METHODS AND COMPOSITIONS FOR REGULATION OF ZINC FINGER PROTEIN EXPRESSION

• US 20150267205A1
• Filed: 03/18/2015
• Published: 09/24/2015
• Est. Priority Date: 03/18/2014
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Gregory, Philip D., Zhang, H. Steve, Froelich, Steven
• Granted Patent: US 9,624,498 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 15/86 Viral vectors
• CPC Class Codes: C12N 2830/60 from viruses

Abstract

The present disclosure is in the field of genome engineering, particularly regulating targeted modification of the genome.

Claims

1. A construct comprising a polynucleotide encoding at least one component of a gene modulator that binds to an intended target site, wherein the polynucleotide is operably linked to a self-regulating target site bound by the gene modulator and further wherein binding of the gene modulator to the self-regulating target sequence modulates expression of the polynucleotide.

2. The construct of claim 1, wherein the intended target site is in the genome of a cell.

3. The construct of claim 1, wherein expression of the polynucleotide is decreased upon binding of the gene modulator to the self-regulating target sequence.

4. The construct of claim 1, wherein the self-regulating target site differs from the sequence of the intended target site by at least one base pair.

5. The construct of claim 1, wherein the construct comprises a control element and the self-regulating target site is within the control element.

6. The construct of claim 1, wherein the construct is a viral construct.

7. The construct of claim 6, wherein the viral construct is a lentivirus (LV), an integration defective lentiviral vector (IDLV), an adenovirus or an AAV construct.

8. The construct of claim 1, wherein the gene modulator is a transcription factor or a nuclease.

9. The construct of claim 8, wherein the transcription factor or the nuclease comprises a zinc finger protein, a TALE protein or a CRISPR/Cas system.

10. The construct of claim 8, further comprising a donor nucleic acid.

11. The construct of a claim 1, further comprising at least one additional polypeptide-encoding sequence operably linked to the sequence encoding the gene regulator and the self-regulating target site.

12. The construct of claim 11, wherein the at least one additional polypeptide-encoding sequence is separated from the sequence encoding the gene regulator by a 2A self-cleaving peptide sequence or an IRES site.

13. A cell comprising the construct of claim 1.

14. The cell of claim 13, wherein the gene modulator comprises a nuclease and further wherein the cell comprises a donor nucleic acid that is integrated into the genome of the cell.

15. The cell of claim 14, wherein the donor nucleic acid is in an adeno-associated viral vector (AAV) or is encoded by a plasmid DNA.

16. A method of regulating expression of an exogenous gene modulator introduced into a cell, the methods comprising introducing a construct according to claim 1 into a cell, wherein expression of the gene modulator is modulated upon binding of the gene modulator to the self-regulating target site.

17. The method of claim 16, wherein the cell is a mammalian or plant cell.

18. The method of claim 16, wherein the cell is a stem cell.

19. A pharmaceutical composition comprising a construct according to claim 1.

20. A pharmaceutical composition comprising a cell according to claim 13.

21. A kit comprising the construct of claim 1.

22. The kit of claim 20, further comprising a donor molecule and instructions.