The Gene Transfer and GMP resource (GTGR) supports gene transfer studies in Projects 1 and 2. These projects are highly dependent on achieving efficient gene transfer in primary T lymphocytes. In pre-clinical studies, the GTGR assists or advises Investigators on the following: 1. Construction of recombinant oncoretroviral- and lentiviral- vectors 2. Transfection of vector DMA in packaging cells and selection of producer cell lines 3. Characterization of vector transmission and stability 4. Expansion of independent packaging cell clones and identification of the best clone for the intended target cells 5. Titration of cell-free retroviral stocks 6. Detection of replication-competent retrovirus in cell-free viral stocks and transduced target cells 7. Detection of oncoretroviral and lentiviral vector integration sites by LM-PCR 8. Optimization of T lymphocyte transduction with oncoretroviral and lentiviral vectors 9. Optimization of lymphocyte expansion ex vivo with XcyteTM Dynabeads? or AAPCs that express costimulatory molecules in conjunction with an antigen. In the clinical phase, the specific aims of the GTGR are to carry, out and/or coordinate: 1. the generation of high-titer producer cell clones, master cell banks (MCB) and retroviral stocks for clinical studies 2. the production of clinical viral stocks in semi-closed systems 3. the expansion and transduction of patient cells in semi-closed systems in collaboration with the investigators of each clinical trial 4. the detection of RCR and other biosafety testing in cultured packaging cell clones (MCB), viral stocks and clinical specimens 5. the characterization of the expression of the transgene (L-NGFR or PSMA-CAR) by flow cytometry analysis in transduced patient cells 6. the determination of the vector copy number by Real time PCR in the peripheral blood, bone marrow samples and available tissues of the patients at different time points after infusion of the genetically modified T cells. Vector production and patient T cell transduction in the GTGR decreases the cost of clinical development, ensures high quality and consistency of molecular and cellular processes, and their availability to all - investigators in the program.
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