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

项目摘要 鉴于最近FDA批准了靶向AAV基因治疗平台和小分子剪接， 调节剂作为治疗遗传神经系统疾病，我们的目标是将这些强大的技术应用于 预防隐性遗传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（Familial dysautonomia，FD）FD由ELP 1基因内含子20中的剪接位点突变引起 （以前称为IKBKAP）。作为错误剪接的结果，外显子20被跳跃，突变体 mRNA降解，导致编码蛋白Elp 1水平降低。有趣的是， 突变的前mRNA根据组织类型而变化，神经元最不能剪接突变的前mRNA。 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，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， 表现为视力丧失没有Elp 1基因的小鼠是胚胎致死的，所以到目前为止， 产生研究FD的小鼠模型的两种不同策略：（i）产生条件性敲除小鼠 (CKO)使用细胞类型特异性cre驱动的启动子;和（ii）含有人FD ELP 1的转基因小鼠 剪接突变前一种方法已经产生了重现FD光学的小鼠模型 由于视网膜神经节细胞逐渐死亡而引起的神经病。这些老鼠是一个很好的预- 用于测试基因治疗预防视网膜病变进行性死亡的有效性的临床模型 神经节细胞（Aim 1A）。然而，该模型并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。后一种做法的结果是， 产生包括人FD ELP 1突变基因拷贝的转基因小鼠。这些小鼠 无症状，除非它们与亚型或无效背景小鼠杂交，但这些复合小鼠 通常都病得很重无法持续进行调查在这里，我们将使一个新的“杂交”线交叉在 将人FD ELP 1突变基因导入我们的视网膜特异性CKO系（Pax 6-cre; Elp 1flox/flox），以克服这些主要的 外地的挑战。这样做，我们将产生一个单一的小鼠模型，体现了人类FD光学 神经病变，在其他健康的背景下，并含有剪接位点突变，其可用于 测试各种治疗方法（目标1A，B）。本提案的总体目标是评估和比较 使用以下两种方法在该新模型小鼠视网膜中恢复Elp 1蛋白的正常水平：（i）AAV 2- 玻璃体内注射野生型Elp 1基因的介导的基因治疗（基因再引入），和（ii）一种新的 剪接增强子化合物，其已显示促进突变FD基因中外显子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坏死的死亡。 神经节细胞除了询问两种方法的组合（目的1C）是否会有添加剂外， 对促进视网膜神经节细胞存活的作用，因为它们通过两种不同的途径发挥作用。

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.