Linear donor constructs for targeted integration

• US 20090263900A1
• Filed: 04/13/2009
• Published: 10/22/2009
• Est. Priority Date: 04/14/2008
• Status: Abandoned Application

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Urnov, Fyodor, Holmes, Michael C., Gregory, Philip D., DeKelver, Russell
• Application Number:
  US12/386,059
• Publication Number:
  US 20090263900A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Disclosed herein are linear donor molecules comprising homology arms of 50-750 base pairs (e.g., 50-100 base pairs) flanking one or more sequences of interest. The donor molecules and/or compositions comprising these molecules can be used in methods for targeted integration of an exogenous sequence into a specified region of interest in the genome of a cell.

Claims

1. A linear donor nucleic acid molecule comprising homology arms of between 50 and 750 base pairs and a sequence of interest, wherein the homology arms flank the sequence of interest.

2. The linear donor nucleic acid of claim 1, wherein the homology arms are between 50 and 100 base pairs in length.

3. The linear donor nucleic acid of claim 1, wherein one or more of the base pairs of the homology arms are joined with a phosphorothioate phosphodiester bond.

4. The linear donor nucleic acid of claim 3, wherein the phosphorothioate phosphodiester bonds are positioned at the first and, optionally, second bonds of the 5′

and 3′

ends of the donor nucleic acid.

5. The linear donor nucleic acid of claim 1, further comprising, between the homology arms, a sequence encoding a 2A peptide.

6. The linear donor nucleic acid of claim 1, further comprising, between the homology arms, a sequence comprising an SA site.

7. The linear donor nucleic acid of claim 1, further comprising, between the homology arms, a sequence comprising an IRES sequence.

8. The linear donor nucleic acid of claim 1, wherein the sequence of interest does not encode a polypeptide.

9. The linear donor nucleic acid of claim 1, further comprising a promoter sequence operably linked to the sequence of interest.

10. The linear donor nucleic acid of claim 1, wherein the sequence of interest encodes a polypeptide.

11. The linear donor nucleic acid according to claim 10, wherein the polypeptide is selected from the group consisting of an antibody, an antigen, an enzyme, a growth factor, a cell surface receptor, a nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter gene, a selectable marker, a secreted factor, an epitope tag and functional fragments thereof and combinations thereof.

12. The linear donor nucleic acid of claim 1, wherein the sequence contains a non-coding nucleic acid.

13. The linear donor nucleic acid according to claim 12, wherein the non-coding nucleic acid is selected from the group consisting of a miRNA, and SH-RNA, or siRNA.

14. A method for homology-dependent targeted integration of a sequence of interest into a region of interest in the genome of the cell, the method comprising the steps of:

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

(b) contacting the cell with a donor polynucleotide of claim 1,

wherein binding of the fusion protein to the target site cleaves the genome of the cell in the region of the interest, thereby resulting in homology-dependent targeted integration of the sequence of interest into the genome of the cell.

15. A method for homology-dependent targeted integration of a sequence of interest into a cell, the method comprising:

(a) expressing a first fusion protein in the cell, the first fusion protein comprising a first DNA-binding domain and a first cleavage half-domain, wherein the first DNA-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 DNA-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 a polynucleotide comprising a donor nucleic acid according to 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 homology-dependent integration of the donor nucleic said into the genome of the cell.

16. The method of claim 14, wherein at least one DNA-binding domain is a zinc finger binding domain.

17. The method of claim 14, wherein at least one DNA-binding domain is a meganuclease DNA-binding domain.

18. The method of claim 14, wherein the sequence of interest from the integrated donor nucleic acid expresses a polypeptide.

19. The method of claim 14, wherein the sequence in interest from the integrated donor comprises a non-coding nucleic acid sequence.

20. The method of claim 14, wherein the cleavage domain is from a meganuclease.

21. The method of claim 14, wherein the first and second cleavage half-domains are from a Type IIS restriction endonuclease.

22. The method according to claim 21, wherein the Type IIS restriction endonuclease is selected from the group consisting of FokI and StsI.

23. The method according to claim 14, wherein the cell is arrested in the G2 phase of the cell cycle.

24. 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.

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

26. The method according to claim 14, wherein the cell is a human cell.

27. The method according to claim 14, wherein the cell is a plant cell.