Using functional homology of RP1 isoforms to guide alternative therapeutic strategies

摘要 遗传性视网膜营养不良(IRD)与多种表型有关，影响个体从 从儿童早期到成年后期。视网膜色素变性(RP)是最常见的IRD，其特征是 进行性视网膜变性，常导致完全失明。目前，已有60个基因被 被鉴定为含有导致反相的突变。视网膜色素变性1(RP1)基因的突变是第三个 RP的最常见原因。RP1是一种微管相关蛋白，定位于 感光细胞外节和RP1突变导致常染色体显性遗传和常染色体隐性遗传 RP通过显性负向机制实现。这些特性使RP1成为基因治疗的一个有吸引力的靶点。而当 腺相关病毒(AAV)介导的基因传递已经得到了很好的研究，并被证明是一种强大的 有效的基因治疗方法，RP1太大，无法包装到AAV衣壳中，需要 确定可供选择的战略。使用RNA-Seq，我们已经全面描述了视网膜 小鼠和人类的转录本，确定了RP1(Nrp1)的一个保守的次要异构体。Nrp1 包含一个开放阅读框架，大约是主要RP1亚型长度的一半，使其易于 打包成AAV。重要的是，它维持了对微管结合至关重要的双重皮质区。我们的 初步研究表明，Nrp1在体外定位于微管，在体内定位于轴丝， 这表明它在功能上类似于典型的RP1。我们假设Nrp1在功能上是同源的 可作为治疗RP1相关RP的另一种基因治疗策略。在AIM 1，我们将全面描述Nrp1在体外和体内系统中的功能。这将提供 任何IRD基因的次要异构体的首批功能特征之一。在目标2中，我们将继续我们的 通过建立转基因小鼠模型来完成对Nrp1的表征。这将有两个目的：1) 研究Nrp1的内源性表达，以及2)用于确定Nrp1在卵巢癌中的治疗潜力 下一个目标。在目标3中，我们将通过与我们开发的转基因系杂交来测试Nrp1的治疗潜力。 目的2采用显性和隐性Rp1突变模型。通过使用转基因小鼠模型引入Nrp1， 与使用AAV相比，我们将能够更好地确定整体的功能和形态救援 眼睛将表达Nrp1，而不是在AAV注射部位单独表达。这项建议是一项 确定Nrp1的功能和治疗潜力的重要第一步。此外，这项工作将作为 用于其他IRD基因的模板，这些基因太大而无法打包到AAV中。

Abstract Inherited retinal dystrophies (IRDs) are associated with a wide range of phenotypes affecting individuals from early childhood to late adulthood. Retinitis pigmentosa (RP), the most common IRD, is characterized by progressive retinal degeneration, often leading to complete loss of vision. Currently, 60 genes have been identified to harbor mutations that lead to RP. Mutations in the Retinitis pigmentosa 1 (RP1) gene are the third most common cause of RP. RP1 is a microtubule associated protein that localizes to the axoneme of photoreceptor outer segments, and mutations in RP1 cause both autosomal dominant and autosomal recessive RP via a dominant negative mechanism. These properties make RP1 an attractive target for gene therapy. While adeno-associated virus (AAV)-mediated gene delivery is well-studied, and has been shown to be a robust and effective gene therapy method, RP1 is too large to be packaged into the AAV capsid, necessitating the identification of an alternative strategy. Using RNA-Seq, we have comprehensively characterized the retinal transcriptomes from both mouse and human, identifying a conserved minor isoform of RP1 (nRP1). nRP1 contains an open reading frame that is roughly half the length of the major RP1 isoform, making it amenable to packaging into AAV. Importantly, it maintains the doublecortin domains vital for microtubule binding. Our preliminary studies have shown that nRP1 localizes to microtubules in vitro and to the axoneme in vivo, suggesting it is functionally similar to the canonical RP1. We hypothesize that nRP1 is functionally homologous to canonical RP1, and it can be used as an alternative gene therapy strategy to treat RP1-associated RP. In Aim 1, we will comprehensively characterize the function of nRP1 in both in vitro and in vivo systems. This will provide one of the first functional characterizations of a minor isoform of any IRD gene. In Aim 2, we will continue our complete characterization of nRP1 by generating a transgenic mouse model. This will serve two purposes: 1) to study endogenous expression of nRP1, and 2) to be used to determine the therapeutic potential of nRP1 in the next Aim. In Aim 3, we will test the therapeutic potential of nRP1 by crossing our transgenic line developed in Aim 2 with a dominant and recessive Rp1-mutant model. By introducing nRP1 using a transgenic mouse model, rather than using AAV, we will be able to better determine functional and morphological rescue, as the whole eye will be expressing nRP1, rather than isolated expression at the site of an AAV injection. This proposal is an important first step in determining the function and therapeutic potential of nRP1. Further, this work will serve as a template for other IRD genes that are too large to be packaged into AAV.