Molecular Profiling of Rods to Identify New Therapies for Retinal Degenerations

杆状细胞的分子分析以确定视网膜变性的新疗法

• 批准号: 8988570
• 负责人: David WU
• 金额: $ 22.2万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8988570
• 关键词: Address; Advisory Committees; Area; Behavioral; Behavioral Assay; Biological Assay; Candidate Disease Gene; Cessation of life; Clinical Trials; Collaborations; Communities; Cone; Data; Disease; Down-Regulation; Ear; Education; Electroporation; Evaluation; Eye; Faculty; Fellowship; Gene Expression; Genes; Genetic; HDAC4 gene; Health; Hypoxia Inducible Factor; Immunohistochemistry; In Situ Hybridization; Institutes; Intervention; Investigation; Lead; Massachusetts; Mediating; Medical; Mentors; Mentorship; Molecular; Molecular Biology; Molecular Biology Techniques; Molecular Profiling; Mus; Mutation; Operative Surgical Procedures; Ophthalmology; Pathway interactions; Phenotype; Photoreceptors; Plasmids; Positioning Attribute; Principal Investigator; Process; Program Development; Proteins; RNA; Research; Research Personnel; Research Training; Residencies; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinitis Pigmentosa; Scientist; Secondary to; Testing; Therapeutic; Training; Training Programs; Transcript; Transduction Gene; Translational Research; Up-Regulation; Viral Vector; Vision; Vision research; Wild Type Mouse; Work; adeno-associated viral vector; career; design; human disease; immunocytochemistry; improved; interest; medical schools; mouse model; next generation sequencing; novel therapeutics; overexpression; photoreceptor degeneration; pre-doctoral; professor; programs; protective effect; research study; retinal rods; screening; tool; transcriptome; transcriptome sequencing; vector; virology

DESCRIPTION (provided by applicant): This proposal describes a 5 year training program for the development of an academic career in molecular retinal therapeutics. The principal investigator has recently completed an ophthalmology residency and vitreoretinal surgical fellowship. The proposed translational research program will identify changes in rod photoreceptor gene expression secondary to degeneration that are amenable to intervention and then develop vectors that target the most promising of these candidate genes. The program will require expertise in a wide range of molecular biology techniques including cutting-edge tools like RNA-seq. Dr. Constance Cepko, a recognized expert in the application of molecular biology, genetics, and virology to retinal research, will mentor the principal investigator in this new area of investigation. As Professor of Genetics and Ophthalmology at Harvard Medical School and Howard Hughes Medical Institute Investigator, Dr. Cepko has trained over 55 pre- and postdoctoral trainees, many of whom have gone on to academic faculty positions. An advisory committee f accomplished scientists in the fields of next-generation sequencing, mouse models of retinal degeneration, and viral vectors will provide additional scientific and career mentorship. Finally, the training program will take place in the very rich vision research and scientific communities of the Massachusetts Eye and Ear Infirmary and Harvard Medical School, offering many additional opportunities for education and collaboration. Research will focus on identifying molecular pathways in rod photoreceptors from rd1 retinas that may influence rod, and indirectly cone photoreceptor survival. Electroporation of histone deacetylase 4 (HDAC4) into an rd1 retina was recently found to protect rods from degeneration. Although cones were not electroporated, those in the vicinity of electroporated rods also survived. Understanding the protective mechanisms of HDAC4 may identify molecular pathways capable of slowing photoreceptor death across multiple mutations or even other diseases. The proposed experiments use RNA-seq to conduct transcriptome profiling of rods from three groups - wild type mice, rd1 mice, and rd1 mice electroporated with HDAC4. Gene expression altered by the disease process and reversed in rods from HDAC4-electroporated rd1 retinas will highlight candidate genes of interest. Immunocytochemistry, in situ hybridization, and quantitative PCR will independently confirm changes in gene expression found during transcriptome analysis. The most promising candidate genes will be cloned into plasmids for electroporation into rods. The constructs producing the best rescue phenotype will be incorporated into adeno-associated viral vectors (AAV) for stable pan-retinal gene transduction. Visual function of mice receiving AAV vectors can be assayed through behavioral assays and ERG. The proposed research and training program addresses important scientific questions while preparing the principal investigator for a career of independent investigation in this area of translational research.

描述（由申请人提供）：该提案描述了一个为期5年的培训计划，旨在发展分子视网膜治疗学的学术生涯。首席研究员最近完成了眼科住院医师和玻璃体视网膜外科奖学金。拟议的转化研究计划将确定杆状光感受器基因表达继发于变性的变化，这些变化是可干预的，然后开发针对这些候选基因中最有希望的载体。该计划将需要广泛的分子生物学技术方面的专业知识，包括RNA-seq等尖端工具。constanance Cepko博士是公认的分子生物学、遗传学和病毒学应用于视网膜研究的专家，他将在这一新的研究领域指导首席研究员。作为哈佛医学院遗传学和眼科学教授和霍华德休斯医学研究所研究员，Cepko博士培养了超过55名博士后和博士生，其中许多人已经成为学术教职员工。一个由下一代测序、视网膜变性小鼠模型和病毒载体领域有成就的科学家组成的咨询委员会将提供额外的科学和职业指导。最后，培训计划将在马萨诸塞眼耳医院和哈佛医学院非常丰富的视觉研究和科学界进行，为教育和合作提供了许多额外的机会。研究将集中于确定rd1视网膜中杆状光感受器中可能影响杆状光感受器和间接锥状光感受器存活的分子途径。电穿孔的