Construction and analysis of recombinant AAV-Epo vectors.

(A) Diagrammatic representation of recombinant AAV vectors used for this study. The AAV-CMVEpo and AAV-HREEpo virus vectors differ only in the nature of the promoter sequence. ITR indicates AAV-2–inverted terminal repeats; CMV, immediate/early promoter enhancer elements from CMV; HRE, hypoxia responsive promoter; mEpo, murine erythropoietin; SV40 (pA), polyadenylation signal from the SV40 virus; stuffer DNA, fragment of the 3′ β-galactosidase gene to ensure genome size is larger than 4 kb. The configuration of the OBHRE promoter is boxed below: the PGK HRE trimer encompassing −307/−290 of murine PGK is shown in bold, the core HIF-1 consensus binding site is underlined, and the minimal SV40 promoter is shown in gray. (B) Proliferation of splenocytes incubated with supernatants from HT1080 cells transfected with pCMV/HRE-Epo plasmids. The assay shows that the increased proliferation of the splenocyte cells when exposed to the supernatant from the pHRE-Epo–transfected cells exposed to hypoxia. Negative control represents untreated cells. The rhEpo is the recombinant human Epo positive control used in this assay at a concentration of 0.5 U/mL. Data are the mean relative light units per second ± SD of 3 samples. (C) Hypoxia-regulated Epo expression is maintained in a rAAV vector. T47D cells were transduced with rAAV vectors, AAV-CMVEpo, and AAV-HREEpo at a multiplicity of infection (MOI) of 10. Supernatants were harvested 1 day (gray bars) and 4 days (white bars) after hypoxia treatment and were analyzed in an Epo ELISA assay to show the hypoxia-mediated expression of Epo was transient. Data are the mean mIU/mL Epo values ± SD of 3 samples.