NOVEL RNAi MOLECULE DELIVERY PLATFORM BASED ON SINGLE-siRNA AND shRNA NANOCAPSULES

Abstract

Novel siRNA and shRNA nanocapsules and delivery methods are disclosed herein. These siRNA and shRNA nanocapsules and delivery methods are highly robust and effective. This invention provides a platform for RNAi delivery with low toxicity and long intracellular half-life for practical therapeutic applications.

1 . A method of treating a subject having a disease or disorder or at risk of developing a disease or disorder, wherein the disease or disorder is characterized by over expression of a gene, the method comprising administering to a subject in need thereof, a polymer nanocapsule comprising a polymer shell and a nucleic acid selected from the group consisting of: an siRNA, an shRNA expression DNA cassette, and a dsRNA, 
       wherein the polymer shell comprises
 a. one or more positively charged monomers selected from the group consisting of: N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)acrylamide, N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)methacrylamide, N-(3-((4-aminobutyl)amino)propyl)acrylamide, N-(3-((4-aminobutyl)amino)propyl)methacrylamide, N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide, N-(2-((2-aminoethyl)(methyl)amino)ethyl) methacrylamide, N-(piperazin-1-ylmethyl)acrylamide, N-(piperazin-1-ylmethyl)methacrylamide, N-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide, N-(2-(bis(2-aminoethyl)amino)ethyl)methacryl amide, (3-acrylamidopropyl) trimethylammonium hydrochloride, and 2-aminoethyl methacrylate; 
 b. one or more crosslinkers selected from the group consisting of: 1,3-glycerol dimethacrylate, glycerol 1,3-diglycerolate diacrylate, N,N′-bis(acryloyl)cystamine, bis[2-(methacryloyloxy)ethyl]phosphate, N,N′-Methylenebisacrylamide, bisacryloylated polypeptide, and 
 c. one or more neutral monomers selected from the group consisting of: N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)acrylamide, acrylamide, N-(hydroxymethyl)acrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 
 
       and wherein the polymer shell encapsulates the nucleic acid. 
     
     
         2 . The method of  claim 1  wherein the siRNA, shRNA expression DNA cassette, or dsRNA knocks down or decreases expression of the gene. 
     
     
         3 . The method of  claim 1 , wherein the one or more crosslinkers comprise:
 a. a degradable crosslinker selected from the group consisting of: 1,3-glycerol dimethacrylate, glycerol 1,3-diglycerolate diacrylate, N,N′-bis(acryloyl)cystamine, bis[2-(methacryloyloxy)ethyl]phosphate and bisacryloylated polypeptide; and   b. a non-degradable crosslinker, wherein the non-degradable cross linker is N,N′-methylenebisacrylamide,   
       wherein the ratio of degradable crosslinker to non-degradable crosslinker is selected from the ratios comprising 1:0, 3:2, 2:3, or 1:4. 
     
     
         4 . The method of  claim 1 , wherein all of the crosslinkers are selected from the group consisting of: 1,3-glycerol dimethacrylate, glycerol 1,3-diglycerolate diacrylate, N,N′-bis(acryloyl)cystamine, bis[2-(methacryloyloxy)ethyl]phosphate and bisacryloylated polypeptide. 
     
     
         5 . The method of  claim 1 , wherein the one or more positively charged monomers is selected from the group comprising N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)methacrylamide, N-(3-((4-aminobutyl)amino)propyl)acrylamide, N-(3-((4-aminobutyl)amino)propyl)methacrylamide, N-(2-((2-aminoethyl)(methyl)amino)ethyl)acrylamide, N-(2-((2-aminoethyl)(methyl)amino)ethyl) methacrylamide, N-(piperazin-1-ylmethyl)acrylamide, N-(piperazin-1-ylmethyl)methacrylamide, N-(2-(bis(2-aminoethyl)amino)ethyl)acrylamide, and N-(2-(bis(2-aminoethyl)amino)ethyl)methacrylamide. 
     
     
         6 . The method of  claim 1 , wherein the polymer shell comprises N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)acrylamide; 1,3-glycerol dimethacrylate; and N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)acrylamide. 
     
     
         7 . The method of  claim 1 , wherein the polymer shell comprises N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)acrylamide; glycerol 1,3-diglycerolate diacrylate; and acrylamide. 
     
     
         8 . The method of  claim 1 , wherein the polymer shell comprises N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)acrylamide; 1,3-glycerol dimethacrylate; and N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)acrylamide. 
     
     
         9 . The method of  claim 1 , wherein the polymer shell comprises N-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)acrylamide; glycerol 1,3-diglycerolate diacrylate; and acrylamide. 
     
     
         10 . The method of  claim 1 , wherein the polymer shell has a diameter of approximately 20 nm to 250 nm. 
     
     
         11 . The method of  claim 1 , wherein the polymer nanocapsule is conjugated to a targeting agent. 
     
     
         12 . The method of  claim 1 , wherein the disease or disorder is a cellular proliferative and/or differentiative disorder, an immune or immunodeficiency disorder, a viral infection, a neurological or neurodegenerative disorder. 
     
     
         13 . The method of  claim 12 , wherein the disease or disorder is cancer, pachyonychia congenital, age-related macular degeneration, choroidal neovascularization, metastatic melanoma, metastatic melanoma without CNS metastases, chronic myeloid leukemia, solid tumors, advanced solid tumors, optic atrophy, non-arteric anterior ischemic optic neuropathy, pancreatic cancer, pancreatic ductal adenocarcinoma, diavetic macular edema, hypercholesterolemia, colorectal cancer with hepatic metastases, pancreatic cancer with hepatic metastases, gastric cancer with hepatic metastases, breast cancer with hepatic metastases ovarian cancer with hepatic metastases, preeclampsia, neuroblastoma, ocular hypertension, open angle glaucoma, glaucoma, ocular pain, dry eye syndrome, kidney injury, acute renal failure, delayed graft function, complications of kidney transplant, TBX3 overexpression, and diabetic retinopathy. 
     
     
         14 . The method of  claim 1 , wherein the disease or disorder is cancer. 
     
     
         15 . The method of  claim 1 , wherein the disease or disorder is a viral infection. 
     
     
         16 . The method of  claim 15  wherein the viral infection is a retroviral viral infection. 
     
     
         17 . The method of  claim 1 , wherein the retroviral infection is HIV or AIDS infection.