Delivery of mrna for the augmentation of proteins and enzymes in human genetic diseases

Abstract

Disclosed herein are compositions and methods of modulating the expression of gene or the production of a protein by transfecting target cells with nucleic acids. The compositions disclosed herein demonstrate a high transfection efficacy and are capable of ameliorating diseases associated with protein or enzyme deficiencies.

1 . A composition for modulating the expression of a protein in a target cell, wherein said composition comprises at least one RNA molecule and a transfer vehicle. 
     
     
         2 . (canceled) 
     
     
         3 . The composition of  claim 1 , wherein the RNA molecule comprises at least one modification which confers stability to the RNA molecule. 
     
     
         4 . The composition of  claim 3 , wherein the RNA molecule comprises a modification of the 5′ untranslated region of said RNA molecule. 
     
     
         5 - 8 . (canceled) 
     
     
         9 . The composition of  claim 3 , wherein the RNA molecule comprises a modification of the 3′ untranslated of said RNA molecule. 
     
     
         10 - 12 . (canceled) 
     
     
         13 . The composition of  claim 1 , wherein the transfer vehicle is a liposome. 
     
     
         14 . The composition of  claim 1 , wherein the transfer vehicle is a lipid nanoparticle. 
     
     
         15 . The composition of  claim 1 , further comprising an agent for facilitating transfer of the RNA molecule to an intracellular compartment of the target cell. 
     
     
         16 . The composition of  claim 15 , wherein the agent is selected from the group consisting of a protein, a peptide, an aptamer, and an oligonucleotide. 
     
     
         17 . The composition of  claim 1 , further comprising a ligand capable of enhancing affinity of the composition for the target cell. 
     
     
         18 . (canceled) 
     
     
         19 . The composition of  claim 17 , wherein said ligand is selected from the group consisting of apolipoprotein-B and apolipoprotein-E. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The composition of  claim 1 , wherein said transfer vehicle comprises one or more cationic lipids. 
     
     
         24 . The composition of  claim 1 , wherein said transfer vehicle comprises one or more non-cationic lipids. 
     
     
         25 . The composition of  claim 1 , wherein said transfer vehicle comprises one or more PEG-modified lipids. 
     
     
         26 . The composition of  claim 1 , wherein said transfer vehicle comprises CHOL, DOPE, DLinDMA and DMG-PEG-2000. 
     
     
         27 . The composition of  claim 1 , wherein said transfer vehicle comprises ICE, DOPE and DMG-PEG-2000. 
     
     
         28 . The composition of  claim 1 , wherein the transfer vehicle comprises one or more lipids selected from the group consisting of ICE, DSPC, CHOL, DODAP, DOTAP and C8-PEG-2000 ceramide. 
     
     
         29 . The composition of  claim 14 , wherein the transfer vehicle comprises DSPC, CHOL, DODAP and C8-PEG-2000 ceramide. 
     
     
         30 - 31 . (canceled) 
     
     
         32 . A composition for increasing expression of a urea cycle enzyme in a target cell, the composition comprising an mRNA and a transfer vehicle, wherein the mRNA encodes a urea cycle enzyme and wherein the mRNA comprises a modification, wherein the modification confers stability to the mRNA. 
     
     
         33 . The composition of  claim 32 , wherein the modification comprises an alteration of a 5′ untranslated of said mRNA. 
     
     
         34 - 37 . (canceled) 
     
     
         38 . The composition of  claim 32 , wherein the modification comprises an alteration of a 3′ untranslated region of said mRNA. 
     
     
         39 - 41 . (canceled) 
     
     
         42 . The composition of  claim 32 , wherein the urea cycle enzyme is selected from the group consisting of ornithine transcarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). 
     
     
         43 . The composition of  claim 32 , wherein following expression of said urea cycle enzyme by said target cell, said urea cycle enzyme is secreted from said target cell. 
     
     
         44 . The composition of  claim 32 , wherein said transfer vehicle comprises one or more cationic lipids. 
     
     
         45 . The composition of  claim 32 , wherein said transfer vehicle comprises one or more non-cationic lipids. 
     
     
         46 . The composition of  claim 32 , wherein said transfer vehicle comprises one or more PEG-modified lipids. 
     
     
         47 . The composition of  claim 32 , wherein said transfer vehicle comprises CHOL, DOPE, DLinDMA and DMG-PEG-2000. 
     
     
         48 . The composition of  claim 32 , wherein said transfer vehicle comprises ICE, DOPE and DMG-PEG-2000. 
     
     
         49 . The composition of  claim 32 , wherein the transfer vehicle comprises one or more lipids selected from the group consisting of ICE, DSPC, CHOL, DODAP, DOTAP and C8-PEG-2000 ceramide. 
     
     
         50 . The composition of  claim 49 , wherein the transfer vehicle comprises DSPC, CHOL, DODAP and C8-PEG-2000 ceramide. 
     
     
         51 . The composition of  claim 32 , wherein the transfer vehicle is a liposome. 
     
     
         52 . The composition of  claim 32 , wherein said transfer vehicle is a lipid nanoparticle. 
     
     
         53 - 54 . (canceled) 
     
     
         55 . The composition of  claim 32 , further comprising a ligand capable of enhancing affinity of the composition for the target cell. 
     
     
         56 . (canceled) 
     
     
         57 . The composition of  claim 55 , wherein said ligand is selected from the group consisting of apolipoprotein-B and apolipoprotein-E. 
     
     
         58 - 60 . (canceled) 
     
     
         61 . The composition of  claim 32 , wherein said target cell is a hepatocyte. 
     
     
         62 . (canceled) 
     
     
         63 . A method of treating a subject, wherein the subject has a protein deficiency, the method comprising administering a composition comprising an mRNA and a transfer vehicle, wherein the mRNA encodes a functional protein. 
     
     
         64 . (canceled) 
     
     
         65 . The method of  claim 63 , wherein said functional protein is secreted from said target cell upon expression of said mRNA. 
     
     
         66 . The method of  claim 63 , wherein the mRNA encodes a functional urea cycle enzyme. 
     
     
         67 . The method of  claim 66 , wherein the urea cycle enzyme is selected from the group consisting of OTC, CPS1, ASS1, ASL, and ARG1. 
     
     
         68 . The method of  claim 63 , wherein the modification comprises an alteration of a 5′ untranslated region of said mRNA. 
     
     
         69 - 72 . (canceled) 
     
     
         73 . The method of  claim 63 , wherein the modification comprises an alteration of a 3′ untranslated region of said mRNA. 
     
     
         74 - 76 . (canceled) 
     
     
         77 . The method of  claim 63 , wherein said transfer vehicle comprises one or more cationic lipids. 
     
     
         78 . The method of  claim 63 , wherein said transfer vehicle comprises one or more non-cationic lipids. 
     
     
         79 . The method of  claim 63 , wherein said transfer vehicle comprises one or more PEG-modified lipids. 
     
     
         80 . The method of  claim 63 , wherein said transfer vehicle comprises CHOL, DOPE, DLinDMA and DMG-PEG-2000. 
     
     
         81 . The method of  claim 63 , wherein said transfer vehicle comprises ICE, DOPE and DMG-PEG-2000. 
     
     
         82 . The method of  claim 63 , wherein the transfer vehicle is a liposome. 
     
     
         83 . The method of  claim 63 , wherein said transfer vehicle is a lipid nanoparticle. 
     
     
         84 - 87 . (canceled) 
     
     
         88 . The method of  claim 63 , wherein said composition comprises a ligand capable of enhancing the affinity of said composition for a target cell of said subject, and wherein said ligand is selected from the group consisting of apolipoprotein-B and apolipoprotein-E. 
     
     
         89 - 92 . (canceled) 
     
     
         93 . A method of expressing a functional protein in a target cell wherein the target cell is deficient in said functional protein, the method comprising contacting the target cell with a composition comprising an mRNA and a transfer vehicle, wherein the mRNA encodes said functional protein and wherein the mRNA comprises a modification, wherein the modification confers stability to the mRNA. 
     
     
         94 . The method  claim 93 , wherein following expression of said mRNA by a target cell a functional protein is produced. 
     
     
         95 . The method of  claim 94 , wherein said functional protein is secreted from said target cell. 
     
     
         96 . The method of  claim 93 , wherein said deficient functional protein is a urea cycle enzyme. 
     
     
         97 . The method of  claim 96 , wherein the urea cycle enzyme is selected from the group consisting of OTC, CPS1, ASS1, ASL, and ARG1. 
     
     
         98 . The method of  claim 93 , wherein the modification comprises an alteration of a 5′ untranslated region of said mRNA. 
     
     
         99 - 102 . (canceled) 
     
     
         103 . The method of  claim 93 , wherein the modification comprises an alteration of a 3′ untranslated region of said mRNA. 
     
     
         104 - 106 . (canceled) 
     
     
         107 . The method of  claim 93 , wherein said transfer vehicle comprises one or more cationic lipids. 
     
     
         108 . The method of  claim 93 , wherein said transfer vehicle comprises one or more non-cationic lipids. 
     
     
         109 . The method of  claim 93 , wherein said transfer vehicle comprises one or more PEG-modified lipids. 
     
     
         110 . The method of  claim 93 , wherein said transfer vehicle comprises CHOL, DOPE, DLinDMA and DMG-PEG-2000. 
     
     
         111 . The method of  claim 93 , wherein said transfer vehicle comprises ICE, DOPE and DMG-PEG-2000. 
     
     
         112 . The method of  claim 93 , wherein the transfer vehicle is a liposome. 
     
     
         113 . The method of  claim 93 , wherein said transfer vehicle is a lipid nanoparticle. 
     
     
         114 - 115 . (canceled) 
     
     
         116 . The method of  claim 93 , wherein the composition further comprises a ligand capable of enhancing affinity of the composition for the target cell. 
     
     
         117 . The method of  claim 116 , wherein said ligand is selected from the group consisting of apolipoprotein-B and apolipoprotein-E. 
     
     
         118 . The method of  claim 117 , wherein said target cell expresses one or more low density lipoprotein receptors. 
     
     
         119 - 122 . (canceled) 
     
     
         123 . The method of  claim 93 , wherein the transfer vehicle comprises one or more lipids selected from the group consisting of ICE, DSPC, CHOL, DODAP, DOTAP and C8-PEG-2000 ceramide. 
     
     
         124 . The method of  claim 93  wherein the transfer vehicle comprises DSPC, CHOL, DODAP and C8-PEG-2000 ceramide. 
     
     
         125 - 126 . (canceled)