Long-TermIn VivoExpression of the MFG-ADA Retroviral Vector in Rhesus Monkeys Transplanted with Transduced Bone Marrow Cells

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Abstract
We have tested the recombinant human adenosine deaminase (hADA) retroviral vector MFG-ADA for its efficacy in transducing hemopoietic stem cells of nonhuman primates and its expression level in the hematopoietic system. The percentage of provirus-positive granulocytes 1 year after transplantation of bone marrow transduced with MFG-ADA was 0.1%, which was equivalent to previously obtained results with the hADA virus-producing cell line POC-1. However, in MFG-ADA monkeys, significantly more peripheral blood mononuclear cells carried the hADA gene (1% versus 0.1%). Human ADA expression levels in peripheral blood mononuclear cells were not different between POC-1 and MFG-ADA monkeys using samples with equal numbers of provirus copies per cell. In contrast, in total red blood cell lysates of MFG-ADA monkeys, the hADA expression was higher (~10-fold) and could be detected longer (20 weeks and up to more than 1 year after bone marrow transplantation in 2 monkeys) than in POC-1 monkeys that were only positive for up to 12 weeks at the most. At 3 years after bone marrow transplantation, the MFG-ADA provirus could still be detected in 0.1% of bone marrow cells and peripheral blood cells and in 1% of cultured T cells. These results show that MFG-ADA virus can give rise to long-term in vivo expression of hADA in the primate hematopoietic system. However, transduction efficiencies remain low. Success of gene therapy relies on several factors, an important one being the vector of choice. In this study we have used the retrovirus-producing cell line MFG-ADA for the transduction of rhesus monkey bone marrow grafts. Long-term follow up showed the presence of the provirus in peripheral blood mononuclear cells, granulocytes, and bone marrow. The highest frequency of transduced cells was found in the T cell lineage. In general, the frequencies of MFG-ADA-carrying cells were low in all lineages, confirming that it is difficult to transduce primate stem cells. However, functional human adenosine deaminase (hADA) enzyme activity was detected in peripheral blood mononuclear cells and erythrocytes long after transplantation. This demonstrates the usefulness of the MFG-ADA vector in bone marrow gene therapy for ADA¯SCID.

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