Enzyme replacement therapy, the intravenous administration of purified preparations of active, wild-type enzymes, has been partially successful but is still an inadequate therapeutic modality for lysosomal storage diseases. For instance, in the treatment of Gauchet disease, the partial success stems from the fact that the administered soluble lysosomal protein is collected from the serum, internalized and concentrated by a variety of cell types, particularly the macrophages, the cell exhibiting the most severe pathology. However, the frequency of enzyme treatment (biweekly) and the cost (ca. $100,000.00 per year per patient) as well as the potential for complications stemming from the life-long series of injections, limit the usefulness of this approach. However, the ability of many cell-types to endocytose and target extracellular lysosomal proteins to their lysosomes and the observation that cells over- expressing lysosomal proteins secrete the overproduced protein, suggest that a properly glycoslylated lysosomal enzyme produced and secreted by one cell type might be passed to and internalized by a second cell. Such an intercellular transfer process and be incorporated into a strategy for gene therapy. This strategy is most easily envisaged in a confined compartment of the body where the producer cells and the target cell are in intimate contact. The CNS is an important example of such a compartment. Here astrocytes, neurons, oligodendrocytes, and microglia (macrophages) are all in close contact. There are many examples of factors secreted by one CNS cell type influencing the behavior, proliferation or differentiation of a second. In such a setting, cells engineered to secrete critical proteins may very well affect the enzyme deficiencies of a second cell type. We propose to explore this possibility with transgenic mice engineered to over-express alpha N- acetyl galactosaminidase or acid sphingomyelinase in their neurons, oligodendrocytes or astrocytes. For these purposes we will create high efficiency, cell type specific expression cassettes from genes encoding the neurofilament subunits (neuron specific), myelin basic protein (oligodendrocyte specific) and glial fibrillary acid protein (astrocyte specific) genes that we have studied extensively. In all three cases, the first generation cassettes have already been fashioned and have been shown to be completely cell-type specific in transgenic mice. In all three cases, the level of expression of the transgene was close to that of the endogenous, parent gene which encodes a major structural proteins. In the proposed study we will trim cassettes of unwanted extraneous sequences to make them more compact and, in the case of the neuron specific cassette, flank it with insulator sequences so that it will be less influenced by alien enhancer elements when inserted in the host chromosome or viral delivery systems. These cassettes will be used to drive the expression of acid sphingomyelinase or alpha N-acetyl galactosaminidase cDNA's in transgenic mice. We are concerned that the massive expression of the cDNA's may by and of itself, be deleterious to the transgenic animal. Consequently, we will examine these transgenic animals in depth for such effects. We will also examine them for evidence of secretion of the lysosomal proteins and re-absorption by bystander cells. Finally, together with our colleagues Drs. Desnick and Schuchman, we will cross these transgenic mice with mice that are deficient in acid sphingomyelinase or N-acetyl galactosaminidase in order to test the strategy that secretion of a critical enzyme by bystander cells will ameliorate the effects of a genetic deficit in second cell- type.
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