The over goal of this research is to engineer a herpes simplex virus type l (HSV-1) replication defective mutant capable of stable transfer and expression of the murine beta-glucuronidase gene in the brains of a mouse model of the lysosomal storage disease mucopolysaccharidosis type VH (Sly syndrome). High level enzyme expression in brain should be therapeutic since enzyme should be released and taken up by neighboring brain cells in an active form thus obviating the need for vector infection of all brain cells. The design of the HSV vector will take advantage of and improve on the large capacity of HSV for foreign DNA and the latency specific expressiOn system that naturally evolved in the HSV life cycle. Four related aims are proposed to achieve this goal: (i) Recombinant promoters will be constructed using the HSV latency promoter/neuronal specific elements in combination with strong proximal and/or basal control elements in order to achieve vigorous foreign gene expression during latency. The design of these promoters will be determined by experiments to elucidate the identity of neuronal specific elements and how these elements functionally interact or cooperate with basal and/or general transcription factors. (ii) Constitutive (e.g. GAL4:VPI6 fusion gene ) and drug-inducible autogene (VPI6:GAL4:HBD fusion gene) systems will be developed to amplify or regulate gene expression from promoters carrying the GAL4 DNA binding elements in cis, such that sustained expression from the vector can be achieved or induced by a drug capable of crossing the blood-brain barrier, RU486. (iii) Vectors with greatly packaging capacity extending up to 40kb will be constructed. This will enable the transduction of large genomic genes as well as multiple cDNAs. These vectors will be defective in two essential genes and therefore be completely defective. In addition, the system has been engineered to eliminate the generation of wild-type recombinants when the vector stock is being prepared on transformed laboratory cell lines, and will also eliminate the potential for the generation of replication proficient virus carrying the transducted gene, which could potentially arise by recombination with latent wild-type virus in vivo. (iv) The best features of these vectors will be combined to express '3- glucuronidase in a murine model of mucopolysacchridosis type VII. This will involve the transduction of both the cDNA and genomic gene encoding beta- glucuronidase and the subsequent analysis for appropriate and therapeutic levels of gene expression.
