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

Abstract

Provided herein is chitosan dually derivatized with arginine and gluconic acid; and methods of making and using the same, e.g., for gene delivery in vivo.

1 - 14 . (canceled) 
     
     
         15 . A method for modulating an immune response in a patient in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic nucleic acid to a target tissue in the patient, wherein said administering comprises contacting said target tissue with a dually derivatized (DD) chitosan nucleic acid polyplex, said DD chitosan nucleic acid polyplex comprising a chitosan-derivative nanoparticle and said therapeutic nucleic acid, wherein the chitosan-derivative nanoparticle comprises chitosan functionalized with arginine (Arg) and gluconic acid. 
     
     
         16 . The method according to  claim 15 , wherein said nanoparticle comprises arginine at an initial concentration of about 10% to about 55%, or about 8% to about 30%. 
     
     
         17 . The method according to  claim 16 , wherein said nanoparticle comprises gluconic acid at a final functionalization of about 3% to about 10%. 
     
     
         18 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex has a combined degree of functionalization with said arginine and said gluconic acid of 1-60%, or 1-30%. 
     
     
         19 . The method according to  claim 15 , wherein the amine to phosphate ratio of said DD-chitosan nucleic acid polyplex is between 2 to 100, between 2 to 50, between 2 to 30, or between 2 to 15. 
     
     
         20 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex has a molar ratio of said arginine to said gluconic acid of between 100:1 and 1:100, between 50:1 and 1:50, between 10:1 and 1:10, between 5:1 and 1:5, or between 2:1 and 1:2. 
     
     
         21 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex is isotonic. 
     
     
         22 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex comprises chitosan molecules having an average molecular weight of less than 110 kDa before functionalization with said gluconic acid and said arginine, less than 50 kDa before functionalization with said gluconic acid and said arginine, less than 30 kDa before functionalization with said gluconic acid and said arginine, or less than 10 kDa before functionalization with said gluconic acid and said arginine. 
     
     
         23 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex has an average polydispersity index (PDI) of less than 0.5. 
     
     
         24 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex increases in average diameter by less than 100% at room temperature for 6 hours. 
     
     
         25 . The method according to  claim 15 , wherein said DD-chitosan nucleic acid polyplex has a therapeutic nucleic acid concentration between about 0.5 mg/ml and about 1.5 mg/ml, and is substantially free of precipitated polyplex. 
     
     
         26 . The method of  claim 15 , wherein the therapeutic nucleic acid encodes a cytokine. 
     
     
         27 . The method of  claim 26 , wherein the cytokine stimulates the proliferation of immune cells that can inhibit a hyperproliferative disorder. 
     
     
         28 . The method of  claim 15 , wherein the therapeutic nucleic acid encodes a therapeutic antibody or antigen-binding fragment thereof. 
     
     
         29 . A dually derivatized-chitosan compound of Formula I wherein chitosan is coupled to arginine and gluconic acid: 
       
         
           
           
               
               
           
         
         wherein n is an integer of 1 to 2000, 
         α is the functionalization degree of arginine, 
         β is the functionalization degree of gluconic acid; and 
         each R 1  is independently selected from hydrogen, acetyl, Formula (II), and Formula (III). 
       
       
         
           
           
               
               
           
         
       
     
     
         30 . A nanoparticle comprising the compound according to  claim 29 . 
     
     
         31 . A composition comprising the nanoparticle according to  claim 30  wherein said chitosan is complexed with a nucleic acid to form a dually derivatised (DD) chitosan nucleic acid polyplex. 
     
     
         32 . A method for modulating an immune response in a patient in need thereof, the method comprising administering to said patient a composition according to  claim 31 , wherein said nucleic acid encodes an immunomodulatory cytokine. 
     
     
         33 . A method for initiating or increasing an immune response to a molecule of interest in a patient in need thereof, the method comprising administering to said patient a composition according to  claim 31 , wherein said nucleic acid encodes an epitope of the molecule of interest. 
     
     
         34 . A method for treating a hyperproliferative disorder, for example a neoplasm, in a patient in need thereof, the method comprising administering to said patient a composition according to  claim 31 , wherein said nucleic acid encodes an immunomodulatory cytokine, or a therapeutic antibody or antigen-binding fragment thereof. 
     
     
         35 . The method according to  claim 34  wherein the hyperproliferative disorder is a neoplasm selected from a neoplasm located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.