Correction of α-l-Fucosidase Deficiency in Fucosidosis Fibroblasts by Retroviral Vector-Mediated Gene Transfer

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Abstract
A full-length cDNA clone encoding the lysosomal hydrolase α-l-fucosidase was cloned into two retroviral vectors, one using the human cytomegalovirus immediate-early promoter for expression, and the other, the retroviral long terminal repeat (LTR). High-titer amphotropic virus was produced for both constructs by infection of PA317 cells, and used to efficiently transduce the α-l-fucosidase gene into both human and canine fucosidosis fibroblasts. This resulted in correction of the α-l-fucosidase enzyme deficiency characteristic of these fibroblasts. The high levels of recombinant enzyme produced corrected the degradative defect normally seen in these cells, enabling them to catabolize efficiently the accumulated storage product present in lysosomes. Therefore, these retroviral constructs should allow us to start evaluating the value of gene therapy in treating the central nervous system pathology associated with fucosidosis and other lysosomal storage disorders in humans, using a canine model of fucosidosis. Gene therapy has become a viable possibility for treating certain genetic diseases, such as the lysosomal storage disorders, which can be cured by reconstitution of single gene products and which have been shown to respond to bone marrow transplantation. With this aim in sight, we have successfully genetically transferred the coding portion of the gene for α-l-fucosidase, a lysosomal enzyme, into human and canine fucosidosis fibroblasts. This was achieved by the use of retroviral vectors capable of efficient transfer of exogenous genetic material into cells in culture. In addition, the enzyme produced by these constructs is capable of correcting the biochemical defect by its ability to turn over stored fucoside substrates in these fucosidosis cells.

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