Methods and compositions for the specific inhibition of alpha-1 antitrypsin by double-stranded RNA

Patent metadata

Field	Value
Publication number	US-9458457-B2
Application number	US-201414323299-A
Country	US
Kind code	B2
Filing date	Jul 3, 2014
Priority date	Jul 3, 2013
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

This invention relates to compounds, compositions, and methods useful for reducing α-1 antitrypsin target RNA and protein levels via use of dsRNAs, e.g., Dicer substrate siRNA (DsiRNA) agents.

First claim

We claim:

1. A modified nucleic acid comprising an oligonucleotide strand of 19-53 nucleotides in length, wherein said oligonucleotide strand is complementary to SEQ ID NO: 1080 along at least 19 consecutive nucleotides of said oligonucleotide strand length and reduces α-1 antitrypsin target mRNA expression when said nucleic acid is introduced into a mammalian cell.

2. A double stranded nucleic acid (dsNA) comprising first and second nucleic acid strands comprising RNA, wherein said first strand is 15-53 nucleotides in length and said second strand of said dsNA is 19-53 nucleotides in length, wherein said second oligonucleotide strand is complementary to SEQ ID NO: 1080 along at least 19 consecutive nucleotides of said second oligonucleotide strand length and reduces α-1 antitrypsin target mRNA expression when said double stranded nucleic acid is introduced into a mammalian cell.

3. The dsNA of claim 2 comprising a duplex region selected from the group consisting of at least 25 base pairs; 19-21 base pairs and 21-25 base pairs.

4. The dsNA of claim 2 , wherein said second oligonucleotide strand comprises 1-5 single-stranded nucleotides at its 3′ terminus.

5. The dsNA of claim 4 , wherein said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand are 1-3 nucleotides in length.

6. The dsNA of claim 4 , wherein said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand are 1-2 nucleotides in length.

7. The dsNA of claim 4 , wherein said nucleotides of said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand comprise a modified nucleotide.

8. The dsNA of claim 7 , wherein said modified nucleotide of said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand is a 2′-O-methyl ribonucleotide.

9. The dsNA of claim 7 , wherein all nucleotides of said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand are modified nucleotides.

10. The dsNA of claim 7 , wherein said 1-5 single-stranded nucleotides of said 3′ terminus of said second strand is two nucleotides in length and comprises a 2′-O-methyl modified ribonucleotide.

11. The dsNA of claim 2 , wherein said first strand is 25-53 nucleotides in length.

12. The dsNA of claim 2 , wherein said second strand is 25-53 nucleotides in length.

13. The dsNA of claim 2 , wherein starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide.

14. The dsNA of claim 13 , wherein said modified nucleotide residue of said 3′ terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule.

15. The dsNA of claim 14 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide.

16. The dsNA of claim 2 , wherein 3′ terminus of said first strand and 5′ terminus of said second strand form a blunt end.

17. The dsNA of claim 2 , wherein said first strand is 25 nucleotides in length and said second strand is 27 nucleotides in length.

18. The dsNA of claim 2 , wherein said second strand comprises SEQ ID NO: 288.

19. The dsNA of claim 2 , wherein said first strand comprises SEQ ID NO: 90.

20. The dsNA of claim 2 comprising a pair of first strand/second strand sequences of SEQ ID NO: 90 and SEQ ID NO: 288.

21. The dsNA of claim 2 , wherein said dsNA comprises a modified nucleotide.

22. The dsNA of claim 2 , wherein the first and second strands are joined by a chemical linker.

23. The dsNA of claim 2 , wherein 3′ terminus of said first strand and 5′ terminus of said second strand are joined by a chemical linker.

24. The dsNA of claim 2 , wherein a nucleotide of said second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

25. The dsNA of claim 2 comprising a modified nucleotide selected from the group consisting of a 2′-O-methyl ribonucleotide, a 2′-methoxyethoxy, a 2′-fluoro ribonucleotide, a 2′-allyl, a 2′-O-[2-(methylamino)-2-oxoethyl], a 4′-thio, a 4′-CH 2 —O-2′-bridge, a 4′-(CH 2 ) 2 —O-2′-bridge, a 2′-LNA, a 2′-amino, a 2′-O-(N-methlycarbamate), a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-amino ribonucleotide, a locked nucleic acid and an unlocked nucleobase analog (UNA).

26. The dsNA of claim 2 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

27. The dsNA of claim 2 comprising a modification selected from the group consisting of a morpholino nucleic acid and a peptide nucleic acid (PNA).

28. The dsNA of claim 2 , wherein said dsNA is attached to a dynamic polyconjugate (DPC).

29. Composition comprising the dsNA of claim 2 , wherein said dsNA is administered with a DPC, where the dsNA and DPC are optionally not attached.

30. The dsNA of claim 2 , wherein said dsNA is attached to a moiety selected from the group consisting of a GalNAc moiety, a cholesterol and a cholesterol targeting ligand.

31. A method for reducing expression of a target α-1 antitrypsin gene in a mammalian cell comprising contacting a mammalian cell in vitro with a dsNA of claim 2 in an amount sufficient to reduce expression of a target α-1 antitrypsin mRNA in said cell.

32. The method of claim 31 , wherein target α-1 antitrypsin mRNA expression is reduced by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.

33. The method of claim 31 , wherein α-1 antitrypsin mRNA levels are reduced by an amount (expressed by %) of at least 90% at least 8 days after said cell is contacted with said dsNA.

34. The method of claim 31 , wherein α-1 antitrypsin mRNA levels are reduced by an amount (expressed by %) of at least 70% at least 10 days after said cell is contacted with said dsNA.

35. A method for reducing expression of a target α-1 antitrypsin mRNA in a mammal comprising administering a nucleic acid of claim 1 to a mammal in an amount sufficient to reduce expression of a target α-1 antitrypsin mRNA in the mammal.

36. The method of claim 35 , wherein said nucleic acid is formulated in a lipid nanoparticle (LNP).

37. The method of claim 35 , wherein said nucleic acid is administered at a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said mammal per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.1 to 2.5 micrograms per kilogram.

38. The method of claim 35 , wherein α-1 antitrypsin

Assignees

• Dicerna Pharmaceuticals Inc

Inventors

• Brown Bob D
• Dudek Henryk T

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P1/16

  for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics · CPC title

• C12N2310/321

  2'-O-R Modification · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title