title: Tightly regulated long-term erythropoietin expression in vivo using tet-inducible recombinant adeno-associated viral vectors.
abstract: Recombinant adeno-associated viral (rAAV) vectors containing an improved tetracycline (tet) system of transcriptional regulation are an efficient strategy for the control of long-term therapeutic gene expression. In vivo studies with the original tet-off and tet-on vectors, while promising, have failed to demonstrate complete repression in the uninduced state. To address this issue, we incorporated the tTS(kid) fusion of the tet repressor and a KRAB-derived transcriptional silencer into the tet-on system in the context of rAAV vectors. The tTS(kid) repressor and rtTA activator were expressed constituitively from a regulator vector, and the repressor and an erythropoietin (Epo) transgene were expressed inducibly via a second vector. Following intramuscular co-injection of these vectors, we observed repeated induction of serum Epo protein following drug administration and undetectable levels after its withdrawal. Four cycles of regulation were achieved over a 32-week period. Thus, the tet-on system plus the tTS(kid) repressor delivered via nonpathogenic rAAV vectors is a powerful tool for controlling the in vivo expression of therapeutic transgenes. In a clinical setting, the repressor could provide a mechanism for abolishing transgene expression if it were no longer needed or if the safety of a patient became compromised.
authors.affiliation: Department of Pharmacology, Chiron Corporation, Emeryville, CA 94608, USA. katherine_rendahl@cc.chiron.com
authors.forename: Katherine G
authors.initials: KG
authors.lastname: Rendahl
authors.forename: Dulce
authors.initials: D
authors.lastname: Quiroz
authors.forename: Martha
authors.initials: M
authors.lastname: Ladner
authors.forename: Mazie
authors.initials: M
authors.lastname: Coyne
authors.forename: Jon
authors.initials: J
authors.lastname: Seltzer
authors.forename: William C
authors.initials: WC
authors.lastname: Manning
authors.forename: Jaime A
authors.initials: JA
authors.lastname: Escobedo
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000187:drug effects / Q000235:genetics*; D004921:Erythropoietin / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors; D006400:Hematocrit; D051379:Mice; D008810:Mice, Inbred C57BL; D009132:Muscles; D012097:Repressor Proteins; D013752:Tetracycline / Q000494:pharmacology; D019076:Transgenes
chemical_list: D012097:Repressor Proteins; D004921:Erythropoietin; D013752:Tetracycline