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

• 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
• 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' 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

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.

项目总结 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 预防遗传性隐性遗传性视神经病变和失明 疾病，家族性自主神经障碍(FD)。由ELP1基因第20内含子的剪接点突变引起的FD (以前称为IKBKAP)。作为错误剪接的结果，外显子20被可变地跳过，突变体 信使核糖核酸降解，导致编码蛋白Elp1水平降低。有趣的是，能够拼接 突变的前-mRNA根据组织类型的不同而不同，神经元拼接突变的前-mRNA的能力最低 MRNA.虽然大多数临床缺陷是由于感觉和自主神经的破坏 神经系统，随着患者进入青少年，他们的黄斑视网膜神经节细胞逐渐死亡， 表现为视力丧失。Elp1基因为空的小鼠是胚胎致死的，所以到目前为止，能量场已经 两种不同的策略来产生小鼠模型来研究FD：(I)产生条件性基因敲除小鼠 使用细胞型特异性cre驱动启动子的(CKO)；和(Ii)含有人FD ELP1的转基因小鼠 剪接突变。前一种方法已经产生了概括FD视觉的小鼠模型 视网膜神经节细胞进行性死亡引起的神经病。这些小鼠是一只优秀的小鼠 检测基因治疗预防视网膜进行性死亡有效性的临床模型 神经节细胞(目标1A)。然而，该模型不适合于测试拼接的有效性 增强子化合物，因为它缺乏FD剪接突变。后一种方法的结果是 产生包含人类FD ELP1突变基因副本的转基因小鼠。这些老鼠是 没有症状，除非它们杂交到低形态或零背景小鼠，但这些复合小鼠 通常都病得太重了，无法持续调查。在这里，我们将通过杂交创造一个新的“杂交”品系 人FD ELP1基因突变到我们的视网膜特异性CKO系(Pax6-cre；Elp1flx/Flox)以克服这些主要问题 战场上的挑战。在这样做的过程中，我们将生成一个显示人类FD视觉的单一鼠标模型 神经病，在其他方面是健康的背景，并包含剪接位点突变，可用于 测试各种治疗方法(Aim1A、B)。这项提案的总体目标是评估和比较 用两种方法恢复这种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基因的介导性基因治疗(基因再导入)，以及(Ii)一种新的 剪接增强剂化合物，已被证明促进突变的fd基因的外显子20的包含 在视网膜中，通过饮食口服。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)是否会有相加 对促进视网膜神经节细胞存活的影响，因为它们通过两种不同的途径发挥作用。

项目成果

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

• DOI: 10.1038/s41598-023-45376-w
• 发表时间: 2023-10-30
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Schultz, Anastasia;Cheng, Shun-Yun;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, Frances
• 通讯作者: Lefcort, Frances