Retrovirus-Mediated Gene Transfer into Rhesus Monkey Hematopoietic Stem Cells: The Effect of Viral Titers on Transduction Efficiency

Abstract

We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until >1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for >2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.

Overview summary

Many disorders could potentially be treated with bone marrow gene therapy. Previously, we have accomplished genetic modification of hematopoietic stem cells of nonhuman primates by transduction with retroviral vectors. However, only few stem cells became transduced. Here we describe an attempt to increase the transduction efficiency by increasing the retrovirus-to-stem cell ratio. To this end, a cell line shedding higher titers of recombinant retroviruses as compared to the previously used cell line POC-1 was generated. The data obtained confirm that our procedures result in long-term persistence and expression of the newly introduced gene in the hematopoietic system of rhesus monkeys in vivo. Surprisingly, however, co-cultivation of bone marrow with the novel cell line did not increase the transduction efficiency of long-term repopulating stem cells as compared to that obtained in studies using POC-1 cells. We conclude that factors other than the retrovirus titer were limiting transduction efficiency of primate hematopoietic stem cells.