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

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

Efficient gene transfer with adeno-associated virus-based plasmids complexed to cationic liposomes for gene therapy of human prostate cancer.

We have shown previously that treatment of rats bearing the Dunning R3327 MatLyLu prostatic tumor with human interleukin 2 (IL-2) gene-modified tumor cell preparations induces potent antitumor immunity in the animal. To test the clinical feasibility of using genetically modified tumor vaccines for the treatment of prostate cancer, we have explored the use of a simplified gene delivery system based on liposomes to introduce and express the IL-2 gene in the Dunning rat R3327 MatLyLu prostatic tumor cell line (MatLyLu) and in short-term cultures of primary human prostatic tumor cells. Liposome-DNA complexes containing the adeno-associated virus inverted terminal repeats exhibited 3-10-fold higher levels of gene transfer and IL-2 expression than did liposome complexes with non-adeno-associated virus containing plasmids. Single transfections resulted in IL-2 expression for an extended period of time that exceeded severalfold the amount of IL-2 secreted from retrovirally transduced MatLyLu cells. X-irradiation of cells (4000 rads) prior to transfection did not affect cytokine secretion, indicating that liposome-mediated gene transfer does not depend on cell proliferation. High levels of gene transfer and IL-2 expression were also achieved in short-term cultures of primary human prostatic tumor cells established from tumor specimens obtained following radical prostatectomy of cancer patients. Depending on the type of liposome used, IL-2 levels secreted from the human prostatic tumor cells were comparable to or exceeded the levels of IL-2 secreted from retrovirally transduced MatLyLu cells, which induced antitumor immunity in the rat model. The ability to culture and expand ex vivo human prostatic tumor cells, and the use of a simple and highly efficient gene transfer method to generate genetically modified tumor vaccines, set the stage for clinical exploration of gene-based immunotherapy of prostate cancer