HIGH-EFFICIENCY RETROVIRAL VECTOR-MEDIATED GENE-TRANSFER INTO HUMAN PERIPHERAL-BLOOD CD4(-LYMPHOCYTES() T)

Genetic modification of peripheral blood T lymphocytes has been propos ed as a therapeutic strategy for treating congenital disorders, cancer and viral diseases. Central to all T lymphocyte-based gene therapy st rategies is the ability to efficiently and stably deliver genes into p rimary T lymphocytes. In this study, we sought to increase the gene tr ansfer efficiency in CD4(+) peripheral blood T lymphocytes using proce dures which could be utilized in clinical applications. In order to qu antify the gene transfer efficiently in primary CD4(+) T cell, a high- titer retroviral vector which efficiently expresses a truncated versio n of the human low-affinity nerve growth factor receptor (Delta LNGFR) was constructed. Transduced cells were then accurately enumerated wit h immunofluorescence staining and fluorescence activated cell sorting (FACS) analysis and rapidly isolated at high purity for further analys is. 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 per ipheral blood CD4(+) T lymphocytes.