Compositions and methods of altering cholesterol levels

Abstract

The present invention relates to compositions, methods and kits using polynucleotides, primary transcripts and mmRNA molecules.

1 . A composition comprising a synthetic polynucleotide encoding PCSK9 negative a low density lipoprotein receptor (LDLR) having one or more amino acids substituted such that the LDLR mutant is deficient in proprotein convertase subtilisin/kexin type 9 (PCSK9) binding, in an acceptable diluent or carrier. 
     
     
         2 . (canceled) 
     
     
         3 . The composition of  claim 1 , wherein the synthetic polynucleotide comprises:
 (a) a first region of linked nucleosides, said first region encoding at least one cholesterol regulating polypeptide;   (b) a first flanking region located at the 5′ terminus of said first region comprising;
 (i) a sequence of linked nucleosides selected from the group consisting of the native 5′ untranslated region (UTR), SEQ ID NO: 1 and functional variants thereof; 
   (c) a second flanking region located at the 3′ terminus of said first region comprising;
 (i′) a sequence of linked nucleosides selected from the group consisting of the native 3′ UTR, any of the forgoing comprising one or more microRNA or microRNA binding site or microRNA seeds and functional variants or combinations thereof; and 
 (ii′) a 3′ tailing sequence of linked nucleosides; 
   
       wherein the first region of linked nucleosides comprises at least a first modified nucleoside. 
     
     
         4 - 6 . (canceled) 
     
     
         7 . A method of treating a disease or disorder in a subject in need thereof comprising administering to said subject the composition of  claim 1 . 
     
     
         8 - 16 . (canceled) 
     
     
         17 . A method of modulating cholesterol levels in plasma of a subject comprising contacting said subject with the composition of  claim 1 . 
     
     
         18 . (canceled) 
     
     
         19 . 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. 
     
     
         20 . The polynucleotide of  claim 19 , wherein the LDLR mutant has two or more amino acids substituted such that the LDLR mutant is deficient in PCSK9 binding, wherein the two or more substituted amino acids correspond to any two or more 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. 
     
     
         21 . The polynucleotide of  claim 19 , wherein the substituted amino acids comprise one or more of N316A, E317A, D331A, Y336A, L339D, D331E, or any combination thereof. 
     
     
         22 . The polynucleotide of  claim 19 , 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. 
     
     
         23 . The polynucleotide of  claim 19  which comprises:
 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. 
 
     
     
         24 . A composition comprising the polynucleotide of  claim 19  and an excipient. 
     
     
         25 . The composition of  claim 24 , wherein the polynucleotide is formulated in a lipid nanoparticle. 
     
     
         26 . A method of treating or reducing the incidence of a disease or disorder in a human subject in need thereof comprising administering to the subject the composition of  claim 24 . 
     
     
         27 . The method of  claim 26 , 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. 
     
     
         28 . The method of  claim 26 , wherein the administration lowers the cholesterol levels in the plasma of the subject. 
     
     
         29 . The method of  claim 26 , wherein the subject has a polymorphism in CYP7A1. 
     
     
         30 . The method of  claim 26 , wherein the mRNA is fully modified with 1-methylpseudouridine, pseudouridine, or the combination thereof. 
     
     
         31 . The method of  claim 26 , further comprising administering to said subject a synthetic polynucleotide encoding CYP7A1. 
     
     
         32 . A method of lowering cholesterol levels and/or low-density lipoprotein (LDL) levels in plasma of a human subject comprising administering to said subject the composition of  claim 24 . 
     
     
         33 . The method of  claim 32 , wherein the cholesterol levels in plasma are lowered by at least 20% at 24 hours following administration of the polynucleotide to the subject. 
     
     
         34 . 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 19 .