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

基本信息

批准号：
10396036
负责人：
Michael Farkas
金额：
$ 30.91万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10396036
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 Lentivirus 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 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

项目摘要

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.

抽象的遗传性视网膜营养不良 (IRD) 与影响个体的多种表型相关童年早期到成年晚期。色素性视网膜炎 (RP) 是最常见的 IRD，其特征是进行性视网膜变性，通常导致视力完全丧失。目前，已有60个基因被经鉴定含有导致 RP 的突变。第三个是视网膜色素变性 1 (RP1) 基因突变 RP 的最常见原因。 RP1 是一种微管相关蛋白，定位于轴丝光感受器外节和 RP1 突变导致常染色体显性和常染色体隐性遗传 RP 通过显性负机制。这些特性使 RP1 成为基因治疗的有吸引力的靶点。尽管腺相关病毒（AAV）介导的基因传递已被充分研究，并已被证明是一种稳健且可靠的方法。有效的基因治疗方法，RP1太大而无法包装到AAV衣壳中，需要确定替代策略。使用 RNA-Seq，我们全面表征了视网膜来自小鼠和人类的转录组，鉴定出 RP1 (nRP1) 的保守次要亚型。 nRP1 包含一个开放阅读框，其长度大约是主要 RP1 同工型的一半，使其适合打包成AAV。重要的是，它保留了对微管结合至关重要的双皮质素结构域。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，这表明它在功能上与规范的 RP1 相似。我们假设 nRP1 在功能上是同源的与经典 RP1 相比，它可以作为治疗 RP1 相关 RP 的替代基因治疗策略。瞄准 1，我们将全面表征nRP1在体外和体内系统中的功能。这将提供任何 IRD 基因的次要亚型的第一个功能特征之一。在目标 2 中，我们将继续我们的通过生成转基因小鼠模型来完成 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 在下一个目标。在目标 3 中，我们将通过与我们开发的转基因品系杂交来测试 nRP1 的治疗潜力。目标 2 使用显性和隐性 Rp1 突变模型。通过使用转基因小鼠模型引入 nRP1，我们将能够更好地确定功能和形态的救援，而不是使用 AAV 眼睛将表达 nRP1，而不是在 AAV 注射部位单独表达。该提案是一个确定 nRP1 的功能和治疗潜力的重要第一步。此外，这项工作将作为其他 IRD 基因的模板太大而无法包装到 AAV 中。