Development of Gene Therapy for Hereditary Deafness using Rational Protein Engineering

利用合理蛋白质工程开发遗传性耳聋基因疗法

基本信息

批准号：
10649587
负责人：
DAVID P COREY
金额：
$ 62.29万
依托单位：
MASSACHUSETTS EYE AND EAR INFIRMARY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-17 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10649587
关键词：
Antibodies Binding Biological Assay Biophysics Birth Blindness C-terminal CDH23 gene Cells Clinic Clinical Treatment Clinical Trials Cochlea Code Cryoelectron Microscopy Dependovirus Development Dimerization Disease Ear Elasticity Electron Microscopy Engineering Equilibrium Evaluation Extracellular Domain Eye Genes Genetic Hair Cells Hearing Histopathology Human In Vitro Inherited Injections Knock-out Knockout Mice Knowledge Labyrinth Length Love Macaca fascicularis Measures Mechanical Stress Mendelian disorder Methods Molecular Motors Morphology Motor Mus Mutate Mutation N-terminal Negative Staining Neonatal Nonmuscle Myosin Type IIA PCDH15 gene Parents Pathology Patients Performance Persons Phenotype Protein Chemistry Protein Engineering Proteins Retinal Diseases Running Single-Gene Defect Structural Models Structural Protein Structure Testing Toxic effect Usher Syndrome Variant Vestibular Hair Cells Wild Type Mouse X-Ray Crystallography adeno-associated viral vector biophysical properties congenital deafness deafness dimer efficacy testing equilibration disorder experimental study gene therapy genetic deafness hearing impairment hereditary blindness hereditary hearing loss improved in silico light microscopy link protein mechanotransduction models and simulation molecular dynamics mouse model mutant nanoscale neonatal mice nervous system disorder nonhuman primate novel preclinical evaluation preservation protein purification rapid technique simulation structural biology success therapeutic gene vestibulo-ocular reflex

项目摘要

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

项目摘要耳聋和失明是最常见，最毁灭性的神经系统疾病之一。很少致命的是，他们将患者与生活中的世界和他们所爱的人分开。在许多情况下，单个基因缺陷导致死亡和失明的结合，通常具有遗传的突变基因拷贝来自父母。 Usher综合征是由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。一个基因，PCDH15，当突变时会导致usher综合征1F，表现为深远的先天性死亡，平衡障碍和进行性失明。没有治疗。新方法正在迅速开发用于治疗单基因疾病的基因治疗，这是改变患者生活的方法。在这里，我们建议为Usher综合征1F制定基因治疗策略，并启动其通往该策略治疗疾病的不同病理的诊所。已发现与腺相关病毒（AAV）载体对内部基因治疗既有效又有效 EAR及其在开发基因添加疗法中的应用来治疗1F的USHER综合征是一种有吸引力的方法。但是，将近6 kb的PCDH15编码序列太大而无法适合AAV，其中具有容量约为4.7 kb。大多数PCDH15都类似于11个珠子的“珠链”结构，具有绑定域在n-和c末端。我们已经开发了保留结合域的新颖的“迷你PCDH15”构造但是缺少4-5个珠子，因此适合AAV矢量。它们在体外表现出正常的结合；至少两个在我们的PCCDH15敲除鼠标模型中救助听证会。我们将使用这些新的Mini-PCDH15结构 PCDH15敲除小鼠中的救助听力和前庭功能。我们还将在计算机和体外雇用结构生物学和蛋白质化学，以仔细表征每个成功的迷你PCDH15，并将其用于工程迷你PCDH15S的迭代优化。最后，我们将测试最佳性能的迷你PCDH15 作为临床前评估的一部分，非人类灵长类动物耳朵中的定位和毒性。因为内耳的机械应力比眼睛更大，所以工程化的迷你PCDH15 成功挽救听力或平衡的构造也有可能停止甚至扭转患者的渐进视力丧失。此外，我们在本研究中开发的方法使用了理性，迭代，基于结构的小型基因方法开发USHER 1F疗法将适用于许多其他死亡具有不适合AAV的编码序列的基因。 1