993. Optical Bioluminescence and Immunologic Analyses of Lentiviral Vectors Administered In Vivo: Transcriptional Targeting through an MHCII Promoter Yields Persistent Transgene Expression and Lack of CTL Stimulation in Immunocompetent Mice Indicating Tolerance

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In vivo administration of lentiviral vectors is a promising novel approach for genetic vaccination and for gene replacement therapy. Bio-distribution studies have shown that i.v. systemic administration of lentiviral vectors produces conspicuous gene marking of antigen presenting cells (APCs) in the liver and spleen. Expression and presentation of immunogenic antigens by APCs may explain the potential of lentiviral vectors for vaccination and the poor persistent expression in immunocompetent animals. Based on this rationale, we attempted to improve the biosafety and immunization potential of lentiviral vectors by targeting the transgene expression to APCs through the Major Histocompatibility Complex class II (MHCII) promoter. We produced third generation self-inactivating vectors with the MHCII or with the CMV promoter as ubiquitous expression control. Ex vivo transduction of MHCII+ and MHCII- cell lines and primary cells using GFP and luciferase as reporter genes confirmed the expression and selectivity of vectors with the MHCII promoter. Nu/Nu nude and C57BL/6 mice (n=3 per group) were injected i.v. with 2 mcg p24 equivalent of vectors expressing firefly luciferase (LV-CMV-LUC or LV-MHCII-LUC) and were analyzed by optical imaging on weeks 1, 2, 4, 8, 12, 16 and 24 after injections. Statistical analyses used a linear regression model comparing the overall differences (bioluminescence signal detected in liver and spleen) between the LV-CMV-LUC and LV-MHCII-LUC injected groups in early (1-8 weeks) and late (8-24 weeks) periods. Nude mice injected with LV-CMV-LUC and LV-MHCII-LUC showed increase in bioluminescence signal during the first 8 weeks, which maintained stable for the late period. Notably, a significant drop in bioluminescence signal occurred for LV-CMV-LUC expression in C57BL/6 mice throughout the observation periods (slope= -21+/-8, p=0.0112), whereas for LV-MHCII-LUC a much lower drop was observed (slope= -8+/-7.4, p=0.2827) and the signal persisted for 24 weeks. Subcutaneous administration of vectors produced similar results. Additionally, we performed immunologic studies with vectors expressing the melanoma-associated antigen TRP2. I.v. vaccination of C57BL/6 mice with LV-CMV-TRP2 produced high frequencies of TRP-2 specific CTL responses (4.5% - 12.5% of circulating CD8+ cells, measured by flow cytometry after anti-TRP-2 tetramer staining or intracellular IFN-gamma staining) and significant (p=0.0365) protection against B16 melanoma challenge. Vaccination with LV-MHCII-TRP2 yielded baseline results comparable to the placebo vaccination group. In summary, these results indicate that transgene expression regulated through the MHCII promoter may induce a tolerogenic effect that would be unsuitable for use in vaccination strategies, but might allow more persistent expression in gene replacement therapy.