Compositions and Methods of Altering Cholesterol Levels

Abstract

The present invention relates to compositions, methods and kits using polynucleotides, primary transcripts and mmRNA molecules. The present invention also relates to compositions and methods for altering cholesterol levels using polynucleotides, primary transcripts and mmRNA molecules.

1 . A composition comprising;
 (a) a first polynucleotide encoding a LDLR mutant having one or more amino acids substituted such that the LDLR mutant is deficient in PCSK9 binding, and   (b) a second polynucleotide encoding a CYP7A1 protein, and   (c) an excipient.   
     
     
         2 - 6 . (canceled) 
     
     
         7 . A method comprising contacting a hepatocyte in a human subject with the composition of  claim 1 . 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The method of  claim 7 , wherein the human subject has a polymorphism in CYP7A1. 
     
     
         12 . A method of treating a disease or disorder in a human subject in need thereof comprising administering, to said subject the composition of  claim 1 . 
     
     
         13 . The method of  claim 12  wherein the disease or disorder is selected from the group consisting of fatty liver disease, hepatocellular carcinoma, NASH, steatosis, familial hypercholesterolemia (FH), hypercholesterolemia, and aberrant lipoprotein profile. 
     
     
         14 - 17 . (canceled) 
     
     
         18 . A method of lowering cholesterol levels in plasma of a human subject comprising contacting said subject with the composition of  claim 1 . 
     
     
         19 . (canceled) 
     
     
         20 . The composition of  claim 1 , wherein the LDLR mutant has two or more amino acids substituted such that the LDLR mutant is deficient in PCSK9 binding. 
     
     
         21 . The composition of  claim 1 , wherein the one or more substituted amino acids correspond to amino acid 316 of SEQ ID NO:19, amino acid 317 of SEQ ID NO:19, amino acid 331 of SEQ ID NO:19, amino acid 336 of SEQ ID NO:19, amino acid 339 of SEQ ID NO:19, or any combination thereof. 
     
     
         22 . The composition of  claim 20 , wherein the two or more substituted amino acids correspond to any two of amino acid 316 of SEQ ID NO:19, amino acid 317 of SEQ ID NO:19, amino acid 331 of SEQ ID NO:19, amino acid 336 of SEQ ID NO:19, and amino acid 339 of SEQ ID NO:19. 
     
     
         23 . The composition of  claim 21 , wherein the substituted amino acids comprise one or more of amino acid N316A, E317A, D331A, Y336A, L339D, D331E, or any combination thereof. 
     
     
         24 . The method of  claim 12 , wherein the half-life of receptors that bind LDL on the surface of cells in the subject is longer after the administration compared to the half-life of receptors that bind LDL on the surface of cells in a subject contacted with a polynucleotide encoding wild-type LDLR consisting of the sequence set forth as SEQ ID NO: 19. 
     
     
         25 . The method of  claim 18 , wherein the half-life of receptors that bind LDL on the surface of cells in the subject is longer after the administration compared to the half-life of receptors that bind LDL on the surface of cells in a subject contacted with a polynucleotide encoding wild-type LDLR consisting of the sequence set forth as SEQ ID NO: 19. 
     
     
         26 . A polynucleotide comprising an mRNA encoding a LDLR mutant having one or more amino acids substituted such that the LDLR mutant is deficient in PCSK9 binding, wherein the one or more amino acids correspond to amino acid 316 of SEQ ID NO: 19, amino acid 317 of SEQ ID NO:19, amino acid 331 of SEQ ID NO:19, amino acid 336 of SEQ ID NO:19, amino acid 339 of SEQ ID NO:19, or any combination thereof. 
     
     
         27 . The polynucleotide of  claim 26 , wherein the LDLR mutant has two or more amino acids substituted such that the LDLR mutant is deficient in PCSK9 binding. 
     
     
         28 . The polynucleotide of  claim 27 , wherein the two or more substituted amino acids correspond to any two of amino acid 316 of SEQ ID NO:19, amino acid 317 of SEQ ID NO:19, amino acid 331 of SEQ ID NO:19, amino acid 336 of SEQ ID NO:19, and amino acid 339 of SEQ ID NO:19. 
     
     
         29 . The polynucleotide of  claim 26 , wherein the substituted amino acids comprise one or more of N316A, E317A, D331A, Y336A, L339D, D331E, or any combination thereof. 
     
     
         30 . The polynucleotide of  claim 29 , wherein the half-life of the LDLR mutant encoded by the polynucleotide is longer than the half-life of wild-type LDLR consisting of the sequence set forth as SEQ ID NO: 19. 
     
     
         31 . The polynucleotide of  claim 26  which comprises:
 (i) a first region of linked nucleosides encoding the LDLR mutant; 
 (ii) a first flanking region located at the 5′ terminus of said first region comprising a sequence of linked nucleosides comprising a 5′ untranslated region (UTR); 
 (iii) a second flanking region located at the 3′ terminus of said first region comprising a sequence of linked nucleosides comprising a 3′ UTR; and 
 (iv) a 3′ tailing sequence of linked nucleosides. 
 
     
     
         32 . A composition comprising the polynucleotide of  claim 26  and an excipient. 
     
     
         33 . A method of treating a disease or disorder in a subject in need thereof comprising administering to the subject the composition of  claim 32 . 
     
     
         34 . The method of  claim 33  wherein the disease or disorder is selected from the group consisting of fatty liver disease, hepatocellular carcinoma, NASH, steatosis, familial hypercholesterolemia (FH), hypercholesterolemia, and aberrant lipoprotein profile. 
     
     
         35 . A method of lowering cholesterol levels in plasma of a subject comprising contacting said subject with the composition of  claim 32 . 
     
     
         36 . A method of increasing expression of receptors that bind LDL on the surface of a cell comprising contacting the cell with the polynucleotide of  claim 26 ,
 wherein the LDLR mutant is expressed on the surface of the cell and wherein the half-life of the receptors that bind LDL on the surface of the cell is longer after the contacting than the half-life of the receptors that bind LDL in a cell contacted with an mRNA encoding wild-type LDLR consisting of the sequence set forth in SEQ ID NO: 19.   
     
     
         37 . A method of increasing expression of receptors that bind LDL on the surface of a cell comprising contacting the cell with the polynucleotide of  claim 26 ,
 wherein the LDLR mutant is expressed on the surface of the cell and wherein the number of the receptors that bind LDL on the surface of the cell in the presence of PCSK9 is increased after the contacting compared to the number of the receptors that bind LDL on the surface of a cell contacted with mRNA encoding wild-type LDLR consisting of the sequence set forth in SEQ ID NO: 19 in the presence of PCSK9.