Rescue of photoreceptor synapses

拯救感光器突触

基本信息

批准号：
9165264
负责人：
Sheila A Baker
金额：
$ 22.88万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9165264
关键词：
Address Adult Affect Age Area Behavioral Assay Biological Models Blindness Cell Culture Techniques Cells Characteristics Clinical Trials Communication Data Defect Development Disease Effectiveness Electroporation Engineering Experimental Models Failure Functional disorder Gene Delivery Gene Expression Gene Transduction Agent Gene therapy trial Genes Genetic Recombination Goals Health Human Image Immunohistochemistry Individual Inherited Knockout Mice Knowledge Leber&apos s amaurosis Life Maintenance Measures Mediating Methods Mus Mutate Mutation Natural regeneration Neurons Night Blindness Organelles Patients Photoreceptors Physical shape Physiological Plasmids Predisposition Presynaptic Terminals Proteins RNA Splicing Reporting Research Retina Retinal Retinal Degeneration Retinal Detachment Retinal Diseases Retinitis Pigmentosa Science Signal Transduction Stem cells Synapses Synaptic plasticity Tamoxifen Testing Therapeutic Studies Time Trans-Splicing Translating Variant Viral Vision Visual Visual system structure Work adeno-associated viral vector base effective therapy gene therapy human disease in vivo inducible gene expression insight intein mouse model neurotransmission neurotransmitter release preclinical study presynaptic prevent regenerative relating to nervous system restoration ribbon synapse success synaptogenesis therapeutic development tool treatment strategy vector voltage

项目摘要

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.

第一个视觉突触的通讯由 L 型电压门控 Cav1.4 Ca2+ 介导渠道。这些通道集中在带下方的突触末端，采用强直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。 Cav1.4 的损失可表现为多种视觉疾病，包括先天性静止性夜盲症或视锥杆营养不良。我们之前已经验证了 Cav1.4 敲除小鼠中 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是成熟个体所需要的。在目标 1 中，我们将测试以下情况下突触救援的效率：突触发生正常完成后，这些小鼠中会诱导 Cav1.4 表达。这将通过使用从质粒中他莫昔芬诱导基因表达的策略来完成电穿孔至小鼠光感受器中，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表达对于基础或临床前研究而言，电穿孔的有限效率抑制了该领域的进展。病毒介导的基因表达在视网膜中非常有效且安全 AAV 是首选载体。然而，AAV 不具备表达大数据的能力。基因如 Cav1.4。在目标 2 中，我们将开发多个表达 AAV 片段的 AAV 载体 Cav1.4 通过反式内含肽剪接进行翻译后重组并测试该策略在成年小鼠光感受器中实现 Cav1.4 表达的有效性。该领域的成功不仅会加速 Cav1.4 的研究，而且可以应用于其他领域大基因是视网膜基因治疗的主要候选者，但目前还不能住宿于AAV。这项工作共同探索了再生能力光感受器突触和 AAV 在视网膜基因治疗中的用途的扩展，将极大地努力挽救和恢复视力。