Correction of alpha-1-antitrypsin genetic defects using spliceosome mediated RNA trans splicing

• US 20060234247A1
• Filed: 01/21/2005
• Published: 10/19/2006
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: VIRxSYS Corporation
• Original Assignee: VIRxSYS Corporation
• Inventors: Mitchell, Lloyd G., Puttaraju, Madaiah, McGarrity, Gerard J., Garcia-Blanco, Mariano A., Temple, Gary F., Otto, Edward, Cote, Colette, Mansfield, S. Gary
• Granted Patent: US 8,053,232 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention provides methods and compositions for generating novel nucleic acid molecules through targeted spliceosomal mediated RNA trans-splicing. The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with a SERPINA1 target precursor messenger RNA molecule (target pre-mRNA) and mediate a trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA). In particular, the PTMs of the present invention include those genetically engineered to interact with SERPINA1 target pre-mRNA so as to result in correction of SERPINA1 genetic defects responsible for AAT deficiency. The PTMs of the invention may also comprise sequences that are processed out of the PTM to yield duplex siRNA molecules directed specifically to mutant SERPIN A1 mRNAs. Such duplexed siRNAs are designed to reduce the accumulation of toxic AAT protein in liver cells. The methods and compositions of the present invention can be used in gene therapy for correction of SERPINA1 disorders such as AAT deficiency.

Claims

1. A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 3′

splice region comprising a branch point and a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

4. The cell of claim 1 wherein the nucleic acid molecule further comprises a 5′

donor site.

5. The cell of claim 1 wherein the 3′

splice region further comprises a pyrimidine tract.

6. The cell of claim 1, 2 or 3 wherein said nucleic acid molecule further comprises a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5′

splice site.

7. The cell of claim 1, 2 or 3 wherein the nucleic acid molecule further comprises nucleotide sequence encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

2. A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

3. A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 5′

splice site;

c) a spacer region that separates the 5′

splice site from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

8. A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 3′

splice region comprising a branch point and a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

11. The cell of claim 8 wherein the nucleic acid molecule further comprises a 5′

donor site.

12. The cell of claim 8 wherein the 3′

splice region further comprises a pyrimidine tract.

9. A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

10. A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 5′

splice site;

c) a spacer region that separates the 5′

splice site from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

13. The cell of claim 8, 9, or 10 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′

splice region.

14. The cell of claim 8, 9 or 10 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

15. A method of producing a chimeric RNA molecule in a cell comprising:

contacting a target SERPINA1 pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises;

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 3′

splice region comprising a branch point and a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.

18. The method of claim 15 wherein the nucleic acid molecule further comprises a 5′

donor site.

19. The method of claim 15 wherein the 3′

splice region further comprises a pyrimidine tract.

20. The method of claim 15, 16 or 17 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′

splice region.

21. The method of claim 15, 16 or 17 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

16. A method of producing a chimeric RNA molecule in a cell comprising:

contacting a target SERPINA1 pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises;

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.

17. A method of producing a chimeric RNA molecule in a cell comprising:

contacting a target SERPINA1 pre-mRNA expressed within the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises;

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within the cell;

b) a 5′

splice site;

c) a spacer region that separates the 5′

splice site from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

22. A nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell;

b) a 3′

splice region comprising a branch point and a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

25. The nucleic acid molecule of claim 22 wherein the nucleic acid molecule further comprises a 5′

donor site.

26. The nucleic acid molecule of claim 22 wherein the 3′

splice region further comprises a pyrimidine tract.

27. The nucleic acid molecule of claim 22, 23, or 24 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′

splice region.

28. The nucleic acid of claim 22, 23, or 24 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

23. A nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell;

b) a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

24. A nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell;

b) a 5′

splice site;

c) a spacer region that separates the 5′

splice site from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

29. A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell;

b) a 3′

splice region comprising a branch point and a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

32. The vector of claim 29 wherein the nucleic acid molecule further comprises a 5′

donor site.

33. The vector of claim 29 wherein the nucleic acid molecule further comprises a pyrimidine tract.

34. The vector of claim 29, 30 or 31 wherein the nucleic acid molecule further comprises a safety nucleotide sequence comprising one or more complementary sequences that bind to one or more sides of the 3′

splice region.

35. The vector of claim 29, 30, or 31 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

36. The vector of claim 29-35 wherein said vector is a viral vector.

37. The vector of claim 29-35 wherein expression of the nucleic acid molecule is controlled by a liver cell specific promoter.

30. A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell;

b) a 3′

splice acceptor site;

c) a spacer region that separates the 3′

splice region from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

31. A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell;

b) a 5′

splice site;

c) a spacer region that separates the 5′

splice site from the target binding domain; and

d) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

38. A method for correcting a SERPINA1 genetic defect in a subject comprising administering to said subject a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 pre-mRNA expressed within a cell wherein said pre-mRNA is encoded by a gene containing a SERPINA1 genetic defect; and

b) a nucleotide sequence to be trans-spliced to the target pre-mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide;

wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

39. A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 mRNA expressed within the cell;

b) a sequence having ribozyme activity; and

c) a nucleotide sequence to be trans-spliced to the target mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide.

40. The cell of claim 39 wherein the nucleic acid molecule further comprises nucleotide sequence encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

41. A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 mRNA expressed within the cell;

b) a sequence having ribozyme activity; and

c) a nucleotide sequence to be trans-spliced to the target mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide.

42. The cell of claim 41 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

43. A method of producing a chimeric RNA molecule in a cell comprising:

contacting a target SERPINA1 mRNA expressed in the cell with a nucleic acid molecule wherein said nucleic acid molecule comprises;

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 mRNA expressed within the cell;

b) a sequence having ribozyme activity; and

c) a nucleotide sequence to be trans-spliced to the target mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide.

44. The method of claim 43 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

45. A nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 mRNA expressed within a cell;

b) a sequence having ribozyme activity; and

c) a nucleotide sequence to be trans-spliced to the target mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide.

46. The nucleic acid of claim 45 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

47. A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 mRNA expressed within a cell;

b) a sequence having ribozyme activity; and

c) a nucleotide sequence to be trans-spliced to the target mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide.

49. The vector of claim 47 or 48 wherein said vector is a viral vector.

50. The vector of claim 47 or 48 wherein expression of the nucleic acid molecule is controlled by a liver cell specific promoter.

48. The vector of claim 48 wherein the nucleic acid molecule further comprises a nucleotide sequence capable encoding a siRNA capable of binding to a mutant SERPINA1 transcript.

51. A method for correcting a SERPINA1 genetic defect in a subject comprising administering to said subject a nucleic acid molecule comprising:

a) one or more target binding domains that target binding of the nucleic acid molecule to a SERPINA1 mRNA expressed within a cell wherein said mRNA is encoded by a gene containing a SERPINA1 genetic defect;

b) a sequence having ribozyme activity and

c) a nucleotide sequence to be trans-spliced to the target mRNA wherein said nucleotide sequence encodes a SERPINA1 polypeptide.