Gene Therapy for Hearing and Balance Disorders

项目摘要 该项目将开发一种基因治疗策略来治疗与3A型Usher综合征相关的听力损失 (USH3A)。这将涉及在USH3A小鼠模型中优化病毒载体，测试最好的 用于非人类灵长类动物和人类毛细胞的有效性和耳蜗毒性的载体以及测试序列 人类USH3A基因(CLRN1)的变异与致病性。在项目完成后，我们将 能够要求与食品和药物管理局召开IND前会议，以确定具体要求 用于临床前测试。 目的1测试不同的AAV载体对小鼠耳蜗毛细胞的基因传递。三个新的AAV- 相关载体在转导耳蜗毛细胞方面显示出良好的前景，我们将对它们进行全部测试，以确定 最好的。然后，我们将在Clrn1-/-和N48K小鼠模型中测试AAV介导的毛细胞功能的恢复。 最后，我们将测试AAV介导的Clrn1表达对听力的毒性。 目的2是测试新型AAV载体在体外培养的人毛细胞中的效果，并在非人类中进行测试 灵长类载体圆窗给药的效率、毒性和免疫原性 膜注射。我们将测试目标1中最好的两个载体，表达GFP来评估病毒的程度 毛细胞转导。我们还将描述GFP-和CLRN1-的毒性和系统分布。 编码AAV载体。免疫反应可能会干扰后续的AAV注射，或者可能导致 对内耳的毒性，所以我们将确定AAV注射到耳朵是否会导致中和 抗体或T细胞反应。 在目标3中，我们将描述人类对外淋巴中适用的血清型的预先存在的免疫，使用 在其他听力障碍的手术中收集的外淋巴。我们还将确定是否有一个 外淋巴液抗体效价与血清抗体效价的相关性。许多变种已经在 CLRN1基因是致病基因，但并非全部都是已知的致病基因。确定USH3A中CLRN1中的哪些变体 患者是致病的，我们将建立一种利用Clrn1-/-小鼠的互补试验，使用AAV来表达 Clrn1具有等同于未知意义的人类变异的特定突变，并评估 通过电生理学和电子显微镜进行功能挽救。

Project Summary This project will develop a gene therapy strategy to treat hearing loss associated with Usher Syndrome type 3A (USH3A) in human. This will involve optimizing viral vectors in mouse models of USH3A, testing the best vectors for efficacy and cochlear toxicity in non-human primates and in human hair cells, and testing sequence variants in the human USH3A gene (CLRN1) for pathogenicity. At the completion of the project, we will be able to request a pre-IND meeting with the Food and Drug Administration to determine specific requirements for preclinical testing. Aim 1 is to test different AAV vectors for gene delivery to cochlear hair cells in mouse. Three new AAV- related vectors have shown promise in transducing cochlear hair cells, and we will test them all to identify the best. We will then test AAV-mediated restoration of hair cell function in the Clrn1-/- and N48K mouse models. Finally, we will test toxicity to hearing of AAV-mediated Clrn1 expression. Aim 2 is to test the efficacy of novel AAV vectors in human hair cells in vitro, and to test, in non-human primates, the efficiency, toxicity and immunogenicity of these vectors administered by round window membrane injection. We will test the two best vectors from Aim 1, expressing GFP to assess the degree of viral transduction of hair cells. We will also describe toxicity and systemic distribution of GFP- and CLRN1- encoding AAV vectors. An immune response might interfere with subsequent AAV injections or could lead to toxicity in the inner ear, so we will determine whether AAV injection into the ear induces neutralizing antibody or T-cell response. In Aim 3 we will characterize human pre-existing immunity against applicable serotypes in perilymph, using perilymph collected during surgery for other auditory disorders. We will also determine whether there is a correlation between antibody titers in perilymph and serum. Many variants have been identified in the CLRN1 gene but not all are known to be pathogenic. To determine which variants in CLRN1 in USH3A patients are pathogenic, we will set up a complementation assay using the Clrn1-/- mouse, use AAV to express Clrn1 with specific mutations equivalent to human variants of unknown significance, and assess degree of functional rescue with electrophysiology and electron microscopy.