The Retroviral Core is designed to support gene transfer studies in Projects 1, 2 3 and 4. These projects are highly dependent on achieving efficient retroviral-mediated gene transfer into cells of hematopoietic origin, including primary T-lymphocytes, dendritic cells and CD34+ hematopoietic origin, including primary T-lymphocytes, dendritic cells and CD34= hematopoietic cells. The proposed studies all require well- characterized and periodically tested high-titer producers cell lines and cell free retroviral stocks, as well as detection of replication-competent retrovirus (RCR). The centralized generation of high-quality producer cell lines retroviral stocks and biosafety testing is cost effective, permits standardization of procedures, and facilitates the exchange of information and expertise between the four projects. In the pre-clinical phase of investigation, the specific aims of the Retroviral Core are: 1. construction of recombinant oncoretroviral of lentiviral vectors; 2. Transfection of vector DNA in packaging cells and selection of recombinant oncoretroviral or lentiviral vectors; 2. transfection of vector DNA in packaging cells and selection of producer cell lines; 3. characterization of vector transmission in standard cell lines; 4. expansion of independent clones and identification of the best clone for the intended target cells; 5. titration of cell-free viral stocks; 6. clones and identification of the best clone for the intended target cells. In the clinical phase of investigation, the specific aims of the Retroviral Core in the clinical phase are to carry out and/or coordinate: 1. the generation of high-titer producers, master cell banks and viral stocks for clinical studies; 2. the development of scaled up procedures for the production of 5 to 15 liter batches of clinical viral stocks in semi-closed systems; 3. the development of scaled up procedures for the transduction of lymphocytes, dendritic cells and CD34+ progenitor cells in semi- closed systems, in collaboration with the investigators of each clinical trials in projects 1, 2 and 3; 4. execute and quantify transduction of patient cells, in collaboration with the investigators of each clinical trial in projects 1, 2 and 3. Our ability to execute most of the production and transduction in the Retroviral Core will greatly decrease the cost of generating high-quality material and therefore the cost of clinical investigation. M. Sadelain, M.D., Ph.D. is the principal investigator of the Retroviral Core and I. Riviere, Ph.D. is the co-principal investigator. In the Gene Transfer and Cell Engineering Facility at MSKCC, they are currently assisted by two senior Research Assistants with considerable experience in cell culture, virology, and molecular techniques and by a Quality Assurance/Quality Control specialist, who will review and assess all quality control issues related to the manufacturing and the release of the clinical vector and patient products. 2.5 additional technical positions (FTE) will be needed for the development and execution of the clinical protocols fin the program project.
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