|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
To develop and improve gene therapy vectos and gene transfer protocols targeting hematopoietic cells, it is essential to accurately assess gene transfer into the targets such as hematopoietic stem cells and to quantitatively track transgene expression in vivo. For this aim, we constructed a retrovirus vector which enables cell marking with green fluorescent protein (GFP) and introducing a therapeutic gene simultaneously.
In the present study, we constructed a bicistronic retrovirus vector containing the human CD24 gene as the first cistron and the picornavirus-derived internal ribosome entry site (IRES)-controlled the enhanced GFP (EGFP) gene as the second cistron (MSCV/CD24-IRES-EGFP). When we transduced Ba/F3 murine pre-B cell line with MSCV/CD24-IRES-EGFP, expression of CD24 and EGFP was stably sustained for more than 6 months without selection. This vector also transduced primary murine bone marrow cells successfully. CD24/EGFP expression was confirmed in the bone marrow cells just after .ex vivo manipulation, as well as erythroid and granulocyte colonies derived from the infected bone marrow. Above all, MSCV/CD24-IRF,S-EGEP transduced long-term repopulating murine hematopoletic stem cells. MSCV/CD24-IRES-EGFP-transduced bone marrow cells were transplanted into lethally irradiated recipient mice, and transgene expression was monitored. Stable expression of CD24 and EGEP was demonstrated in the peripheral blood for 6 months, and in the secondary recipients for another 6 months. Detailed analysis of the lymphohematopoietic tissues (bone marrow, peripheral blood, thymus and spleen) in the primary recipients revealed normal hematopoietic differentiation in all the examined lineages (B- and T-lymphoid, erythroid, granulocytic and monocytic). Taken together, MSCV/CD24-JRES-EGFP vector successfully transduded tine murine heinatopoietic stem cells capable of self-renewal and multilineage differentiation.