Vectors conditionally expressing protein
This invention relates to the field of therapeutics. Disclosed are methods of conditionally expressing erythropoietin under the control of an ecdysone receptor-based gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals. The methods of the invention cause an in vivo increase in the expression of erythropoietin and an increase in the hematocrit or volume percentage of red blood cells in blood after administration of the ligand.
1. A method of inducing, regulating, or enhancing erythropoietin (EPO) expression in a mammal, wherein the method comprises
(a) administering an adeno-associated virus to the mammal, wherein the virus comprises a polynucleotide encoding EPO; and
(b) administering an activator ligand which induces EPO expression from the virus polynucleotide encoding EPO,
wherein the adeno-associated virus is administered intramuscularly,
wherein the adeno-associated virus further comprises an ecdysone receptor (EcR)-based gene switch, wherein the gene switch comprises at least one transcription factor sequence operably linked to a first promoter, wherein at least one transcription factor encoded by the at least one transcription factor sequence is a ligand-dependent transcription factor,
wherein the adeno-associated virus further comprises a second promoter operably linked to the polynucleotide encoding EPO, wherein the second promoter is activated by the at least one ligand-dependent transcription factor following administration of activator ligand,
wherein EPO expression is increased in the mammal, and
wherein the hematocrit or volume percentage of red blood cells in blood is increased in the mammal by at least 25%, compared with the hematocrit or volume percentage of red blood cells prior to administration of ligand.
2. The method of claim 1 , wherein the mammal is human.
3. The method of claim 1 , wherein activator ligand is administered in a dose or doses sufficient to induce or maintain EPO expression levels within a normal physiologic range.
4. The method of claim 1 , wherein the polynucleotide encoding EPO comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6.
5. The method of claim 1 , wherein the polynucleotide encoding EPO comprises an amino acid sequence at least 95% identical to SEQ ID NO 6.
6. The method of claim 1 , wherein the polynucleotide encoding EPO comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8.
7. The method of claim 1 , wherein the polynucleotide encoding EPO comprises an amino acid sequence at least 95% identical to SEQ ID NO: 8.
8. The method of claim 1 , wherein the hematocrit or volume percentage of red blood cells in blood is increased in the mammal by at least 40%, compared with the hematocrit or volume percentage of red blood cells prior to administration of ligand.
9. The method of claim 1 , wherein the second promoter is a constitutive promoter.
10. The method of claim 1 , wherein the second promoter is a cytomegalovirus (CMV) promoter.
11. The method of claim 1 , wherein the second promoter is an elongation factor 1 alpha (EF1) promoter.
12. The method of claim 1 , wherein the gene switch is the RheoSwitch® gene switch.
13. The method of claim 1 , wherein the ligand is a diacylhydrazine ligand.
14. The method of claim 1 , wherein the ligand is selected from the group consisting of RG-115819, RG-115932 and RG-115830.
15. The method of claim 1 , wherein the ligand is RG-11593.
16. The method of claim 1 , wherein the ligand is administered at a dose of about 1 to about 50 mg/kg/day.
17. The method of claim 16 , wherein the ligand is administered at a dose of about 30 mg/kg/day.
18. The method of claim 1 , wherein about 1.0×10 9 to about 1×10 13 viral particles are administered to the mammal per cycle of vector administration.
19. The method of claim 18 , wherein about 1.0×10 11 viral particles are administered to the mammal per cycle of vector administration.
20. The method of claim 1 , wherein the adeno-associated virus is administered following a pre-administration of hyaluronidase.
21. The method of claim 1 , wherein the polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first therapeutic switch promoter and a second transcription factor sequence under the control of a second therapeutic switch promoter, wherein the proteins encoded by the first transcription factor sequence and the second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.
22. The method of claim 21 , wherein the first transcription factor sequence comprises a nucleic acid encoding for a VP-16 transactivation domain and a retinoic X receptor (RXR) protein.
23. The method of claim 22 , wherein the second transcription factor sequence comprises a nucleic acid encoding for a GAL-4 DNA binding domain and Ecdysone receptor (EcR) protein.
24. The method of claim 21 , wherein the first transcription factor sequence comprises a nucleic acid encoding for a fusion protein comprising the transcription activation domain of HSV-VP 16 and a chimeric RXR derived from human sequence and a locust migration ultraspiracle polypeptide (Lm USP).
25. The method of claim 24 , wherein the second transcription factor sequence comprises a nucleic acid encoding for a fusion protein comprising the DNA binding domain of yeast Gal4 and the DEF domains of the mutagenized ecdysone receptor from the insect Choristoneura fumiferana.
26. The method of claim 21 , wherein the both the first therapeutic switch promoter and the second therapeutic switch promoter are constitutive promoters.
27. The method of claim 21 , wherein the first therapeutic switch promoter is a CMV promoter.
28. The method of claim 21 , wherein the second therapeutic switch promoter is an EF1 promoter.