GDNF and ribozymes for retinitis pigmentosa

常染色体显性色素性视网膜炎（ADRP）是一组显性遗传性退行性视网膜疾病。ADRP是一种典型的慢性神经退行性疾病，其特征是杆状光感受器细胞的进行性凋亡丧失，通常导致缓慢但不可避免地进展为失明。在之前的资助期内，腺相关病毒（AAV）传递的核酶被用来阻断一种啮齿动物ADRP模型的这种退化。结果显示，治疗组视棒细胞长期存活，视觉功能得以保存

Autosomal Dominant Retinitis Pigmentosa (ADRP) comprises a group of dominantly inherited degenerative retinal diseases. It is prototypic of slow neurodegenerative diseases in that ADRP is characterized by the progressive apoptotic loss of rod photoreceptor cells normally leading to a slow but inexorable progression to blindness. In the previous grant period ribozymes delivered by Adeno-associated virus (AAV) were employed to block this degeneration in one rodent ADRP model. The result was long-term rod cell survival and preservation of visual function in treated eyes. In this project we propose to improve and extend those results in order to propel retinal gene therapy towards a clinical application. For slowly progressive neurodegenerafive diseases it is important to assess the longevity of any therapeutic effect, to determine if efficacy can be further improved to enhance safety and to apply the therapeutic modality to a large animal disease model with human sized eye. To achieve these ends, we propose to: (1) Measure the duration of neural retina protection afforded by AAV-delivered ribozymes in P23H rhodopsin rats, and to initiate therapy in these rats at one year of age when only 5-15% of normal rod function remains in order to model the expected situation in an initial human trial where late-stage RP patients will be the first tested. (2) Improve in vivo ribozyme performance by (a) by increasing ribozyme catalytic activity, stability and expression level, (b) by enhancing the expression of glial cell derived neurotrophic factor (GDNF) from AAV vectors and (c) by determining whether photoreceptor survival is enhanced by expressing GDNF along with the ribozyme relative to single gene therapy. Elements to be tested include ribozyme structural improvements, RNA stability elements, alternative promoters, alternative AAV serotypes and vector capsid mutants that target novel subsets of retinal and retina-associated cell types. Non-overlapping improvements will be combined into a single vector (or combination of vectors) to produce the optimal therapeutic reagent. (3) Test AAV-vectored ribozymes against mutations creating canine models of ADRP and X-linked RP. This includes the T4R rod opsin mutation (substitution of arginine for threonine at position 4 of rod opsin) leading to dominant RP similar to humans with the T4K mutation and mutations in the canine RPGR gene that cause X-linked retinal disease in dogs analogous to that in humans. Our primary aim here will be to determine whether successful therapy in the small rodent eye scales effectively to a human sized eye. If successful, we anticipate that these optimized, more general ribozyme approaches will permit us to attempt gene therapy in humans possessing a wide spectrum of mutations causing RP.