Makoto Otsu

Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia.

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1+/− mice had a significantly reduced number of hematopoietic stem cells (CD34−cKit+ScaI+Lin− cells or CD34−KSL cells) compared with Sf3b1+/+ mice, but hematopoiesis was grossly normal in Sf3b1+/− mice. When transplanted competitively with Sf3b1+/+ bone marrow cells, Sf3b1+/− stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1+/− mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

272. Development of |[ldquo]|Full|[rdquo]|-Closed Sterile and Automatic Retroviral Gene Transfer System for Clinical Gene Therapy

Manipulating large number of cultured cells for gene therapy has risk of microbiological contamination constantly. It would become more serious under the situation like re-infusion of such cells to the compromised host. Our clinical protocol, HSV-TK gene transfer into donor lymphocytes for control of allogeneic Graft-versus-leukemia and Graft-versus-host-disease (Japanese trial), is one of such sensitive protocol by meaning of immunological conditions of patients and manipulation steps. To avoid those problems we are developing manipulation protocol for retroviral gene transfer to human T lymphocytes using automatic cell processor CytoMateTM, magnetic cell selection system IsolexTM and CO2 permeable cell culture bag OpticytoTM. All of manipulations, except centrifugation for gene transfer, had been performed in full-closed sterile conditions. Function and sterility of final products, HSV-TK gene transferred lymphocytes, were evaluated by comparing with non-closed system, using centrifugation tubes and flasks.

330. Genetic Modification of Murine Embryonic Stem Cells by the Gene Silencing Resistance Retroviral Vector GCDsap

In the last ASGT meeting, we presented that the retroviral vector GCDsap with high resistance to gene silencing maintained expression of the transgene (EGFP) in mouse embryonic stem (ES) cells over 60 days and the expression was continued even after differentiation of the transduced ES cells into three germ layers of teratomas in NOD/SCID mice. In this study, to assess the ability of the vector to maintain the transgene expression through ontogeny, the transduced ES cells were injected into C57BL/6 mouse 3.5 days blastocytes and chimeric mice were generated. The birth ratio of chimeric mice determined by coat color was 67% (24 out of 36 mice) and 17 out of 24 mice (71%) highly expressed EGFP in their tissues, including hematopoietic cells, neurons, muscles, and intestinal tracts. Furthermore, such high expression of EGFP was transmitted through F1 (47/95 = 49%) to F2 mice (32/56 = 58%) generated by mating with C57BL/6 mice. Although the mean intensity of EGFP varied among the mice, some had 100% of EGFP expressing cells in their peripheral blood. Interestingly, a chimera mouse gave rise to F1 mice that were divided into three groups by the expression patterns in their peripheral blood. Southern blot analyses revealed that the chimeric mouse had 8 copies of proviruses integrated in its chromosomes and each F1 group showed an identical integration pattern of proviruses. This was applicable to F2 mice, in particular, F2 mice borne by F1 mice with the highest EGFP expression also showed high EGFP expression and their integration patterns were identical to that of the F1, suggesting that maintenance of the transgene expression was greatly influenced by the integration sites as well as vector structures. Taken together, it is likely that GCDsap provided a useful tool not only for future regenerative medicine, but also for analysis of epigenetic changes in chromosomes during gonogenesis.