Rescue of photoreceptor synapses

第一视觉突触的信息传递由L型电压门控钙通道Cav1.4Ca~(2+)介导 频道。这些通道集中在突触终末的功能区下，一个 使用紧张性神经递质释放的突触的细胞器特征。基因突变 Cav1.4抑制神经传递，但也可以阻止突触发育。损失Cav1.4 可表现为各种视觉疾病，包括先天性静止性夜盲或 视锥-杆状营养不良。我们已经在Cav1.4基因敲除小鼠中验证了Cav1.4的恢复 突触发生可以防止这些缺陷。但光感受器突触疾病的治疗 对于成熟的个体来说是必要的。在目标1中，我们将测试突触救援的效率，当 在正常情况下，突触发生完成后，Cav1.4在这些小鼠中被诱导表达。这将是 是通过使用他莫昔芬诱导的从质粒中表达基因的策略来实现的 电穿孔进入小鼠光感受器，然后是形态和功能 成年期形成的突触的特征。无论何时使用Cav1.4表达式 是基础或临床前研究所需要的，电穿孔的有限效率抑制了 这一领域的进展。病毒介导的基因表达在视网膜中非常有效和安全 AAV是首选载体。然而，AAV没有能力表达大的 像Cav1.4这样的基因。在目标2中，我们将开发多个AAV载体来表达 Cav1.4通过反式内含子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快对Cav1.4的研究，还可能适用于其他 大基因是视网膜基因治疗的主要候选者，但目前还不能 提供AAV住宿。总而言之，这项探索可再生能力的工作 光感受器突触和AAV用于视网膜基因治疗的用途的扩大，将大大 推进挽救和恢复视力的努力。

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.