High-efficiency retroviral vector mediated gene transfer into human peripheral blood CD4⁺ T lymphocytes

Genetic modification of peripheral blood T lymphocytes 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 quantify 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 (ΔLNGFR) was constructed. Transduced cells were then accurately enumerated with immunofluorescence staining and fluorescence activated cell sorting (FACS) 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.