Abstract
Genetic modification of peripheral blood T lymphocytes has been proposed as a therapeutic strategy for treating congenital disorders, cancer and viral diseases. Central to all T lymphocyte-based gene therapy strategies is the ability to efficiently and stably deliver genes into primary T lymphocytes. In this study, we sought to increase the gene transfer efficiency in CD4+ peripheral blood T lymphocytes using procedures which could be utilized in clinical applications. In order to quantity the gene transfer efficiency in primary CD4+ T cells, a high-titer retroviral vector which efficiently expresses a truncated version of the human low-affinity nerve growth factor receptor (delta LNGFR) was constructed. Transduced cells were then accurately enumerated with immunofluorescence staining and fluorescence activated cell sorting (FACS) analysis and rapidly isolated at high purity for further analysis. Using this system, a supernatant-based gene transfer procedure was developed which routinely yields gene transfer efficiencies of 25-40% into a wide repertoire of both freshly obtained and cryopreserved peripheral blood CD4+ T lymphocytes.
