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Severe progressive fatal neurological degeneration occurs in fucosidosis, a storage disease. Bone marrow transplantation into affected dogs has shown that haematopoietic stem cells can provide enzyme producing daughter cells to the central nervous system, altering disease course. This makes canine fucosidosis an ideal large animal model for gene therapy. Fucosidosis affected allogeneic or autologous canine marrow was transduced ex vivo by cocultivation, then transplanted into fucosidosis affected dogs conditioned with total lymphoid irradiation. The vectors were Moloney murine leukaemia virus based. Transduction efficiency was increased with multiple cytokines in short term marrow culture. Despite high levels of transduction, proviral sequence was detected 2 months post transplant in only one dog. Early or total graft failure occurred in all transplants. We believe lack of engraftment could be caused by differentiation or change of repopulating ability of marrow cells occurring with multiple cytokine mixes in culture media.

Fucosidosis is a lysosomal storage disorder caused by deficiency of alpha-L-fucosidase. A biochemically and clinically well characterized canine model of fucosidosis exists in a colony of English Springer Spaniels. To facilitate its use as a model for gene therapy and enzyme replacement therapy in lysosomal storage disorders displaying neurological symptoms, isolation of the canine alpha-L-fucosidase cDNA was undertaken. Both the nucleotide sequence and the predicted amino acid sequence of canine fucosidase show high levels of identity with the human and rat sequences. Fucosidosis dogs were found to have a greatly reduced level of alpha-L-fucosidase mRNA when compared with normal dogs by Northern blot analysis. Direct PCR sequencing of products generated from cDNA demonstrated a 14-bp deletion in mRNA from affected dogs. This deletion creates a frameshift mutation and introduces a premature translation termination codon at amino acid position 152 and was shown to correspond to a deletion of the last 14 base pairs of exon 1 of the canine alpha-L-fucosidase gene. Rapid PCR-based screening for the mutation has now been performed on genomic DNA from dogs within the colony, enabling detection of both carriers and homozygotes.

A full-length cDNA clone encoding the lysosomal hydrolase alpha-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 alpha-L-fucosidase gene into both human and canine fucosidosis fibroblasts. This resulted in correction of the alpha-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.