Therapeutic strategies for mitigating loss of retinal ganglion cells in familial dysautonomia

减轻家族性自主神经功能障碍患者视网膜神经节细胞丢失的治疗策略

基本信息

批准号：
10093053
负责人：
Frances Lefcort
金额：
$ 18.64万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10093053
关键词：
5&apos Splice Site Age Automobile Driving Autonomic nervous system Blindness Blood Brain Cell Survival Cessation of life Clinic Clinical Clinical Research Cytoprotection Data Defect Diet Disease Embryo Enhancers Exons Familial Dysautonomia Family Generations Genes Genetic Goals Human Hybrids In Vitro Individual Introns Kinetins Knockout Mice Life Mediating Messenger RNA Methods Modeling Modification Mus Mutate Mutation Nervous system structure Neurodegenerative Disorders Neurons Oral Pathway interactions Patients Penetration Peripheral Phenotype Point Mutation Pre-Clinical Model Preclinical Testing Proteins Publishing Quality of life RNA Splicing Reporting Retina Retinal Degeneration Retinal Dystrophy Retinal Ganglion Cells Sensory Spinal Muscular Atrophy Splice-Site Mutation Technology Teenagers Testing Therapeutic Tissues Transgenes Transgenic Mice Translations Treatment Protocols Viral Wild Type Mouse Work base cell type clinical investigation compare effectiveness conditional knockout disease phenotype effectiveness evaluation effectiveness testing exon skipping ganglion cell gene correction gene therapy improved in vivo insight intravitreal injection mRNA Precursor macula mouse model mutant nervous system disorder novel optic nerve disorder pre-clinical prevent promoter protein complex recessive genetic trait restoration retinal ganglion cell degeneration retinal neuron reverse genetics small molecule targeted treatment tool

项目摘要

PROJECT SUMMARY Given the recent FDA approval of targeted AAV gene therapy platforms and of small-molecule splicing modulators as treatments for genetic neurological disorders, our goal is to apply these powerful technologies to prevent the progressive optic neuropathy and blindness that develops in patients with the genetic recessive disease, Familial dysautonomia (FD). FD results from a splice site mutation in intron 20 of the gene ELP1 (formerly called IKBKAP). As a consequence of the mis-splicing, exon 20 is variably skipped, the mutant mRNA degraded, resulting in reduced levels of the encoded protein, Elp1.. Interestingly, the ability to splice the mutated pre-mRNA varies according to tissue type, with neurons least capable of splicing the mutated pre- mRNA. While the majority of the clinical deficits are due to the devastation of the sensory and autonomic nervous systems, as patients enter their teens, their macular retinal ganglion cells progressively die, manifesting as visual loss. Mouse that are null for Elp1 are embryonic lethal so the field has, until now, taken two distinct strategies to generate mouse models to investigate FD: (i) generation of conditional knock-out mice (CKO) using cell-type specific cre-driven promoters; and (ii) transgenic mice that contain the human FD ELP1 splicing mutation. The former approach has generated mouse models that recapitulate the FD optic neuropathy that results from the progressive death of retinal ganglion cells. These mice are an excellent pre- clinical model for testing the effectiveness of gene therapy for preventing the progressive demise of retinal ganglion cells (Aim 1A). However this model does not lend itself to testing the effectiveness of splicing enhancer compounds since it lacks the FD splicing mutation. The latter approach has culminated in the generation of transgenic mice that include copies of the human FD ELP1 mutated gene. These mice are asymptomatic unless they are crossed to a hypomorph or null background mouse, but these compound mice are typically too sick to investigate consistently. Here we will make a new “hybrid” line by crossing in the human FD ELP1 mutated gene into our retina-specific CKO line (Pax6-cre;Elp1flox/flox) to overcome these major challenges to the field. In so doing, we will generate a single mouse model that manifests the human FD optic neuropathy, in an otherwise healthy background, and contains the splice site mutation, which can be used to test a variety of therapeutic approaches (Aim1A, B). The overall aim of this proposal is to assess and compare two methods for restoring normal levels of the Elp1 protein in this new model mouse retinae using: (i) AAV2- mediated gene therapy (gene reintroduction) of the wild type Elp1 gene injected intravitreously, and (ii) a novel splicing enhancer compound that has been shown to promote the inclusion of exon 20 in the mutant FD gene in the retina, delivered orally through diet. Our goal is to test which method best mitigates the death of retinal ganglion cells in addition to interrogating whether a combination of both methods (Aim 1C) will have additive effects on promoting the survival of retinal ganglion cells, given they work via two distinct pathways.

项目概要鉴于 FDA 最近批准了靶向 AAV 基因治疗平台和小分子剪接调节剂作为遗传性神经系统疾病的治疗方法，我们的目标是将这些强大的技术应用于预防遗传隐性患者发生的进行性视神经病变和失明家族性自主神经功能障碍 (FD) 是由 ELP1 基因内含子 20 的剪接位点突变引起的。（以前称为 IKBKAP）由于错误剪接，外显子 20 被不同程度地跳过，即突变体。 mRNA 降解，导致编码蛋白 Elp1 水平降低。突变的前 mRNA 根据组织类型而变化，神经元剪接突变的前 mRNA 的能力最弱。虽然大多数临床缺陷是由于感觉和自主神经的破坏造成的。神经系统，当患者进入青少年时，他们的黄斑视网膜神经节细胞逐渐死亡， Elp1 缺失的小鼠是胚胎致死的，因此迄今为止，该领域尚未取得进展。两种不同的策略来生成用于研究 FD 的小鼠模型：(i) 生成条件性基因敲除小鼠 (CKO) 使用细胞类型特异性的 cre 驱动启动子；以及 (ii) 含有人 FD ELP1 的转基因小鼠前一种方法已经生成了重现 FD 光学的小鼠模型。视网膜神经节细胞进行性死亡导致的神经病变这些小鼠是极好的预治疗对象。用于测试基因治疗预防视网膜进行性死亡有效性的临床模型然而，该模型不适合测试剪接的有效性。增强子化合物，因为它缺乏 FD 剪接突变。产生含有人类 FD ELP1 突变基因的转基因小鼠。无症状，除非它们与亚型或零背景小鼠杂交，但这些复合小鼠通常病得太重而无法持续进行调查，在这里我们将通过交叉来制作新的“混合”品系。将人类 FD ELP1 突变基因引入我们的视网膜特异性 CKO 系（Pax6-cre；Elp1flox/flox）以克服这些主要问题在此过程中，我们将生成一个表现人类 FD 光学的小鼠模型。神经病变，在其他健康的背景下，并包含剪接位点突变，可用于测试各种治疗方法（Aim1A、B）该提案的总体目标是评估和比较。使用以下两种方法恢复该新模型小鼠视网膜中 Elp1 蛋白的正常水平：(i) AAV2- 玻璃体内注射野生型 Elp1 基因介导的基因治疗（基因再引入），以及 (ii) 一种新型剪接增强剂化合物已被证明可以促进突变型 FD 基因中外显子 20 的包含我们的目标是测试哪种方法最能减轻视网膜的死亡。神经节细胞除了询问两种方法的组合（目标 1C）是否会产生累加性鉴于它们通过两种不同的途径发挥作用，因此对促进视网膜神经节细胞的存活有影响。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Reduction of retinal ganglion cell death in mouse models of familial dysautonomia using AAV-mediated gene therapy and splicing modulators.

使用 AAV 介导的基因治疗和剪接调节剂减少家族性自主神经失调小鼠模型中的视网膜神经节细胞死亡。

DOI：
发表时间：
2023-10-30
期刊：
影响因子：
4.6
作者：
Schultz, Anastasia;Cheng, Shun;Kirchner, Emily;Costello, Stephanann;Miettinen, Heini;Chaverra, Marta;King, Colin;George, Lynn;Zhao, Xin;Narasimhan, Jana;Weetall, Marla;Slaugenhaupt, Susan;Morini, Elisabetta;Punzo, Claudio;Lefcort, France
通讯作者：
Lefcort, France