Compositions and methods for inhibiting gene expression of alpha-1 AntiTrypsin
View Patent ↗The invention relates to a RNA interference triggers for inhibiting the expression of an AAT gene through the mechanism of RNA interference. The invention also relates to a pharmaceutical composition comprising the AAT RNAi trigger together with an excipient capable of improving delivery of the RNAi trigger to a liver cell in vivo. Delivery of the AAT RNAi trigger to liver cells in vivo provides for inhibition of AAT gene expression and treatment of alpha 1-antitrypsin deficiency and associated diseases.
1. An RNA interference (RNAi) trigger molecule for inhibiting the expression of an alpha-1 antitrypsin gene in a cell in vivo, wherein the RNAi trigger molecule comprises a sense strand sequence that is 18-24 nucleotides in length and an antisense strand sequence that is 19-24 nucleotides in length, wherein the antisense strand sequence and the sense strand sequence are fully complementary over a core stretch of at least 18 consecutive nucleotides, wherein the antisense strand sequence comprises in order the nucleobase sequence 5′ GGAACUUGGUGAUGAUAU 3′ (SEQ ID NO: 1), and wherein said SEQ ID NO: 1 is located at nucleotide positions 2-19 from the 5′ terminal end of the antisense strand.
2. The RNAi trigger molecule of claim 1 , wherein the sense strand or the antisense strand further comprises a 3′ extension of 1-5 nucleotides in length.
3. The RNAi trigger molecule of claim 2 , wherein the 3′ extension of the antisense strand comprises dTdT or dTsdT, wherein dT is a deoxythymidine nucleotide and s is a phosphorothioate internucleoside linkage.
4. The RNAi trigger molecule of claim 3 , wherein the 3′ extension of the sense strand comprises Af(invdT), wherein Af is a 2′-deoxy-2′-fluoro adenosine nucleotide, and (invdT) is an inverted deoxythymidine (3′-3′-linked) nucleotide.
5. The RNAi trigger molecule of claim 1 , wherein the sense strand further comprises a 5′ extension of 1-5 nucleotides in length.
6. The RNAi trigger molecule of claim 5 , wherein the 5′ extension of the antisense strand comprises deoxythymidine.
7. The RNAi trigger molecule of claim 5 , wherein the 5′ extension of the sense strand comprises UAU or uAu, wherein U is uracil, A is adenine, and u is 2′-O-methyl modified uracil.
8. The RNAi trigger molecule of claim 1 , wherein a targeting moiety is conjugated to the 5′ end of the sense strand.
9. The RNAi trigger molecule of claim 8 , wherein the targeting moiety comprises N-acetyl-galactosamine.
10. The RNAi trigger molecule of claim 8 , wherein the targeting moiety comprises a cholesterol-triethylene glycol group.
11. The RNAi trigger molecule of claim 1 , wherein the sense strand sequence and antisense strand sequence form a sequence pair consisting of SEQ ID NOs: 1/8.
12. The RNAi trigger molecule of claim 2 , wherein the sense strand sequence and antisense strand sequence form sequence pairs or meroduplexes selected from the group consisting of SEQ ID NOs: 15/23, 15/24, 15/25/41, 15/26/42, and 15/27/43.
13. The RNAi trigger molecule of claim 1 , wherein the sense strand or antisense strand contains one or more modified nucleotide or nucleotide mimic.
14. The RNAi trigger molecule of claim 13 , wherein the sense strand sequence and antisense strand sequence form sequence pairs selected from the group consisting of SEQ ID NOs: 50/62 and 50/63.
15. The RNAi trigger molecule of claim 13 , wherein the sense strand sequence and antisense sequence form sequence meroduplexes selected from the group consisting of SEQ ID NOs: 50/64/81, 50/65/82, and 50/66/83.
16. The RNAi trigger molecule of claim 13 , wherein the antisense strand sequence contains at least one 2′,3′-seco RNA nucleotide mimic.
17. The RNAi trigger molecule of claim 13 , wherein the sense strand sequence and antisense strand sequence form a sequence pair consisting of SEQ ID NOs: 52/63.
18. The RNAi trigger molecule of claim 13 , wherein the modified nucleotide is selected from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a deoxythymidine, an inverted deoxythymidine, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphorothioate linked nucleotide, and a non-natural base comprising nucleotide.
19. A composition comprising the RNAi trigger molecule as defined in claim 13 .
20. The composition of claim 19 , further comprising a pharmaceutically acceptable carrier, stabilizer and/or diluent.
21. A method for inhibiting the expression of an alpha-1 antitrypsin (AAT) gene in a cell, a tissue, or an organism, the method comprising introducing into a cell, tissue, or organism the RNAi trigger molecule as defined in claim 13 .
22. The method of claim 21 , wherein inhibiting expression of an AAT gene in an organism treats, or manages a pathological condition or disease caused by alpha-1 antitrypsin deficiency.
23. The method claim 22 , wherein the pathological condition and disease caused by alpha-1 antitrypsin deficiency is selected from the group consisting of: chronic hepatitis, cirrhosis, hepatocellular carcinoma, and fulminant hepatic failure.
24. The RNAi trigger molecule of claim 16 , wherein the sense strand sequence and antisense strand sequence form a sequence pair consisting of SEQ ID NOs: 51/63.
25. The composition of claim 19 , further comprising a melittin-like protein (MLP) delivery polymer.