1. Replacement gene therapy Tubby-like protein 1 (TULP1) is a photoreceptor specific protein expressed in both rods and cones. Gene defects in Tulp1 cause retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in humans. A knockout mouse model lacking Tulp1 (TULP1/) develops rapid photoreceptor degeneration with severe mislocalization of rod and cone opsins. We evaluated whether adeno-associated virus (AAV)-mediated gene replacement therapy in TULP1/ model could improve photoreceptor survival and recovery of rhodopsin and cone opsin in outer segments of rods and cones respectively. We validated the efficacy of mouse and human TULP1 replacement gene controlled by a promoter derived from the human rhodopsin kinase (RK) gene which is active in both rods and cones. We found substantial rescue of the disease phenotype as a result of transgene expression. This is another gene therapy study in which both rods and cones were targeted successfully with a single photoreceptor-specific promoter. We propose that the vector and construct design used in this study could serve as a prototype for a human clinical trial. In addition to TULP1, we are also continuing our efforts in collaboration with Peter Colosi toward a better understanding of the RPGR isoforms expression in photoreceptors in preparation for future clinical studies. Another ongoing gene therapy effort has been focused on RPGRIP1. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. In collaboration with Drs. Eliot Berson and Michael Sandberg at the Harvard Medical School/Mass. Eye &Ear Infirmary, we are moving forward with preclinical studies to evaluate the potential of this approach for clinical applications. 2. Study of Usher syndrome. Usher syndrome is a genetic disorder affecting both vision and hearing. NHERF-1 (Na+/H+ exchanger regulatory factor 1) is an adaptor protein containing tandem PDZ domains and was previously shown to be localized to the microvilli of retinal pigment epithelium (RPE). In the present study, we determined the subcellular localization of NHERF-1 in the retina and inner ear, and evaluated the potential defects in these two tissues in NHERF-1 knockout mice (NHERF-1/). We found that in the retina, NHERF-1 was concentrated at the RPE microvilli and along the apical processes of Muller glia. In the inner ear, NHERF-1 was associated with the stereocilia of outer hair cells, but was largely absent from the inner hair cells. In NHERF-1/ mice, there was a loss of polarity in melanin granule distribution resembling the Ush1B mutant mouse. Photoreceptor degeneration was mild but noted in older NHERF-1/ mice (at 23 months of age), as indicated by slight thinning of the outer nuclear layer and up-regulation of glial fibrillary acidic protein. Hearing loss was pronounced at 8 months of age as demonstrated by DPOAE testing. By histologic sectioning and scanning EM, there was disorganization of stereocilia and a loss of outer hair cells, which were more severe at the middle to basal turns. Therefore we show that despite its broad tissue distribution, loss of NHERF-1 results in a set of defects confined to the retina and the inner ear. These data suggest that the NHERF-1/ mouse may be a useful model for studying human Usher syndrome. It is further postulated that certain mutations in the NHERF-1 gene in humans may be a cause of Usher syndrome.
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