476. In Vivo Repopulation of Syngenic Hematopoietic Cells Expressing GFP Transfected by Recombinant Sendai Virus–Toward Development of ‘Cytoplasmic Gene Therapy’ for Hematopoietic Disorders
Authors: Kumi Yoshida, Yoshikazu Yonemitsu, Mamoru Haswgawa, Katsuo Sueishi
Journal: Molecular Therapy (2006)

Gene therapy targeting hematopoietic cells is a hopeful strategy to treat inherited hematopoietic disorders. Recent success in clinical gene therapy for X-linked SCID using a retoroviral vector expressing common gamma-chain encouraged physicians, patients and scientists; however serious adverse event, namely T-cell leukemia, related to the insertion of pro-viral genome into the host chromosome mediated by retroviral vector is a hazard to development the gene therapy strategies in this field. We have developed a novel RNA viral vector for efficient gene transfer, namely recombinant Sendai virus (SeV). Essential features of SeV is the cytoplasmic transcription, as well as highly efficient gene transfer to hematopoietic stem-derived from human cord blood preserving their functions to differentiate into the entire hematopoiesis series (Jin C-H et al. Gene Ther 2003).The aim of this study was investigate to dynamic states of reconstituting blood system in syngenic bone marrow transplantation (BMT) mediated by new type of SeV, temperature sensitive mutant SeV deficient to F-gene (tsSeV/dF).Crude bone marrow (BM) cells from female C57BL/6 mice were transferred GFP via SeV/dF or tsSeV/dF, and showed highly efficient gene transfer in hematopoietic stem cells, common myeloid progenitors and common lymphoid progenitors of whole bone marrows (80% in each population). In vivo transplantation of the BM after T-cell depletion to lethal irradiated syngenic recipient mice demonstrated highly efficient repopulation of GFP-positive cells in peripheral blood, spleen, bone marrow, and thymus in use of tsSeV/dF, but not of SeV/dF, at 4 weeks in approximately 50 % of mice (GFP-high group). In these mice, GFP-expressing donor cells exhibited repopulation to all blood lineages, including T cells, in recipient mice; these animals, however, died at 5-8 weeks after transplantation due to pancytopenia. On the other hand, the residual mice demonstrated gradual decrease of the number of GFP-positive cells in peripheral blood, and lived without any serious adverse event (GFP-low group). Neither treatment with FK506 nor similar experiment using BALBc nu/nu mice could rescue the number of GFP-positive cells, suggesting that CTL may not be a significant effector to the repopulating cells.To our best knowledge, this study is first report reconstituting whole hematopoietic series using bone marrow cells gene transferred by a vector with a cytoplasmic transcription system. This could be achieved by tsSeV/dF, but not SeV/dF, suggesting that reduced expression of membranous proteins, probably HN protein, might be critical for repopulation of hematopoietic cells. Further studies, however, assessing the mechanisms regarding the serious adverse event, namely pancytopenia, should be required to overcome the current hazard to development of gene therapy targeting hematopoietic cells using tsSeV. Gene therapy targeting hematopoietic cells is a hopeful strategy to treat inherited hematopoietic disorders. Recent success in clinical gene therapy for X-linked SCID using a retoroviral vector expressing common gamma-chain encouraged physicians, patients and scientists; however serious adverse event, namely T-cell leukemia, related to the insertion of pro-viral genome into the host chromosome mediated by retroviral vector is a hazard to development the gene therapy strategies in this field. We have developed a novel RNA viral vector for efficient gene transfer, namely recombinant Sendai virus (SeV). Essential features of SeV is the cytoplasmic transcription, as well as highly efficient gene transfer to hematopoietic stem-derived from human cord blood preserving their functions to differentiate into the entire hematopoiesis series (Jin C-H et al. Gene Ther 2003). The aim of this study was investigate to dynamic states of reconstituting blood system in syngenic bone marrow transplantation (BMT) mediated by new type of SeV, temperature sensitive mutant SeV deficient to F-gene (tsSeV/dF). Crude bone marrow (BM) cells from female C57BL/6 mice were transferred GFP via SeV/dF or tsSeV/dF, and showed highly efficient gene transfer in hematopoietic stem cells, common myeloid progenitors and common lymphoid progenitors of whole bone marrows (80% in each population). In vivo transplantation of the BM after T-cell depletion to lethal irradiated syngenic recipient mice demonstrated highly efficient repopulation of GFP-positive cells in peripheral blood, spleen, bone marrow, and thymus in use of tsSeV/dF, but not of SeV/dF, at 4 weeks in approximately 50 % of mice (GFP-high group). In these mice, GFP-expressing donor cells exhibited repopulation to all blood lineages, including T cells, in recipient mice; these animals, however, died at 5-8 weeks after transplantation due to pancytopenia. On the other hand, the residual mice demonstrated gradual decrease of the number of GFP-positive cells in peripheral blood, and lived without any serious adverse event (GFP-low group). Neither treatment with FK506 nor similar experiment using BALBc nu/nu mice could rescue the number of GFP-positive cells, suggesting that CTL may not be a significant effector to the repopulating cells. To our best knowledge, this study is first report reconstituting whole hematopoietic series using bone marrow cells gene transferred by a vector with a cytoplasmic transcription system. This could be achieved by tsSeV/dF, but not SeV/dF, suggesting that reduced expression of membranous proteins, probably HN protein, might be critical for repopulation of hematopoietic cells. Further studies, however, assessing the mechanisms regarding the serious adverse event, namely pancytopenia, should be required to overcome the current hazard to development of gene therapy targeting hematopoietic cells using tsSeV.