with DNA were investigated to improve transfection efficiency. The detection of a luciferase pseudo-gene was used to systematically optimize nanoparticle formulations. Transfection efficiency was enhanced by modulating the charge ratio and electrolyte content of nanoparticle formulations.

Effect of electrolyte concentration and N:P charge ratio

[0099] The influence of charge ratio and salt content on nanoparticle transfection efficiency was examined. Chitosan nanoparticles containing the luciferase plasmid were formulated at N:P ratios of 1 :1, 2:1 , 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 and 10:1 using both 25 mM sodium acetate-acetic acid and 50 mM sodium sulphate, 25 mM sodium acetate-acetic acid only, 50 mM sodium sulphate only or no salts. The results indicate that maximum transfection occurred at a charge ratio of 3: 1 (Figure 16). Furthermore, formulations containing only 50 mM sodium sulphate provided enhanced transfection while formulations including 25 mM sodium acetate- acetic acid suppressed transgene expression.

Effect of chitosan type

[00100] The type of chitosan used in the formulation of nanoparticles may influence transfection efficiency. All previous experiments utilized a chitosan chloride salt with a molecular weight of 213 kDa (CL 213). The influence of chitosan type on transfection efficiency was examined with chitosan chloride salt 213 (CL 213), chitosan chloride salt 1 13 (CL1 13), chitosan glutamate salt 213 (G213) and chitosan glutamate 113 (Gl 13). Nanoparticles were formulated using these four different chitosan salts in 50 mM sodium sulphate at charge ratios of 1 :1 , 3:1 , 5:1 7:1 and 10:1. The optimal charge ratio was determined to be 3:1 for all chitosan salt forms (Figure 16). Furthermore, the chitosan chloride (CL 213) provided the highest transfection, while G 213 provided the lowest transgene expression.

Discussion

[00101] Chitosan nanoparticles formulated with chitosan chloride (CL 213) in 25 mM acetate-acetic acid and 50 mM sodium sulphate at an N:P charge ratio of 4:1 were shown in Example 1 to be useful for the gene delivery of FVIII. The purpose of this study was to optimize the chitosan nanoparticle formulation to increase transfection efficiency.

[00102] Chitosan salts used in this study are water soluble and have improved transfection efficiency compared to chitosan base. Nanoparticles formulated with chitosan chloride (CL 213) with 50 mM sodium sulphate at a charge ratio of 3:1 increased transfection by 40% compared to the chitosan chloride (CL 213) in 25 mM acetate-acetic acid and 50 mM sodium sulphate at an N:P charge ratio of 4:1. Peak transgene expression was observed with nanoparticle formulated at a 3:1 charge ratio regardless of electrolyte content. Interestingly, electrolytes modulated nanoparticle properties as sodium sulphate increased transfection while acetic acid suppressed transgene expression. Thus, these electrolytes may modulate chitosan-DNA electrostatic interaction along with their packaging and release characteristics.