Jomary C, Grist J, Milbrandt J, Neal M J, Jones S E
Retinitis Pigmentosa Research Unit, The Rayne Institute, Division of Pharmacology and Therapeutics, Guy's, King's and St Thomas' School of Biomedical Sciences, King's College London, St Thomas' Hospital, London, UK.
Mol Vis. 2001 Feb 23;7:36-41.
Neurturin (NTN) is a potent neuronal survival factor in the central and peripheral nervous systems. We previously described altered expression of mRNAs for NTN and one of its receptor components, GFRa-2 in degenerative retinas of rd/rd mice. Towards assessing the potential for transfer of these genes to counteract retinal degeneration, we examined recombinant adeno-associated virus (rAAV) constructs for expression of NTN and GFRa-2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mice.
The rAAV constructs incorporated epitope tags to facilitate discrimination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was used to drive expression of human GFRa-2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of predegenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography.
Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of alternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed.
Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NTN or GFRa-2, and may therefore depend on additional synergistic factors. Our AAV constructs will facilitate the development of combinatorial approaches to the treatment of central and peripheral neurodegenerations.
神经营养因子（NTN）是中枢和外周神经系统中一种强大的神经元存活因子。我们之前描述了在rd/rd小鼠退化的视网膜中，NTN及其受体成分之一GFRa-2的mRNA表达发生了改变。为了评估这些基因转移以对抗视网膜退化的潜力，我们检测了重组腺相关病毒（rAAV）构建体在体外视网膜细胞中NTN和GFRa-2转基因的表达情况，以及在rd/rd小鼠眼内注射后转基因表达对视网膜功能的影响。
rAAV构建体掺入了表位标签，以促进转基因表达与内源性表达之间的区分。小鼠NTN的表达由CMV启动子驱动，部分小鼠视蛋白启动子用于驱动人GFRa-2的表达。rAAV制剂用于感染小鼠视网膜细胞培养物，并用于对处于退化前期的rd/rd小鼠进行眼内注射。通过免疫荧光分析内源性和转基因表达。通过视网膜电图评估治疗小鼠的光感受器功能。
两种载体在体外和体内均传递并表达了它们的转基因。通过使用不同的启动子在体内实现了差异靶向。在所检测的条件下，未观察到rd光感受器的功能挽救。
视网膜退化的rd模型的治疗似乎不受NTN或GFRa-2表达的简单调节的影响，因此可能取决于其他协同因子。我们的AAV构建体将有助于开发治疗中枢和外周神经退行性变的联合方法。
