Development of Gene Therapy for Hereditary Deafness using Rational Protein Engineering

项目摘要 耳聋和失明是两种最常见和最具破坏性的神经系统疾病。很少 致命的是，它们将病人与他们生活的世界和他们所爱的人分开，一辈子。在许多情况下， 耳聋和失明是由单一基因缺陷引起的，通常是遗传的突变基因拷贝 都是父母的Usher综合征是一种毁灭性的遗传性耳聋和失明， 九个基因中的任何一个其中一个基因PCDH 15突变后会导致1F型Usher综合征，表现为 严重先天性耳聋、平衡障碍和进行性失明。没有治疗方法。新方法 正迅速发展成为治疗单基因疾病的基因疗法，这些方法改变了病人的生活。 在这里，我们建议开发Usher综合征1F型的基因治疗策略，并启动其通往 临床治疗疾病的不同病理。 腺相关病毒（Adeno-associated virus，AAV）载体已被发现用于内分泌系统的基因治疗是高效和有效的。 耳及其在开发基因添加疗法治疗Usher综合征1F型中的应用， 有吸引力的方法。然而，几乎6 kb的PCDH 15编码序列太大而不适合AAV，其具有 容量约为4.7kb。大多数PCDH 15类似于11个珠子的“珠链”结构，具有结合结构域 在N-和C-末端。我们已经开发了新的“mini-PCDH 15”结构， 但缺少4-5个珠粒，因此适合于AAV载体。它们在体外显示正常结合;至少两个 在我们的Pcdh 15敲除小鼠模型中进行听力拯救。我们将使用这些和新的mini-PCDH 15构建体， Pcdh 15基因敲除小鼠的听力和前庭功能恢复。我们还将在计算机和体外 结构生物学和蛋白质化学来仔细表征每一个成功微型PCDH 15， 迭代优化设计的mini-PCDH 15。最后，我们将测试性能最佳的mini-PCDH 15， 在非人灵长类动物耳中的定位和毒性，作为其临床前评价的一部分。 由于PCDH 15在内耳中的机械应力大于在眼睛中的机械应力，因此工程化的mini-PCDH 15 成功挽救听力或平衡的结构也有可能阻止甚至逆转听力或平衡。 患者视力逐渐下降。此外，我们在这项研究中开发的方法使用理性的，迭代的， 基于结构微基因方法开发Usher 1F疗法将适用于许多其他耳聋 编码序列不适合AAV的基因。 1

PROJECT SUMMARY Deafness and blindness are two of the most common and most devastating neurological disorders. Seldom fatal, they separate patients from the world they live in and the people they love, for a lifetime. In many cases, combined deafness and blindness result from a single gene defect, usually with a mutant gene copy inherited from both parents. Usher syndrome is a devastating hereditary deafness and blindness caused by mutation of any of nine genes. One gene, PCDH15, when mutated, causes Usher syndrome type 1F, manifesting as profound congenital deafness, balance disorder and progressive blindness. There is no treatment. New methods are rapidly being developed for gene therapy to treat monogenic disorders, methods that change patient lives. Here, we propose to develop a gene therapy strategy for Usher Syndrome Type 1F and to initiate its path to the clinic to treat different pathologies of the disease. Adeno-associated virus (AAV) vectors have been found to be efficient and effective for gene therapy in the inner ear and their application in development of gene addition therapies to treat Usher syndrome type 1F is an attractive approach. However, the PCDH15 coding sequence of almost 6 kb is too large to fit in AAV, which has a capacity of ~4.7 kb. Most of PCDH15 resembles a “bead chain” structure of 11 beads, with binding domains at N- and C-terminal ends. We have developed novel “mini-PCDH15” constructs that retain the binding domains but lack 4-5 beads, and that consequently fit in AAV vectors. They show normal binding in vitro; at least two rescue hearing in our Pcdh15-knockout mouse model. We will use these and new mini-PCDH15 constructs to rescue hearing and vestibular function in Pcdh15-knockout mice. We will also employ in silico and in vitro structural biology and protein chemistry to carefully characterize each successful mini-PCDH15, and use that for iterative optimization of engineered mini-PCDH15s. Finally, we will test the best performing mini-PCDH15 for localization and toxicity in non-human primate ear as part of their preclinical evaluation. Because mechanical stress on PCDH15 is greater in the inner ear than in the eye, the engineered mini-PCDH15 constructs that successfully rescue hearing or balance will also have the potential to halt or even reverse the progressive vision loss in patients. In addition, methods we develop in this study using a rational, iterative, structure-based mini-gene approach to develop Usher 1F therapy will be applicable to many other deafness genes with coding sequences that do not fit in AAV. 1