Fig. 4.

Evaluation of rAAV-CMV-EGFP-HSV1-TK and rAAV-CMV-HSV1-TK in vitro. a Diagrammatic representation of rAAV6.2 expressing HSV1-TK. rAAV-CMV-EGFP-HSV1-TK (left) and rAAV-CMV-HSV1-TK (right) plasmids were constructed as described (Methods). rAAV6.2 expressing the respective plasmids was produced by the Horae Gene Therapy Center. b Detection of EGFP and HSV1-TK proteins by immunoblot. rAAV6.2 bearing EGFP-HSV1-TK (top) or HSV1-TK alone (bottom) versus no virus control was used to infect the indicated B-cell lines as listed in the right margin GAPDH was used as a loading control. EGFP and HSV1-TK were coordinately expressed (top) when rAAV6.2-EGFP-TK was transduced. The pattern of TK1 expression was unchanged when rAAV6.2TK1 alone was transduced. The quantity of EGFP protein detected in each line by immunoblot paralleled the relative fluorescence of the same lines (Figs. 1, 2), highlighting the reproducibility of rAAV6.2 transduction. c Functionality of rAAV6.2 encoding HSV1-TK. 143B TK cells transduced with either rAAV6.2 HSV1-TK or rAAV6.2 EGFP-HSV1-TK (top panels) were visualized by confocal microscopy. Cells were next incubated with 10uM ganciclovir for 72 h (bottom panels) and again visualized. The bottom left panel shows that only rounded and dying cells remain. The bottom right panel confirms that virtually all fluorescent cells have been eliminated. d Loss of viability among rAAV6.2 HSV1-TK transduced cells incubated with ganciclovir. Representative B-cell lines and 143B TK-cells (control) were transduced with rAAV6.2-HSV1-TK. Twenty-four hours later cells were incubated with 10uM ganciclovir for 72 h at which time an MTT-based assay was performed to measure cell viability. Results are displayed as percent survival (y-axis) of representative B-cell lines (x-axis). Inverse correlation of viability with percentage fluorescence of the identical cell lines displayed in Figs. 1, 2 underscores the reproducibility of rAAV6.2 transduction of specific B-cell lines