Circulating α-galactosidase a derived from transduced bone marrow cells: Relevance for corrective gene transfer for fabry disease

Fabry disease is caused by a deficiency of the lysosomal enzyme α- galactosidase A (α-gal A). We previously engineered a retrovirus encoding human α-gal A and demonstrated enzymatic correction of patient cells. Further, we demonstrated metabolic cooperativity, in that corrected cells secrete α-gal A that can be taken up and utilized by bystander cells in vitro. In the present study, we created a system to examine and quantitate this phenomenon in vivo. To differentiate from endogenous α-gal A, we constructed a retroviral vector (pUMFG/α-gal A/FLAG) containing a fusion form of α-gal A with a specific tag sequence at the carboxy terminus. The catalytic activity of the fusion protein was identical to wild-type α-gal A. The fusion protein was overexpressed in and secreted by transduced patient cells. In uptake studies, the fusion protein was detected in the lysosome- enriched fraction of recipient cells. We then examined the effectiveness of the pUMFG/α-g A/FLAG retroviral vector in vivo. Murine bone marrow (BM) cells were transduced and transplanted into irradiated hosts. After 9 weeks, proviral DNA was detected by PCR in peripheral blood and BM mononuclear cells. More importantly, specific fusion protein enzymatic activity could be demonstrated in those cells and in plasma. Thus, we have demonstrated that overexpressed α-gal A enters the circulation from transduced BM cells and is stable over a significant period of time.