TRANSDUCED FIBROBLASTS AND METACHROMATIC LEUKODYSTROPHY LYMPHOCYTES TRANSFER ARYLSULFATASE A TO MYELINATING GLIA AND DEFICIENT CELLS IN-VITRO

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease, cau sed by deficiency of arylsulfatase A (ASA), that manifests primarily i n the white matter of the nervous system. Currently, no specific treat ment exists that will reverse its fatal outcome. Replacement therapy h as been hampered by the blood-brain barrier (BBB). To circumvent this problem we designed an ex vivo gene therapy strategy that includes the retrovirus-mediated ASA transduction of cells, such as activated lymp hocytes, that are able to traverse the BBB or other membranes of the C NS. For this purpose, two recombinant retroviruses based on the pLXSN vector were produced, containing the wild-type ASA cDNA or a pseudodef iciency ASA cDNA, which encodes a smaller enzyme with normal activity. After transduction, ASA activity increased more than 100-fold in fibr oblasts from an MLD patient. Furthermore, ASA-transduced MLD PBLs expr essed 30 times higher ASA activity when compared with control PBLs. Mo reover, cell culture experiments demonstrated that transduced fibrobla sts could efficiently transfer ASA to deficient cells across a transwe ll barrier, whereas transduced MLD lymphocytes could transfer ASA to d eficient fibroblasts only by direct cell-to-cell contact. Finally, ASA was taken up by normal oligodendrocytes and Schwann cells, the target myelinating glial cells for therapy in MLD. These data suggest possib le short-term strategies for transfer of ASA into the CNS via transduc ed autologous cells while long-term strategies, related to autologous transduced bone marrow transplant, take effect in patients.