Introduction Of Drug Resistance Genes Mediated By Retrovirus To Hematopoietic Progenitor Cells For Chemoprotection

High-dose chemotherapy promised to be an effective approach to improve therapeutic results in a variety of human tumors. Myelosuppression induced by antitumor agents is one of the major side effects that limits the effectiveness of the treatment, a problem that has only partially been solved by the use of hematopoietic growth factors. In fact, presently available growth factors alleviate, but do not prevent, drug梚nduced myelotoxicity, particularly in those situations in which the bone marrow reserve is inadequate because of previous treatment with myelotoxic agents. Introdction of genes into hematopoietic precursors that confer protection against the hematotoxicity of ant itumor agents has been proposed to reduce myelosuppress ion.1.A retroviral bicistronic strategy for efficient coexpression of classl aldehyde dehydrogenase(ALDH1) and multidrug resistance gene(MDR1)Objective:To elucidate the feasibility of coexpression of human ALDH1 and MDR1 by a retroviral bicistronic vector containing an internal ribosome entry site (IRES). Methods: The constructed retroviral MDR1 (GiNa-AIM) was transduced into amphotropic packaging cells PA317 by electroporation. Selection was performed by using l5ng/ml vincristine. The titer of retrovirus was elevated by ping-pong transduction. K562 cells was transfected with retroviral producing cell supernatant, the integration of the construct, expression of transgenes and efficiency of transfection were determined by polymerase chain reaction (PCR) Southern blot, MTT assay, flow cytornetry (FCM), and colony forming assay, respectively. Results: The retroviral titer reached 1.0 ×10 5cfu/ml in the supernatant of the producer cells PA317/ AIM after cross-infection with ecotropic GP+E86 cells. The transfected K562 cells exhibited a 3-10-fold increase of resistance to 4-hydroperoxycyclophosphamide (4-HC) and vincristin (VCR), 62% of which expressed P-gp as shown by FCM. The integration of both ALDH1 and MDR1 was confirmed by PCR and Southern blot analysis. Conclusion: The IRES containing bicistronic retroviral vector allows the functional coexpression of two different kinds of drug resistance genes. This strategy can be applicable to transfer any combination of drug resistance genes and dominantly select transfectant cells in vivo.2.The Supportive Effect of Primarily Cultured Bone MarrowStromal Layers on Retroviral-mediated Transduction of HumanHematopoietic Stem/Progenitor CellsObjective:To elucidate the effect of established primarily cultured bone marrow stromal layers on the gene transduction of human hematopoietic stem/progenitor cells(HSC/HPC). Methods:Mononuclear cells (MNC) from adult bone marrow were isolated by centrifugation on ficoll-hypaque gradients and plated in6stromal culture medium .The cells were incubated until passage no.4 to establish primary stromal layers. The HSC/HPC prestimulated by cytokines were transduced by retroviral supernatant containing MDR1 gene in presence of irradiated strorna-contact support. Transduced cells were plated in a colony-forming assay with and without vincristine(VCR) to assess the extent of transduction. Individual colonies were also analyzed by polymerase chain reaction (PCR) for the presence of provirus. Results: The mixed adherent cell layers were formed when adult bone marrow stromal cells were incubated for four to six weeks, mainly being composed of fibroblasts. In the presence of stroma-contact support, the average of gene transduction efficiency in mobilized peripheral blood and marrow-derived progenitors increased 1. 3-fold to 3. 3-fold measured by colony forming assay and/or PCR, especially higher in marrow progenitors. Conclusion: The presence of bone marrow stroma support in combination with cytokine facilitates augmenting the extent of retroviral-mediated gene transduction.3.Effect of Multidrug Resistance Gene(MDR1) Modulated Bone Marrow Cells on Hematopoietic Reconstitution and ChemoprotectionObjective:To investigate the effect of multi...