Photoreceptor Ciliopathies: RP2, KIF17 and NPHP5

描述（由申请人提供）：感光细胞完整性的挑战涉及蛋白质在一生中从内节段到外节段的生物合成后递送。该应用程序将识别：i）RP 2和ARL 3在蛋白质转运中的作用; ii）由同型二聚体驱动蛋白-II（KIF 17）介导的视杆和视锥鞭毛内转运的差异;以及iii）肾囊蛋白-5在光感受器运输和细胞极性中的功能。目标1A将解决 RP 2、ARL 3和RP 2/ARL 3/UNC 119复合物的生理功能。我们将生成RP 2（G2 A）敲入小鼠模型。RP 2（G2 A）敲入构建体将在内含子1和ATG上游掺入loxP和Frt位点，以允许另外进行RP 2的种系敲除，预期其在雄性小鼠中模拟XLRP。在目标1B中，我们提出产生光感受器特异性ARL 3敲除小鼠以研究ARL 3在视杆细胞和视锥细胞中的作用。RP 2基因敲入突变体、RP 2基因敲除突变体和ARL 3基因敲除突变体小鼠的建立将有助于阐明RP 2和ARL 3在感光细胞生物学中的作用，并为未来的基因治疗提供重要工具。目的2将集中在分子马达移动杆光转导蛋白的鞭毛内运输（IFT）的OS。同源二聚体驱动蛋白-II（KIF 17）在极化IMCD 3细胞和小鼠光感受器中的作用将进行比较和仔细检查。我们将通过与iCre-75小鼠交配产生Kif 17 flox/flox小鼠并特异性地在视杆细胞中缺失KIF 17。基于C. elegans，我们假设部分冗余的KIF 3和KIF 17杆。因此，我们将通过产生视杆细胞特异性KIF 3/KIF 17双敲除来删除视杆细胞中的KIF 3和KIF 17，并研究其后果。目的3将检测编码肾囊蛋白-5的NPHP 5基因，其功能未知。人类中的NPHP 5无效等位基因导致肾单位肾痨（一种髓质囊性肾病）和视网膜色素变性（Senior-Loken综合征）。我们将产生一个杆特异性NPHP 5敲除，小鼠模型，将阐明致病机制的疾病表型。此外，由于已知肾囊蛋白参与控制细胞极性，我们将使用体外肾上皮细胞培养物来探测 控制细胞极化的机制。最后，将设计AAV载体以拯救NPHP 5-RP敲除小鼠。 公共卫生相关性：我们建议产生模拟人类视网膜色素变性（RP）的小鼠模型。第一个目标集中在X连锁RP（XLRP）引起的RP 2基因的突变，未知的功能。XLRP在生命早期表现为夜盲症、视野缺陷和视功能下降的症状。在目标3中，我们专注于RP的组成部分，老年Loken综合征所造成的无效等位基因肾-5。人类中的NPHP 5无效等位基因导致肾单位肾结核（一种髓质囊性肾病）和视网膜色素变性，其组合被称为Senior-Loken综合征。目前还不存在基于RP 2突变的XLRP模型或Senior- Loken综合征模型。一旦产生，它们将成为鉴定导致疾病的机制和开发基因治疗剂的宝贵工具。我们进一步建议研究鞭毛内转运（IFT），这是一种将蛋白质从光感受器的内节传递到外节的途径，通过连接纤毛。IFT在顺行方向（朝向微管正端）运输货物，并在逆行方向（朝向基体和微管负端）返回产物。我们的目标是确定确切的电机（S）是负责纤毛运输的货物杆。

DESCRIPTION (provided by applicant): A challenge to photoreceptor cell integrity concerns the post-biosynthetic delivery of proteins from the inner, to the outer, segments over a lifetime. This application will identify: i) the roles of RP2 and ARL3 in protein transport; ii) differences n rod and cone intraflagellar transport mediated by homodimeric kinesin-II (KIF17); and iii) functions of nephrocystin-5 in photoreceptor trafficking and cell polarity. Aim 1A will address the physiological functions of RP2, ARL3 and the RP2/ARL3/UNC119 complex. We will generate an RP2(G2A) knockin mouse model. The RP2(G2A) knockin construct will incorporate loxP and Frt sites in intron 1 and upstream of ATG to permit, additionally, a germline knockout of RP2 which is expected to mimic XLRP in male mice. In aim 1B, we propose to generate a photoreceptor-specific ARL3 knockout mouse to study the role of ARL3 in rods and cones. Mutant knockin RP2, knockout RP2 and knockout ARL3 mice will elucidate the roles of RP2 and ARL3 in photoreceptor cell biology, and provide important tools for future gene therapy. Aim 2 will focus on molecular motors which move rod phototransduction proteins to the OS by intraflagellar transport (IFT). The role of homodimeric kinesin-II (KIF17) in polarized IMCD3 cells and mouse photoreceptors will be compared and scrutinized. We will generate Kif17flox/flox mice and delete KIF17 specifically in rod photoreceptors by mating with iCre-75 mice. Based on highly conserved IFT pathways in C. elegans, we postulate partial redundancy of KIF3 and KIF17 in rods. Therefore, we will delete both KIF3 and KIF17 in rods by generating a rod-specific KIF3/KIF17 double knockout and study the consequences. Aim 3 will examine the NPHP5 gene that encodes nephrocystin-5, of unknown function. NPHP5 null alleles in human cause nephronophthisis, a medullary cystic kidney disease, and retinitis pigmentosa (Senior-Loken syndrome). We will generate a rod-specific NPHP5 knockout, a mouse model that will elucidate mechanisms causative for the disease phenotype. Further, as nephrocystins are known to be involved in controlling cell polarity, we will use in-vitro renal epithelial cell cultures to probe mechanisms controlling cell polarization. Finally, AAV vectors will be designed to rescue the NPHP5-RP knockout mouse. PUBLIC HEALTH RELEVANCE: We propose to generate mouse models mimicking human retinitis pigmentosa (RP). The first aim focuses on X-linked RP (XLRP) caused by mutations in the RP2 gene, of unknown function. XLRP manifests early in life with symptoms of night blindness, visual field defects and decreased visual function. In aim 3 we focus on the RP component of Senior-Loken syndrome caused by null alleles of nephrocystin-5. NPHP5 null alleles in human cause nephronophthisis (a medullary cystic kidney disease) and retinitis pigmentosa, the combination of which is called Senior-Loken syndrome. Models for XLRP based on mutations in RP2, or models for Senior- Loken Syndrome currently do not exist. Once generated, they will be valuable tools for identification of mechanisms leading to disease and for the development of gene therapy agents. We further propose to study intraflagellar transport (IFT), a pathway that delivers proteins from the inner segment to the outer segment of photoreceptors, passing through a connecting cilium. IFT transports cargo in the anterograde direction (toward microtubule plus ends) and returns products in the retrograde direction (towards the basal body and microtubule minus ends). Our goal is to identify exactly which motor(s) is responsible for ciliary transport of cargo in rods.