Functional Engraftment of Retrovirally Transduced and Pre-Selected Hematopoietic Cells into Partially Ablated Recipient Fabry Mice.

Transplantation of hematopoietic cells can impact clinical outcomes in lysosomal storage disorders (LSDs). This treatment schema will be enhanced by gene therapy. Vector transduced cells in LSDs often secrete the therapeutic lysosomal hydrolase, which can be used functionally by bystander cells leading to systemic improvement. Yet for many LSDs, full marrow suppression to obtain efficient hematopoietic cell engraftment will not likely be part of the clinical regimen. Fabry disease is X-linked and the second-most prevalent LSD. We have examined outcomes in a mouse model of Fabry disease using integrating oncoretroviral vector-transduced hematopoietic cells that have been transplanted into animals receiving 8 different partial conditioning regimens. These regimens include: low-dose whole-body irradiation (single and double course), single limb irradiation, fludarabine treatment, fludarabine with low-dose irradiation, cyclophosphamide treatment, busulfan treatment, and fludarabine plus cyclophosphamide treatment. A minimum of 5 animals was maintained in each group. Donor cells were collected from the bone marrow of male Fabry mice and transduced twice a day for 3 days in the presence of SCF, IL-6, and protamine sulfate. The oncoretroviral vector used for transductions is bicistronic, encoding the cDNA for human a-galactosidase A and a cell surface marker, human CD25. Animals were transplanted on the same day with the same pool of transduced cells. Results are compared against untouched Fabry mice, unconditioned but transplanted Fabry mice, lethally-irradiated transplanted Fabry mice, and also against wild-type age/strain matched controls. Two long-term and comprehensive experiments are detailed. In the first, cells were transduced and directly transplanted. Here animals received 4x10⁵ cells that were 50% positive for huCD25 expression. In the second, transduced cells were pre-selected for functional transgene expression by immuno-absorption methods prior to transplantation. Animals in the second experiment received 7x10⁵ cells that were 98% positive for huCD25 expression. We detail changes to the complete hematopoietic milieu mediated by each conditioning regimen. We track total leukocyte, neutrophil, lymphocyte, erythrocyte, hemoglobin, and platelets counts for each animal in each group. We also tracked immune responses to the corrective but foreign factor. Overall huCD25 expression levels were much higher for all groups in Expt. 2 than Expt. 1. We demonstrate effective long-term engraftment of 180 days with up to 8% functionally transgene-positive cells in the peripheral blood of partially-ablated animals. Furthermore, we demonstrate that these levels of engraftment lead to increased plasma enzyme activity and systemic amelioration of the lysosomal hydrolase deficiency in clinically relevant organs. Analyses of lipid storage in organs are underway. This data provides an important pre-clinical bridge to the adaptation of gene therapy for LSDs targeting hematopoietic cells.