1054. Bio-Distribution Studies of Lentiviral Vectors Injected Intravenously by Dynamic Bioluminescence Imaging Analyses: Development of APC-Transcriptionally Targeted Vectors for Genetic Vaccination Purposes
Authors: Takahiro Kimura, Richard C. Koya, Nori Kasahara, Renata Stripecke
Journal: Molecular Therapy (2005)

Intravenous administration of high titer VSV-G pseudotyped lentiviral vectors (LVs) leads to conspicuous gene transfer into liver, spleen and bone marrow, as previously shown by PCR, immunohistochemistry (IHC) and flow cytometry analyses. Interestingly, antigen presenting cells (APCs) corresponding to dendritic cells (DCs) and hematopoietic precursor cells homing in these tissues seem to be main target cell populations for gene delivery and expression. These results correlate with several reports showing that systemic administration of LVs expressing melanoma-associated antigens induced protective anti-tumoral immune responses. Thus, LVs are currently being explored as tools for genetic immunization, which warrants further understanding regarding the mechanisms of in vivo bio-distribution of these vectors. Furthermore, one of the logical advances toward the safety of LVs for vaccine development is the design of tissue specific promoters to restrict transgene expression APCs. In this study, we aim to characterize transcriptionally targeted LVs in vivo using bioluminescence imaging analyses, which allows to assess dynamically the spatio-temporal pattern of gene expression. Thus, nude mice were initially injected intravenously with a single dose (6 mcg of p24 equivalent) of the LV containing the CMV promoter driving transcription of firefly luciferase. After injecting mice with the luciferin substrate, in vivo whole-body optical imaging was performed using an IVIS bioluminescence optical imaging system at different time points, ranging from 3 days to 48 days post injection. Notably, during the first week the bioluminescence signal was more localized in the liver and spleen, but it gradually it dispersed towards the bones. We hypothesize that the initial gene expression pattern in the liver and spleen is due to their first-pass effect and immunologic sentinel functions, respectively, and that the subsequent shift of expression towards the bones is due to the gene expression by hematopoietic precursors present in the bone marrow that have self-renewal capacity and are able to sustain persistent LV expression. In order to explore and optimize this potential natural tropism of LVs to DCs and hematopoietic precursors for genetic immunization purposes, we are now testing LVs containing regulated promoters to restrict transgene expression in mature APCs. A LV containing the HLA-DRalpha promoter previously shown to confer high specificity of expression in MHCII+ DCs (Cui, Y., et al., Blood, 2002. 99(2): p. 399|[ndash]|408) is currently being evaluated for biodistribution through bioluminescence imaging analyses. In addition, we have generated a LV containing the human CD83 promoter (kindly provided by Dr. A. Steinkasserer), which successfully conferred up-regulation of transgene expression in DCs in vitro upon maturation with LPS or soluble CD40L. With these tools in hands and using a combination of bioluminescence imaging analyses, IHC and flow cytometry analyses, we seek to evaluate the efficacy and biosafety of these APC-transcriptionally targeted LVs for future vaccine development.