Studies of retroviral vectors for in utero gene transfer and investigation of calcium-mediated gene regulation by human T-lymphotropic virus type-1.

摘要

Retrovirus-derived vectors provide efficient means of gene transfer and are potential excellent tools for therapeutic intervention against congenital or inherited disorders by in utero gene therapy and for gene transfer into dividing cells both in vitro and in vivo. To improve biodistribution of retroviral gene transfer vectors and to obtain efficient gene transfer towards hematopoietic stem cells via pre-natal administration, we performed in utero administration of retroviral vectors pseudotyped with different retroviral and non-retroviral envelope glycoproteins. RD114 envelope enhanced the gene transfer ability of retroviral vectors to peripheral blood, thymus, kidney and brain, while amphotropic envelope pseudotyped vectors had significantly higher gene transfer to thymus, spleen, kidney, brain and gonads. The ability of retroviral vectors to transduce myeloid progenitors was highest for ecotropic envelope pseudotype followed by amphotropic, RD114, VSV-G, GALV and rabies. Gene transfer to erythroid progenitors was more efficient with ecotropic pseudotype followed by RD114, VSV-G, GALV, amphotropic and rabies. Although there was a significant reduction in the percentage of colonies containing the transgene 3 months postnatally irrespective of the pseudotype, high level (40%--58%) of gene expression was observed in both erythroid and myeloid colonies. We then used HIV-1 derived lentiviral vectors to investigate the role of human T-lymphotropic virus type-1 (HTLV-1) accessory protein p12I in viral pathogenesis.; HTLV-1 is the etiologic agent of adult T-cell leukemia/lymphoma, an aggressive CD4+ T-lymphocyte malignancy. Activation of T-lymphocytes is required for effective infectivity, but the molecular mechanisms involved in HTLV-1 mediated T-cell activation remain unclear. HTLV-1 encodes various accessory proteins from the pX region of the genome such as p12I. We have earlier demonstrated that p12I localizes in the endoplasmic reticulum, increases intracellular calcium, activates n&barbelow;uclear f&barbelow;actor of a&barbelow;ctivated T&barbelow; cells-mediated transcription and is critical for HTLV-1 infectivity in vivo and in vitro. To further elucidate the role of p12I in regulation of cellular gene expression, we performed gene array analysis on stable p12I expressing Jurkat T-cells. (Abstract shortened by UMI.)