Specialized Resource B- Gene Transfer and Cell Engineering (SR-B). SR-B of ICMIC-3 will support preclinical and clinical gene transfer studies in RP1. It also provides support for engineering tumor cell lines for studies by RP2 and RP4. These projects are highly dependent on achieving efficient gene transfer in either primary T lymphocytes, packaging cell lines or tumor cell lines. In research and pre-clinical studies, the SR-B assists Investigators in the; 1. Construction of recombinant oncoretroviral and lentiviral vectors 2. Transfection or transduction of vector in packaging cells and selection of producer cell lines 3. Characterization of vector transmission and stability 4. Expansion of 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 viral stocks and transduced target cells 7. Detection of oncoretroviral and lentiviral vector integration sites by LM-PCR 8. Optimization of tumor cell lines and T lymphocyte transduction with retroviral vectors 9. Optimization of lymphocyte expansion ex vivo with Dynabeads ClinExvivoCD3/CD28. In the clinical phase, the SR-B will carry out and/or coordinate the: 1. Generation of high-titer producer cell clones, master cell banks (MCB) and retroviral stocks for clinical studies 2. Production of clinical viral stocks 3. Expansion and transduction of patient cells in semi-closed systems in collaboration with the investigators of the clinical trial 4. Detection of RCR and other biosafety testing in cultured packaging cell clones (MCB), viral stocks and clinical specimens 5. Characterization of the expression of the transgene (imaging reporters or PSMA-CAR) by either RT-PCR or flow cytometry analysis in transduced patient cells 6. 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.
This Specialized Resource will provide services and expertise in gene transfer, molecular analysis and cellular processes to Research Projects 1, 2 and 4. Through SR-B, the centralized generation of high-quality producer cell lines, of viral stocks and the development of procedures to efficiently engineer tumor cell lines and transduce patients T cells will permit standardization of procedures and facilitate the exchange of information, reagents and expertise between all the projects. It will also be cost effective.
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