Methods and compositions for targeted integration

• US 20090117617A1
• Filed: 10/23/2008
• Published: 05/07/2009
• Est. Priority Date: 10/25/2007
• Status: Active Grant

• Current Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Yao, Shuyuan, Holmes, Michael C.
• Granted Patent: US 8,936,936 B2
• Time in Patent Office: Days
• US Class Current: 435/69.700
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/15045 Special targeting system for viral vectors
• CPC Class Codes: C12N 2840/203 having an IRES

Abstract

Disclosed herein are methods and compositions for targeted integration of one or more copies of a sequence of interest using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain and integrase defective lentiviral donor constructs.

Claims

1. An integrase-defective lentiviral (IDLV) donor polynucleotide comprising, between the lentivirus LTRs,

(i) first and second nucleotide sequences that are homologous sequences to a region of interest of cellular chromatin of a cell;

and

(ii) an exogenous sequence.

2. The IDLV donor polynucleotide of claim 1, further comprising a sequence encoding a first selectable or screening marker.

3. The IDLV donor polynucleotide of claim 2, wherein the first selectable or screening marker is not between the first and second nucleotide sequences.

4. The IDLV donor polynucleotide of claim 2, further comprising a sequence encoding a second selectable or screening marker.

5. The IDLV donor polynucleotide of claim 4, wherein the second selectable or screening marker is flanked by the first and second nucleotide sequences.

6. The IDLV donor polynucleotide of claim 4, wherein the first and/or second selectable marker is a positive selection marker.

7. The IDLV donor polynucleotide of claim 4, wherein the first and/or second selectable marker is a negative selection marker.

8. The IDLV donor polynucleotide of claim 4, wherein the first and/or second selectable marker is a positive and a negative selection marker.

9. The IDLV donor polynucleotide of claim 8, wherein the positive-negative selection marker is hyg-TK.

10. The IDLV donor polynucleotide of claim 1, wherein the cell is eukaryotic cell.

11. The IDLV donor polynucleotide of claim 10, wherein the cell is a mammalian cell.

12. The IDLV donor polynucleotide of claim 11, wherein the cell is a human cell.

13. The IDLV donor polynucleotide of claim 1, wherein the first and second nucleotides flank the exogenous sequence.

14. A method for controlled, site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, the method comprising:

(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first zinc finger binding domain and a first cleavage half-domain, wherein the first zinc finger binding domain has been engineered to bind to a first target site in a region of interest in the genome of the cell;

(b) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain and a second cleavage half domain, wherein the second zinc finger binding domain binds to a second target site in the region of interest in the genome of the cell, wherein the second target site is different from the first target site; and

(c) contacting the cell with any of the IDLV donor polynucleotides of claim 1;

wherein binding of the first fusion protein to the first target site, and binding of the second fusion protein to the second target site, positions the cleavage half-domains such that the genome of the cell is cleaved in the region of interest, thereby resulting in integration of one or more copies of the exogenous sequence into the genome of the cell in the region of interest.

15. The method according to claim 14, wherein the exogenous nucleic acid sequence comprises a cDNA.

16. The method of claim 14, wherein the region of interest is in a region of the genome that is not essential for viability.

17. The method of any claim 14, wherein the region of interest is in a region of the genome that is transcriptionally active.

18. The method of claim 14, wherein a single copy of the exogenous sequence is integrated into the genome of the cell.

19. The method of claim 14, wherein multiple copies of the exogenous sequence are integrated into the genome of the cell.

20. The method according to claim 14, wherein at least one of the fusion proteins comprises an alteration in the amino acid sequence of the dimerization interface of the cleavage half-domain.

21. The method according to claim 14, wherein the cell is a mammalian cell.

22. The method according to 21, wherein the cell is a human cell.