Integration-site directed vector systems

• US 20070031380A1
• Filed: 08/08/2005
• Published: 02/08/2007
• Est. Priority Date: 08/08/2005
• Status: Active Grant

• Current Assignee: Immusoft Corporation
• Original Assignee: Discovery Genomics, Inc (Immusoft Corporation)
• Inventors: Essner, Jeffrey, Hackett, Perry
• Granted Patent: US 7,919,583 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Some aspects of the application describe materials and methods for making a molecular tether. A molecular tether, in certain embodiments, includes a target-DNA-binding domain having a specific binding affinity for a target-DNA segment in a host chromosome, a carrier-binding domain that specifically binds to a DNA segment on a carrier, and a spacer covalently bonded to the target DNA-binding domain and the carrier-binding domain.

Claims

1. A molecular tether comprising a RecA or Rad51 protein, a carrier-binding domain and a spacer connecting the RecA or Rad51 protein to the carrier-binding domain, wherein the RecA or Rad51 protein is further bound to a single-stranded nucleic acid segment having a sequence for binding to a target DNA segment, and wherein the carrier-binding domain has specific binding affinity for a nucleic acid sequence.

2. The molecular tether of claim 1, wherein the carrier-binding domain comprises one or more zinc fingers, at least one helix-turn-helix motif, or at least one helix-loop-helix motif.

3. The molecular tether of claim 1, wherein the spacer comprises a peptide.

4. A system comprising a molecular tether and a carrier comprising a vector with an expressible nucleic acid segment, with the molecular tether comprising:

a target-DNA-binding domain having a specific binding affinity for a target double-stranded DNA segment;

a carrier-binding domain having a specific binding affinity for a DNA segment located on the carrier; and

a spacer sequence that is covalently bonded to the target-DNA-binding domain and the carrier-binding domain.

5. The system of claim 4, wherein the carrier-binding domain or the target-DNA-binding domain comprises one or more zinc fingers.

6. The system of claim 4, wherein the target-DNA-binding domain or the carrier-binding domain comprises at least one helix-turn-helix motif or at least one helix-loop-helix motif.

7. The system of claim 4, wherein the target-DNA-binding domain or the carrier-binding domain comprises a triple helix-stabilizing protein.

8. The system of claim 7, wherein the triple-helix-stabilizing protein is a RecA protein or a Rad51 protein.

9. The system of claim 7, wherein the triple-helix-stabilizing protein is a RecA or Rad51, and the RecA or Rad51 is associated with an single-stranded nucleic acid segment having a sequence that is matched to the target double-stranded DNA segment'"'"'s sequence.

10. The system of claim 4, wherein the carrier comprises a plasmid.

11. The system of claim 4, wherein the vector comprises a transposon.

12. The system of claim 11, wherein the vector comprises a Sleeping Beauty transposon.

13. The system of claim 14, wherein the spacer comprises a peptide.

14. The system of claim 4, comprising a composition that comprises a mixture of the carrier and the molecular tether.

15. A molecular tether comprising:

a target DNA-binding domain having a specific binding affinity for a target-DNA segment that is double-stranded;

a carrier-binding domain that specifically binds to a DNA segment on a carrier; and

a spacer covalently bonded to the target-DNA-binding domain and the carrier-binding domain.

16. The molecular tether of claim 15, wherein the target-DNA-binding domain comprises a polypeptide and the carrier-binding domain comprises a polypeptide.

17. The molecular tether of claim 15, wherein the target DNA-binding domain or the carrier-binding domain comprises at least one helix-turn-helix motif or at least one helix-loop-helix motifs.

18. The molecular tether of claim 15, wherein the target-DNA-binding domain or the carrier-binding domain comprises one or more zinc fingers.

19. The molecular tether of claim 15, wherein the target-DNA-binding domain or the carrier-binding domain comprises a triple-helix-stabilizing protein.

20. The molecular tether of claim 19, wherein the triple-helix-stabilizing protein comprises a RecA protein or a Rad51 protein.

21. The molecular tether of claim 19, wherein the triple-helix-stabilizing protein is a RecA or Rad51, and the RecA or Rad51 is associated with an single-stranded nucleic acid segment having a sequence that is matched to the target double-stranded DNA segment'"'"'s sequence.

22. The molecular tether of claim 15, wherein the carrier comprises a plasmid.

23. The molecular tether of claim 15, wherein the carrier comprises a transposon.

24. The molecular tether of claim 15, wherein the spacer comprises a peptide.

25. The molecular tether of claim 15, wherein the carrier comprises a viral vector-based genome comprising an expressible DNA segment for insertion into the host.

26. The molecular tether of claim 15, wherein the molecular tether is a polypeptide.

27. A nucleic acid segment comprising an mRNA or a DNA encoding the tethering polypeptide of claim 26.

28. A method for site-directed DNA insertion into a genome, the method comprising:

inserting, within a cell, carrier DNA and a molecular tether wherein the carrier DNA comprises a vector, wherein the molecular tether comprises a target-DNA-binding domain connected via a spacer to a carrier-binding domain.

29. The method of claim 28, wherein either the carrier-binding domain or the target-DNA-binding domain comprises one or more zinc fingers.

30. The method of claim 28, wherein the target-DNA-binding domain or the carrier-binding domain comprises one or more helix-turn-helix motifs.

31. The method of claim 28, wherein the target-DNA-binding domain or the carrier-binding domain comprises a triple-helix-stabilizing protein.

32. The method of claim 31, wherein the triple-helix-stabilizing protein is a RecA protein or a Rad51 protein.

33. The method of claim 31, wherein the triple-helix-stabilizing protein is a RecA or Rad51, and the RecA or Rad51 is associated with an single-stranded DNA segment having a sequence that is matched to the target DNA-binding domain'"'"'s sequence.

34. The method of claim 31, wherein the carrier is a plasmid, a transposon, or a Sleeping Beauty Transposon.

35. The method of claim 31, wherein the spacer comprises a peptide.

36. A method of treatment of a patient comprising administering transformed cells to a patient, wherein the cells have been transformed by the method of claim 28.