Using functional homology of RP1 isoforms to guide alternative therapeutic strategies

利用 RP1 亚型的功能同源性来指导替代治疗策略

• 批准号: 9884767
• 负责人: Michael Farkas
• 金额: $ 32.64万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9884767
• 关键词: Address; Adult; Affect; Alternative Splicing; Antibodies; Binding; Biological Assay; Blindness; CRISPR/Cas technology; Capsid; Data; Dependovirus; Disease; Dominant-Negative Mutation; Eye; Gene Delivery; Gene Expression; Genes; Genome; Goals; Human; In Vitro; Individual; Inherited; Injections; Lead; Length; Mediating; Methods; Microtubule-Associated Proteins; Microtubules; Minor; Modeling; Morphology; Mus; Mutant Strains Mice; Mutation; NRP1 gene; Neural Retina; Open Reading Frames; Pathogenesis; Phenotype; Photoreceptors; Play; Property; Protein Isoforms; Proteins; Research; Retina; Retinal Degeneration; Retinal Diseases; Retinal Dystrophy; Retinitis Pigmentosa; Role; Safety; Site; Subfamily lentivirinae; System; Technology; Testing; Therapeutic; Tissues; Transgenic Mice; Transgenic Organisms; Work; base; early childhood; gene augmentation therapy; gene therapy; in vitro activity; in vivo; mouse model; mutant; mutant mouse model; novel; novel strategies; therapeutic evaluation; tool; transcriptome; transcriptome sequencing

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.

摘要 遗传性视网膜营养不良（IRD）与影响个体的广泛表型相关， 从儿童早期到成年晚期。视网膜色素变性（RP）是最常见的IRD，其特征在于： 进行性视网膜变性，通常导致视力完全丧失。目前，已有60个基因 被鉴定为携带导致RP的突变。视网膜色素变性1（RP 1）基因突变是第三种 RP的最常见原因RP 1是一种微管相关蛋白，定位于神经元的轴丝， 光感受器外节和RP 1突变导致常染色体显性和常染色体隐性 RP通过显性负机制。这些特性使RP 1成为基因治疗的有吸引力的靶点。而 腺相关病毒（AAV）介导的基因递送被充分研究，并且已被证明是一种稳健的且 有效的基因治疗方法，RP 1太大而不能被包装到AAV衣壳中， 确定替代战略。使用RNA-Seq，我们已经全面表征了视网膜 从小鼠和人的转录组，确定一个保守的次要亚型RP 1（nRP 1）。NRP1 包含一个开放的阅读框架，大约是主要RP 1同种型长度的一半，使其适合于 包装成AAV。重要的是，它维持着对微管结合至关重要的doublecortin结构域。我们 初步研究表明nRP 1在体外定位于微管和在体内定位于轴丝， 这表明它在功能上类似于典型的RP 1。我们假设nRP 1在功能上是同源的， 与经典RP 1相比，它可以作为治疗RP 1相关RP的替代基因治疗策略。在Aim中 1，我们将全面表征nRP 1在体外和体内系统中的功能。这将提供 任何IRD基因的次要同种型的第一个功能特征之一。在目标2中，我们将继续 通过生成转基因小鼠模型来完成nRP 1的表征。这样做有两个目的：1） 研究nRP 1的内源性表达，和2）用于确定nRP 1在 下一个目标在目标3中，我们将通过将我们的转基因株系杂交来测试nRP 1的治疗潜力。 目的2建立显性和隐性Rp 1突变体模型。通过使用转基因小鼠模型引入nRP 1， 而不是使用AAV，我们将能够更好地确定功能和形态拯救，作为一个整体， 眼睛将表达nRP 1，而不是在AAV注射部位的孤立表达。这项建议是一项 这是确定nRP 1功能和治疗潜力的重要第一步。此外，这项工作将作为 其他IRD基因的模板太大而不能包装到AAV中。