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

探索新型小鼠模型中的 MYO7A 功能并改进针对 Usher 综合征 1B 的 AAV 双载体基因疗法

基本信息

批准号：
9892873
负责人：
Kaitlyn Rose Calabro
金额：
$ 1.93万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-28 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9892873
关键词：
Affect Animal Model Back Binding Birth Blindness C57BL/6 Mouse Capsid Carrying Capacities Cell Nucleus Choroideremia Clinical Trials Cochlear Implants Complementary DNA Congenital Abnormality Data Defect Dependovirus Disease Dose Engineering Ensure Equilibrium Equine Infectious Anemia Virus Event Exhibits Functional disorder Gene Transduction Agent Genes Genetic Recombination Genetic Transcription Genotype Goals Gold Head Hereditary Disease Human Inherited Injections Knock-out Knockout Mice Leber&apos s amaurosis Length Lentivirus Vector Live Birth MYO7A gene Mediating Methods Modeling Mus Mutation Outcome Measure Patients Phenotype Photoreceptors Production Proteins Reporting Research Retina Retinal Retinal Degeneration Retinal gene therapy Role Safety Split Genes Structure Supplementation System Tail Time Toxic effect Transgenes Usher Syndrome Viral Vector Vision Disorders Visual Fields Work Zinc Fingers adeno-associated viral vector behavior test clinical application deaf deafness disease-causing mutation experience experimental study gene product gene therapy hearing restoration improved in vivo insight mouse model mutant novel recombinase-mediated cassette exchange therapeutic protein transgene expression vector vector genome

项目摘要

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.

本申请的目标是开发一种安全有效的基于双腺相关病毒（AAV）的基因用于治疗 Usher 综合征 1B (USH1B) 的疗法是一种隐性遗传性疾病。表现为先天性耳聋和前庭缺陷、进行性视网膜变性以及视力丧失第一个十年，开发 USH1B 患者视力丧失治疗方法的一个主要障碍是缺乏动物。忠实再现先前描述的自然发生的视网膜表型的模型。菌株仅表现出视网膜功能的细微变化，并且没有视网膜变性，它们也很难发挥作用。由于在具有不同基础的 shaker1 菌株中观察到的 MYO7A 表达水平存在差异为了克服这些限制，我们设计了两种新的 USH1B 小鼠模型，具有 1) 完整的突变。敲除 MYO7A (Myo7a-/-) 或 2) 有条件、仅视网膜敲除 MYO7A (CKO Myo7a-/-) 在目标 1 中，我会。表征这些新颖模型中的视网膜结构和功能，以确定潜在的结果测量 MYO7A 基因治疗为评估视网膜中的基因补充提供了机会。内源性 MYO7A，以获得对该疾病机制的关键见解，并观察任何差异可能只存在于 MYO7A 在小鼠视网膜和人类视网膜中的作用之间，CKO Myo7a-/- 小鼠允许。分离前庭缺陷并首次能够在视觉引导下进行行为测试 USH1B 模型已成为视网膜基因治疗的黄金标准。 MYO7A cDNA 超出其包装容量（约 5kb）为了克服这一障碍，双 AAV 载体平台具有。已经开发出大基因被分成两个 AAV 载体，基因的 5' 和 3' 一半共注射后，5' 和 3'“半部”重组形成全长基因并包装成单独的衣壳。我们已经证明了使用体内递送全长 MYO7A 的能力。然而，我们之前的结果表明，这些双 AAV 载体平台的安全性和有效性仍然存在。双 AAV-MYO7A 会促进视网膜下注射的 C57BL/6 小鼠的毒性，我们认为这是由形成引起的来自 5' 基因“half”的截短蛋白质在目标 2 中，我将通过改变来消除截短蛋白质的形成。 MYO7A 的分裂点并研究 AAV 衣壳突变体以在未重组时“沉默”5' 载体使用 3' 载体时，双 AAV 载体的一个问题是 5' 和 3' 基因之间的重组率较低。我将通过用锌指指导基因半部的串联来提高重组效率。通过将独特的锌指结合域整合到我们的双载体系统中。会将我们的双载体以正确的方向“拉”在一起，从而增加重组和转基因重组率/总体 MYO7A 表达的增加将导致总数的减少。载体剂量，一个附加的安全特性通过提高双 AAV-MYO7A 载体的安全性和效率，我们可以收集必要的 IND 数据以支持 USH1B 基因疗法的临床应用。