Rescue of photoreceptor synapses

第一视觉突触的信息传递由L型电压门控Cav1.4 Ca ~（2+）介导 渠道这些通道集中在带状物下方的突触末端， 利用紧张性神经递质释放的突触特征的细胞器。突变 Cav1.4抑制神经传递，但也可以阻止突触发育。损失Cav1.4 可以表现为各种视觉疾病，包括先天性静止性夜盲症或 视锥杆细胞营养不良我们已经验证了Cav1.4基因敲除小鼠中Cav1.4的恢复， 突触发生阻止了这些缺陷。但是光感受器突触疾病的治疗 是成熟个体所必需的。在目标1中，我们将测试突触拯救的效率， Cav1.4的表达是在突触形成正常完成后在这些小鼠中诱导的。这将 通过使用他莫昔芬诱导的基因从质粒表达的策略来实现 电穿孔到小鼠光感受器中，然后进行形态和功能研究。 成年期形成的突触的特征。无论何时Cav1.4表达 是基础或临床前研究所需的，但电穿孔的有限效率抑制了 这一领域的进步。病毒介导的基因表达在视网膜中非常有效和安全 其中AAV是优选的载体。然而，AAV不具有表达大的 Cav1.4等基因在目标2中，我们将开发多个AAV载体，表达 Cav1.4通过反式内含肽剪接进行翻译后重组，并测试 这一策略在成年小鼠光感受器中实现Cav1.4表达的有效性。 这一领域的成功不仅将加速Cav1.4的研究，而且可以适应其他领域的研究。 大基因是视网膜基因治疗的主要候选者，但目前还不能 容纳在AAV中。总之，这项工作探索的再生能力 光感受器突触和扩大AAV用于视网膜基因治疗的效用，将大大 努力挽救和恢复视力。

Communication at the first visual synapse is mediated by L-type voltage-gated Cav1.4 Ca2+ channels. These channels are concentrated in the synaptic terminal beneath the ribbon, an organelle characteristic of synapses employing tonic neurotransmitter release. Mutation of Cav1.4 inhibits neurotransmission but can also prevent synaptic development. Loss of Cav1.4 can present as a variety of visual diseases, including congenital stationary night blindness or cone-rod dystrophy. We have validated that restoration of Cav1.4 in Cav1.4 knockout mice prior to synaptogenesis prevents these defects. But treatments for photoreceptor synaptic diseases are needed for mature individuals. In Aim 1, we will test the efficiency of synaptic rescue when Cav1.4 expression is induced in these mice after synaptogenesis is normally complete. This will be accomplished by using a strategy of tamoxifen-inducible gene expression from plasmids electroporated into mouse photoreceptors, followed by morphological and functional characterization of the synapses that form in adulthood. Regardless of when Cav1.4 expression is desired for basic or pre-clinical studies, the limited efficiency of electroporation inhibits advances in this field. Viral mediated gene expression is very effective and safe in the retina with AAV being the preferred vector. However, AAV does not have the capacity to express large genes like Cav1.4. In Aim 2, we will develop multiple AAV vectors expressing fragments of Cav1.4 engineered for post-translational recombination by trans-intein splicing and test the effectiveness of this strategy for achieving expression of Cav1.4 in adult mouse photoreceptors. Success in this area will not only accelerate research on Cav1.4 but could be adapted to other large genes that are prime candidates for retinal gene therapy but currently cannot be accommodated in AAV. Together, this work exploring the regenerative capacity of photoreceptor synapses and expansion of the utility of AAV for retinal gene therapy, will greatly advance efforts to save and restore vision.