This project is aimed at understanding and treating autosomal dominant retinitis pigmentosa (ADRP) at a molecular level. Successful gene therapy for ADRP requires: (1) an efficient and cell-type specific gene delivery/expression system, (2) a selective means of inhibiting production of the mutant protein, and (3) valid animal models of ADRP in which to test and optimize (1) and (2). (1) We have made synthetic genes for the several ribozymes (catalytic RNA molecules capable of destroying specific target RNAs). These ribozymes recognize the nucleotide change causing the P23H mutation in one form of ADRP and the S334ter mutation in another. We will test these in vitro and in cultured cells and plan to deliver the most active versions of these ribozymes to the retinas of transgenic rates bearing these mutant forms of rod opsin and exhibiting RP-like symptoms. (2) Using a recombinant Adeno-associated virus (rAAV) in which expression is driven by a portion of the rod opsin promoter, we have achieved predominant (but not absolute) photoreceptor-specific expression of reporter genes in mouse and rate by ocular injection. We propose a systematic study of rAAV constructs containing segments of the opsin regulatory sequence to achieve controllable,, cell-type specific expression of the virally-packaged passenger gene. (3) Transgenic rat lines carrying the P23H or S334ter mutation in the rod opsin gene under control of the opsin promoter exhibit a course of retinal disease remarkably similar to that observed in humans bearing such mutations. We propose to test rAAV-ribozymes in P23H and S334ter in transgenic rats to determine whether the course of the RP-like disease can be ameliorated with a minimum of pathogenic side effects. As an independent test of the efficacy of ribozymes in these models, we will also prepare transgenic mice expressing the P23H ribozymes and measure its activity following matings with P23H transgenic mice. Assays for activity include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretinography. We are testing the specific hypothesis that viral mediated retinal delivery of ribozymes to reduce expression of a dominant negative gene will be therapeutic for ADRP. In a broader view, this systematic approach to optimizing regulated expression in a differentiated tissue should contribute at many levels toward human gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011596-03
Application #
2888540
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1997-07-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Florida
Department
Genetics
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
White, D Alan; Hauswirth, William W; Kaushal, Shalesh et al. (2007) Increased sensitivity to light-induced damage in a mouse model of autosomal dominant retinal disease. Invest Ophthalmol Vis Sci 48:1942-51
Gorbatyuk, M; Justilien, V; Liu, J et al. (2007) Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery. Vision Res 47:1202-8
Gorbatyuk, M; Justilien, V; Liu, J et al. (2007) Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme. Exp Eye Res 84:44-52
Glushakova, Lyudmyla G; Timmers, Adrian M; Issa, Tawfik M et al. (2006) Does recombinant adeno-associated virus-vectored proximal region of mouse rhodopsin promoter support only rod-type specific expression in vivo? Mol Vis 12:298-309
Nusinowitz, S; Ridder 3rd, W H; Pang, J J et al. (2006) Cortical visual function in the rd12 mouse model of Leber Congenital Amarousis (LCA) after gene replacement therapy to restore retinal function. Vision Res 46:3926-34
Glushakova, Lyudmyla G; Timmers, Adrian M; Pang, Jijing et al. (2006) Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors. Invest Ophthalmol Vis Sci 47:3505-13
Gorbatyuk, M S; Pang, J J; Thomas Jr, J et al. (2005) Knockdown of wild-type mouse rhodopsin using an AAV vectored ribozyme as part of an RNA replacement approach. Mol Vis 11:648-56
Liu, Jianwen; Timmers, Adrian M; Lewin, Alfred S et al. (2005) Ribozyme knockdown of the gamma-subunit of rod cGMP phosphodiesterase alters the ERG and retinal morphology in wild-type mice. Invest Ophthalmol Vis Sci 46:3836-44
Hauswirth, William W; Li, Quihong; Raisler, Brian et al. (2004) Range of retinal diseases potentially treatable by AAV-vectored gene therapy. Novartis Found Symp 255:179-88; discussion 188-94
Qi, Xiaoping; Lewin, Alfred S; Hauswirth, William W et al. (2003) Optic neuropathy induced by reductions in mitochondrial superoxide dismutase. Invest Ophthalmol Vis Sci 44:1088-96

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