Exploring MYO7A function in novel mouse models and improving AAV-Dual Vector Gene Therapy for Usher Syndrome 1B

本应用的目标是开发一种安全有效的双重腺相关病毒(AAV)基因 治疗Usher综合征1B(USH1B)USH1B是一种隐性遗传性疾病 出生后出现耳聋和前庭缺陷，进行性视网膜变性和视力丧失 第一个十年。开发USH1B患者视力丧失治疗方法的主要障碍是缺乏动物 真实再现视网膜表型的模型。以前描述过的、自然发生的振动器1 菌株只显示视网膜功能的细微变化，没有视网膜退化。他们也很难工作 由于在Shaker1菌株之间观察到的MYO7A表达水平不同， 突变。为了克服这些限制，我们设计了两种新的USH1B小鼠模型，1)完整 MYO7A基因敲除(MYO7A-/-)或2)条件性纯视网膜基因敲除MYO7A(CKO MYO7A-/-)。在《目标1》中，我将 在这些新的模型中表征视网膜的结构和功能，以确定潜在的结果测量 MYO7A基因治疗。这些小鼠提供了评估在没有NO的视网膜中进行基因补充的机会 内源性MYO7A，以获得对疾病机制的关键见解，并观察 可能存在MYO7A在小鼠和人视网膜中的作用。仅限视网膜，CKO MYO7A-/-小鼠允许 分离前庭缺陷和第一次进行视觉引导行为测试的能力 USH1B模型。AAV已成为视网膜基因治疗的金标准。但是，它的大小 MYO7A基因超过了其包装容量(~5kb)。为了克服这一障碍，双AAV载体平台 其中大基因被分成两个AAV载体，带有基因的5‘和3’半部分 被包装在单独的衣壳里。当共注射时，5‘和3’‘半部分重组形成全长基因和 编码全长蛋白质。我们已经证明了在体内传递全长MYO7A的能力 这些双重AAV载体平台。然而，安全性和有效性方面的担忧依然存在。我们之前的结果表明， 双重AAV-MYO7A促进视网膜下注射C57BL/6小鼠的毒性，我们认为这是由形成引起的 来自5‘基因’一半‘的截短蛋白质。在目标2中，我将通过改变 MYO7A基因的裂解点及AAV衣壳突变体对5‘端载体不重组时的沉默作用 具有3‘向量。双重AAV载体的一个问题是5‘和3’基因之间的低重组率 一半。我将通过用锌指引导基因一半的串联来提高重组效率 通过将独特的锌指结合结构域整合到我们的双载体系统中。我假设这一点 将我们的双重载体以正确的方向“拉”在一起，从而增加重组和转基因。 表情。增加的重组率/总体MYO7A表达将允许总的 载体剂量，增加了安全功能。通过提高双AAV-MYO7A载体的安全性和效率，我们 可以收集必要的IND使能数据，以支持USH1B基因疗法的临床应用。

The goal of this application is to develop a safe and effective dual Adeno associated virus (AAV)-based gene therapy for the treatment of Usher Syndrome 1B (USH1B). USH1B is a recessively inherited disease that presents with deafness and vestibular defects from birth, progressive retinal degeneration, and vision loss within the first decade. A major obstacle to developing treatments for vision loss in USH1B patients is the lack of animal models that faithfully recapitulate the retinal phenotype. Previously characterized, naturally-occurring shaker1 strains display only subtle changes in retinal function and no retinal degeneration. They are also difficult to work with due to the variable MYO7A expression levels observed among shaker1 strains that have different underlying mutations. To overcome these limitations, we engineered two new mouse models of USH1B with 1) a complete knockout of MYO7A (Myo7a-/-) or 2) conditional, retina-only knockout of MYO7A (CKO Myo7a-/-). In Aim 1, I will characterize retinal structure and function in these novel models to identify potential outcome measures for MYO7A gene therapy. These mice provide the opportunity to evaluate gene supplementation in a retina with no endogenous MYO7A, to gain key insights into the mechanism of the disease, and observe any differences that may exist between the roles of MYO7A in mouse vs human retina. Retina only, CKO Myo7a-/- mice allow for separation of the vestibular defect and the ability, for the first time, to conduct visually-guided behavior tests in a model of USH1B. AAV has emerged as the gold standard in retinal gene therapy. However, the size of the MYO7A cDNA exceeds its packaging capacity (~5kb). To overcome this hurdle, dual AAV vector platforms have been developed wherein large genes are split into two AAV vectors, with the 5' and 3' halves of the gene packaged into separate capsids. Upon co-injection, the 5' and 3' `halves' recombine to form full-length gene and encode full length protein. We have already demonstrated the ability to deliver full length MYO7A in vivo using these dual AAV vector platforms. However, safety and efficacy concerns remain. Our previous results show that dual AAV-MYO7A promote toxicity in subretinally injected C57BL/6 mice that we believe is caused by formation of truncated protein from the 5' gene `half'. In Aim 2, I will eliminate formation of truncated protein by changing the split point of MYO7A and investigating AAV capsid mutants to “silence” the 5' vector when it is not recombined with the 3' vector. A concern with dual AAV vectors is the low rate of recombination between the 5' and 3' gene halves. I will increase recombination efficiency by directing concatemerization of gene halves with zinc fingers through the incorporation of unique zinc finger binding domains into our dual vector system. I hypothesize this will “pull” our dual vectors together in their proper orientation, thus increasing recombination and transgene expression. The increased recombination rate/overall MYO7A expression will allow for a reduction in the total vector dose, an added safety feature. By improving the safety and efficiency of dual AAV-MYO7A vectors, we can gather the necessary IND-enabling data to support clinical application of a gene therapy for USH1B.