Development of Gene Therapy for Hereditary Deafness using Rational Protein Engineering

项目总结 耳聋和失明是两种最常见、最具破坏性的神经系统疾病。很少 致命的是，他们将患者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， 混合性耳聋和失明是由单个基因缺陷引起的，通常是遗传的突变基因拷贝。 来自父母双方。Usher综合征是一种由基因突变引起的毁灭性遗传性耳聋和失明。 九个基因中的任何一个。一种名为PCDH15的基因发生突变时，会导致亚瑟综合征1F型，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 正在迅速开发用于治疗单基因疾病的基因疗法，这种方法可以改变患者的生活。 在这里，我们建议开发一种针对Usher综合征1F型的基因治疗策略，并启动其通往 临床治疗本病的不同病理类型。 腺相关病毒(AAV)载体已被发现在体内进行基因治疗是有效和有效的 EAR及其在基因加成疗法治疗Usher综合征1F型中的应用 吸引人的方法。然而，几乎6kb的PCDH15编码序列太大，不适合AAV，它具有 容量约4.7kb。PCDH15的大部分类似于由11个珠子组成的“珠链”结构，带有结合结构域 在N端和C端。我们已经开发出了保留结合结构域的新型“mini-PCDH15”结构 但缺少4-5个珠子，因此适合AAV载体。它们在体外表现出正常的结合；至少有两个 我们的PCDH15基因敲除小鼠模型中的救援听力。我们将使用这些和新的迷你PCDH15构造来 PCDH15基因敲除小鼠的挽救听力和前庭功能。我们还将在硅胶和体外实验中使用 结构生物学和蛋白质化学来仔细描述每个成功的迷你PCDH15的特征，并将其用于 工程小型PCDH15的迭代优化。最后，我们将测试性能最好的迷你PCDH15 非人灵长类动物耳朵的定位和毒性作为其临床前评估的一部分。 由于PCDH15在内耳的机械应力大于眼睛，因此设计的迷你PCDH15 成功挽救听力或平衡的构造也有可能停止甚至逆转 患者进行性视力丧失。此外，我们在本研究中开发的方法使用理性的、迭代的、 基于结构的微基因方法开发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