Targeted integration and expression of exogenous nucleic acid sequences

• US 20070134796A1
• Filed: 07/26/2006
• Published: 06/14/2007
• Est. Priority Date: 07/26/2005
• Status: Abandoned Application

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Urnov, Fyodor, Rebar, Edward, Gregory, Philip, Brennan, Sean, Holmes, Michael
• Application Number:
  US11/493,423
• Publication Number:
  US 20070134796A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for targeted integration of a exogenous sequence into a predetermined target site in a genome for use, for example, in protein expression and gene inactivation.

Claims

1. A method for expressing the product of an exogenous nucleic acid sequence in 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 a polynucleotide comprising an exogenous nucleic acid sequence and a first nucleotide sequence that is homologous to a first sequence in the region of interest;

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 the exogenous sequence into the genome of the cell in the region of interest and expression of the product of the exogenous sequence.

2. The method according to claim 1, wherein the polynucleotide further comprises a second nucleotide sequence that is homologous to a second sequence in the region of interest.

3. The method according to claim 2, wherein the first and second nucleotide sequences flank the exogenous sequence.

4. The method according to claim 1, wherein the region of interest is in a region of the genome that is transcriptionally active and not essential for viability.

5. The method according to claim 4, wherein the region of interest is the human homologue of the murine Rosa26 gene.

6. The method according to claim 4, wherein the region of interest is a CCR5 gene.

7. The method according to claim 1, wherein the first and second cleavage half-domains are from a Type IIS restriction endonuclease.

8. The method according to claim 1, wherein the region of interest comprises a gene.

9. The method according to claim 8, wherein the gene comprises a mutation.

10. The method according to claim 9, wherein the exogenous nucleic acid sequence comprises the wild-type sequence of the gene.

11. The method according to claim 9, wherein the exogenous nucleic acid sequence comprises a portion of the wild-type sequence of the gene.

12. The method according to claim 9, wherein the exogenous nucleic acid sequence comprises a cDNA copy of a transcription product of the gene.

13. The method according to claim 1, wherein the cell is arrested in the G2 phase of the cell cycle.

14. The method according to claim 1, 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.

15. A method for integrating an exogenous sequence into a region of interest in the genome of 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 a polynucleotide comprising an exogenous nucleic acid sequence;

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 the exogenous sequence into the genome of the cell in the region of interest.

16. The method according to claim 15, wherein the integration inactivates gene expression in the region of interest.

17. The method according to claim 15, wherein the exogenous nucleic acid sequence comprises a sequence of between 1 and 50 nucleotides in length.

18. The method according to claim 15, wherein the exogenous sequence comprises a cleavage enzyme recognition site.

19. The method according to claim 18, wherein the cleavage enzyme is a meganuclease.

20. The method according to claim 19, wherein the meganuclease is I-SceI.

21. The method according to claim 19, wherein the meganuclease has been engineered to bind a non-natural target site.

22. The method according to claim 15, wherein the first and second cleavage half-domains are from a Type IIS restriction endonuclease.

23. The method according to claim 15, wherein the cell is arrested in the G2 phase of the cell cycle.

24. The method according to claim 15, 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.