Methods and compositions for regulation of zinc finger protein expression
The present disclosure is in the field of genome engineering, particularly regulating targeted modification of the genome.
1. A construct comprising a polynucleotide encoding at least one component of a non-naturally occurring gene modulator that binds to an intended target site, wherein the polynucleotide is operably linked to a heterologous promoter comprising 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 modulator 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 pharmaceutical composition comprising a construct according to claim 1 .
17. A pharmaceutical composition comprising a cell according to claim 13 .
18. A kit comprising the construct of claim 1 .
19. The kit of claim 17 , further comprising a donor molecule and instructions.
20. A method of regulating expression of an exogenous gene modulator introduced into a cell, the method 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.
21. The method of claim 20 , wherein the cell is a mammalian or plant cell.
22. The method of claim 20 , wherein the cell is a stem cell.