C/EBP ALPHA SARNA COMPOSITIONS AND METHODS OF USE
The invention relates to saRNA targeting a C/EBPα transcript and therapeutic compositions comprising said saRNA. Methods of using the therapeutic compositions are also provided.
1 . A synthetic isolated saRNA which up-regulates expression of C/EBPα gene, wherein the saRNA is at least 80% complement to a region on SEQ ID No. 77, and wherein the saRNA has 14-30 nucleotides.
2 . The saRNA of claim 1 , wherein the saRNA is single stranded.
3 . The saRNA of claim 2 , wherein the saRNA comprises a 3′ overhang.
4 . The saRNA of claim 2 , wherein the saRNA is modified.
5 . The saRNA of claim 4 , wherein the saRNA comprises at least 2 modifications.
6 . The saRNA of claim 4 , wherein the modification comprises any of 2′-F, 2′-OMe, inverted deoxyribose, or phosphorothioate linkage between nucleotides.
7 . The saRNA of claim 2 , wherein the saRNA comprises a sequence selected from SEQ ID No. 35, 37, 39, 41, 43, 45, 47, 49, 93 (AW51) and 109 (CEBPA51).
8 . The saRNA of claim 1 , wherein the saRNA is double-stranded and comprises an antisense strand and a sense strand.
9 . The saRNA of claim 5 , wherein the antisense strand comprises a sequence selected from SEQ ID No. 35, 37, 39, 41, 43, 45, 47, 49, 93 (AW51) and 109 (CEBPA51).
10 . The saRNA of claim 6 , wherein the sense strand comprises a sequence selected from SEQ ID No. 34, 36, 38, 40, 42, 44, 48, 94 and 110.
11 . The saRNA of claim 8 , wherein the saRNA is modified.
12 . The saRNA of claim 11 , wherein the saRNA comprises at least 2 modifications.
13 . The saRNA of claim 11 , wherein the modification may comprise any of 2′-F, 2′-OMe, inverted deoxyribose, or phosphorothioate linkage between nucleotides.
14 . The saRNA of claim 11 , wherein the modification is on the sense strand.
15 . The saRNA of claim 11 , wherein the modification is on both the sense and antisense strand.
16 . A method of up-regulating C/EBPα gene in a cell or up-regulating the expression of a gene selected from, alanine-glyoxylate aminotransferase (AGXT), cytochrome P450 3A4 (CYP3A4), ornithine transcarbamylase (OTC, or hepatocyte nuclear factor 4-alpha (HNF4a) in a cell, comprising administering the saRNA of claim 1 to the cell.
17 . The method of claim 16 , wherein the cell is a proliferating cell.
18 . The method of claim 17 , wherein the cell is a cancer cell.
19 . The method of claim 18 , wherein the cell is a hepatocellular carcinoma (HCC) cell.
20 . The method of claim 16 , wherein the cell is not hyperproliferating cell.
21 . The method of claim 20 , wherein the cell is a primary human hepatocyte cell.
22 . A method of treating liver fibrosis, liver failure, or nonalcoholic steatohepatitis (NASH) of a subject in need thereof comprising administering the saRNA of claim 1 to the subject.
23 . The method of claim 22 , wherein the liver failure is acute liver failure.
24 . The method of claim 22 , wherein the total bilirubin (TBIL) level, circulating alanine aminotransferase (ALT) level, aspartate aminotransferase (AST) level, alkaline phosphatase (ALP) level, gamma-glutamyl-transpeptidase (GGT) level, liver hydroxyproline level, prothrombin time, ammonia, or liver triglyceride (liver TG) level of said subject is decreased.
25 . The method of claim 22 , wherein the serum albumin level, total protein level of said subject is increased.
26 . The method of claim 22 , wherein the fibrous tissue or peudolobule formation of said subject is reduced.
27 . A method of treating type II diabetes or insulin resistance of a subject in need thereof comprising administering the saRNA of claim 1 to the subject.
28 . The method of claim 27 , wherein liver cholesterol level, serum AST level, fasting glucose level, the ratio of triglycerides to HDL-c, or liver to body ratio of said subject is decreased.
29 . The method of claim 27 , wherein the insulin level of said subject is increased.
30 . A method of encapsulating the saRNA of claim 1 in a liposome, comprising:
dissolving the saRNA in a first buffer to for a saRNA solution,
filtering the saRNA solution through a 0.2 μm filter,
mixing the filtered saRNA solution with a lipid solution in an injection module to form a liposome formulation,
adding a second buffer to the liposome formulation.
31 . The method of claim 30 , wherein the first buffer is Na-Acetate/Sucrose.
32 . The method of claim 30 , wherein the pH for the saRNA solution is around 4.0.
33 . The method of claim 30 , wherein the concentration of the saRNA in the saRNA solution is around 2.38 mg/mL.
34 . The method of claim 30 , wherein the lipid solution comprises 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesteryl-hemi succinate (CHEMS), and 4-(2-aminoethyl)-morpholino-cholesterol hemisuccinate (MOCHOL).
35 . The method of claim 34 , wherein the molar ratio of POPC:DOPE:CHEMS:MOCHOL is around 6:24:23:47.
36 . The method of claim 30 , wherein the second buffer has a pH of around 9.
37 . The method of claim 36 , wherein the second buffer is NaCl/Na2HPO4.