Enzyme replacement methodologies applicable to human lysosomal storage diseases with mental retardation will be developed using cats with genetic beta-hexosaminidase deficiency and generalized GM2 gangliosidosis as a model. Purified human placental beta-hexosaminidase will be chemically modified to prevent sequestration by reticuloendothelial cells and to enhance uptake by other cell types. The use of enzyme-bound, non-toxic fragment C of tetanus toxin as a means of targeting to neurons will be explored. Uptake specificity, lysosomal localization and tissue distribution will be assessed in cultured fibroblasts and neural cells and in vivo, respectively. Reversible blood-brain barrier permeability will be obtained by hyperosmotic mannitol infusion; the efficiency of enzyme delivery and physiopathologic consequences will be assessed; retrograde axonal transport will be explored as an alternative to barrier permeabilization. The appropriate combination of methodologies will be applied to affected kittens, and the effect on ganglioside storage, aberrant neuronal morphology and neurologic manifestations will be determined. Complementary DNA encoding the beta-chain of human beta- hexosaminidase will be transfected into feline cultured fibroblasts and neural cells using calcium phosphate aggregates, defective retrovirus vectors, reconstituted viral envelopes and derivates thereof. Efficiency of transfection, state of integration and level of expression will be assessed. If significant expression of the human gene is obtained, transfection of somatic and neural tissues in vivo will be attempted using strategies developed for enzyme replacement. Transfection, integration and expression, as well as effects on ganglioside storage and clinical status will be evaluated.
