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，最毁灭性的神经病学致命，他们将患者与生活中的世界和他们所爱的人分开从单个基因缺陷中结合了耳聋和失明，通常带有柔和的基因副本继承父母双方都是乌瑟综合症。九个基因中的任何一个。深刻的聋哑人，平衡障碍和渐进式失明正在迅速开发基因以信任单基因疾病，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）载体是有效且有效的疗法内部的 EAR和在基因添加基因曲折的发展中的应用中，以信任Usher综合征1F是一种但是，有吸引力的方法。〜4.7 kb的capace。在N和C末端，我们开发了新颖的“ Mini-PCDH15”构造但是缺乏4-5个珠子，并且在AAV载体中均显示出正常的结合在我们的PCDH15敲除鼠标模型中，我们将使用故事Mini-PCDH15构造 PCDH15敲除小鼠的救助听力和前庭功能。结构生物学和蛋白质化学，以仔细表征每个成功的迷你PCDH15，并将其用于最终，我们将测试执行迷你PCDH15的迭代优化作为预先评估的一部分，非人类灵长类动物耳朵中的本地化和毒性。因为眼睛内部的PCDH15上的机械应力更大，因此工程的迷你PCDDH15 成功拯救听力或平衡的构造也有可能停止甚至扭转脚趾的构造逐渐丧失专门的视力，我们在本研究中使用的方法使用，基于结构的小型基因开发USHER 1F疗法将适用于许多其他耳聋具有不适合AAV的编码序列的基因。 1