Integration-site directed vector systems

• US 7,919,583 B2
• Filed: 08/08/2005
• Issued: 04/05/2011
• Est. Priority Date: 08/08/2005
• Status: Active Grant

• Current Assignee: Immusoft Corporation
• Original Assignee: Discovery Genomics, Inc (Immusoft Corporation)
• Inventors: Hackett, Perry B., Essner, Jeffrey
• Primary Examiner(s): BURKHART, MICHAEL D
• Application Number:
  US11/198,987
• Publication Number:
  US 20070031380A1
• Time in Patent Office: 2,066 Days
• Field of Search: None
• US Class Current: 530/350
• 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. An isolated 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, with the carrier-binding domain and the spacer being polypeptides.

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. A purified vector system for targeting a double-stranded target-DNA segment, the 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 comprising a polypeptide that associates with a single-stranded DNA to thereby have a specific binding affinity for a target double-stranded DNA segment;

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

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

4. The system of claim 3, wherein the carrier-binding domain or the target-DNA-binding domain comprises one or more zinc fingers.

5. The system of claim 3, 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.

6. The system of claim 3, wherein the target-DNA-binding domain or the carrier-binding domain comprises a triple helix-stabilizing protein.

7. The system of claim 6, wherein the triple-helix-stabilizing protein is a RecA protein or a Rad51 protein.

8. The system of claim 6, 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.

9. The system of claim 3, wherein the carrier comprises a plasmid.

10. The system of claim 3, wherein the vector comprises a transposon.

11. The system of claim 10, wherein the vector comprises a Sleeping Beauty transposon.

17. The system of claim 3, wherein the carrier comprises a viral vector-based genome comprising an expressible DNA segment.

12. An isolated molecular tether comprising:

a target DNA-binding domain comprising a polypeptide that associates with a single-stranded DNA to thereby have 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

with the spacer and the carrier-binding domains being polypeptides.

13. The molecular tether of claim 12, 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.

14. The molecular tether of claim 12, wherein the target-DNA-binding domain or the carrier-binding domain comprises one or more zinc fingers.

15. The molecular tether of claim 12, wherein the target-DNA-binding domain or the carrier-binding domain comprises a triple-helix-stabilizing protein.

16. The molecular tether of claim 15, wherein the triple-helix-stabilizing protein comprises a RecA protein or a Rad51 protein.

18. A nucleic acid segment comprising an mRNA or a DNA encoding the molecular tether of claim 12.

22. The molecular tether of claim 12 wherein the carrier-binding domain comprises one or more zinc fingers.

19. A purified ex vivo vector system for targeting a double-stranded target-DNA segment in a patient that comprises a purified pharmaceutically acceptable preparation that comprises:

a polypeptide molecular tether specifically bound to a carrier via protein-to-DNA specific binding, with

the carrier comprising;

a Sleeping Beauty transposon vector and a DNA segment for protein-to-DNA specific binding to the tether, and

the molecular tether comprising;

a RecA or Rad51 polypeptide associated with a single-stranded nucleic acid and having a specific binding affinity for a predetermined target double-stranded DNA segment in a chromosome of a patient;

a polypeptide carrier-binding domain with either a helix-loop-helix motif, a helix-turn-helix motif, or at least one zinc finger motif, having a specific binding affinity for the DNA segment on the carrier; and

a spacer sequence that is a polypeptide covalently bonded to the carrier-binding domain and the RecA or Rad51 polypeptide.

20. The system of claim 19 wherein the helix-loop-helix motif is derived from the LexA protein and the system comprises a RecA- LexA fusion polypeptide.

21. The system of claim 19 wherein the system comprises a RecA-Gal4 fusion protein.