application) This application is directed toward achieving gene replacement in mammalian cells to correct genetic deficiency states using F-plasmid-based herpes simplex virus type 1 (HSV) amplicon (famplicon) vectors bearing elements of adenoassociated virus (AAV). Two strategies will be employed: 1) to increase the frequency of homologous recombination at endogenous mutant loci, large exogenous genomic fragments (50-150 kb) will be delivered by famplicons packaged in HSV virions, using a helper virus-free system. 2) to integrate transgene sequences into the AAVS1 site, the ITR elements and rep gene of AAV will be incorporated into famplicon vectors. Model systems will include cultured fibroblasts from patients with autosomal recessive deficiency states, GM1 gangliosidosis, and ataxia telangiectasia (AT), for which the corresponding genes, BGAL and ATM, are 65 and 150 kb in size, respectively. Recovery of function will be assessed at the single cell level by restoration of acidic betagalactosidase activity for GM1, and by decreased sensitivity to gamma irradiation for AT. Gene replacement by genomic integration will also be evaluated in mutant mice with the hypogonadal (hpg) deficiency state using the 8kb mouse gene LHRH- GAP, as a function of incorporation of the human AAVS1 locus into the mouse genome. Conditions will be developed for efficient delivery of transgenes to the hypothalamus by stereotactic injection of vectors in order to try to restore reproductive functions in hpg mice. This application extends existing technologies developed by Dr. Breakefield and Dr. Fraefel in a novel manner to achieve gene correction in normal human cells and in mouse models of human disease.
| Yoshimura, S; Takagi, Y; Harada, J et al. (2001) FGF-2 regulation of neurogenesis in adult hippocampus after brain injury. Proc Natl Acad Sci U S A 98:5874-9 |
| Costantini, L C; Bakowska, J C; Breakefield, X O et al. (2000) Gene therapy in the CNS. Gene Ther 7:93-109 |
