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: 8618153
• 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/8618153
• 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; Gene Expression Profile; Genes; Genetic; 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 HDAC4 protein; human disease; immunocytochemistry; improved; interest; medical schools; mouse model; next generation sequencing; overexpression; photoreceptor degeneration; pre-doctoral; professor; programs; protective effect; public health relevance; research study; retinal rods; screening; tool; 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(由申请者提供)：这份建议书描述了一项为期5年的培训计划，旨在发展分子视网膜治疗的学术生涯。首席研究员最近完成了眼科住院医师和玻璃体视网膜手术奖学金。拟议的翻译研究计划将确定继发于变性的杆状光感受器基因表达的变化，这些变化是可以干预的，然后开发针对这些候选基因中最有希望的基因的载体。该计划将需要广泛的分子生物学技术方面的专业知识，包括像RNA-seq这样的尖端工具。康斯坦斯·切普科博士是分子生物学、遗传学和病毒学应用于视网膜研究的公认专家，他将指导这一新调查领域的首席研究员。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博士已经培训了55名博士后和博士后学员，其中许多人已经进入学术教职。一个由在下一代测序、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顾问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州眼耳医院和哈佛医学院非常丰富的视力研究和科学社区进行，为教育和合作提供许多额外的机会。研究将集中在识别Rd1视网膜视杆感光器中可能影响视杆和间接视锥感光细胞存活的分子通路。电穿孔技术 最近发现，将组蛋白脱乙酰基酶4(HDAC4)注入RD1视网膜可以保护视杆细胞免于退化。尽管锥体没有被电穿孔，但那些位于电穿孔棒附近的锥体也存活了下来。了解HDAC4的保护机制可能会识别能够减缓多个突变甚至其他疾病的光感受器死亡的分子途径。拟议的实验使用RNA-seq对三组小鼠-野生型小鼠、Rd1小鼠和HDAC4电穿孔的Rd1小鼠的Rd1进行转录组图谱分析。疾病过程改变的基因表达，并在HDAC4电穿孔RD1视网膜的视杆中逆转，将突出感兴趣的候选基因。免疫细胞化学、原位杂交和定量聚合酶链式反应将独立地证实转录组分析中发现的基因表达的变化。最有希望的候选基因将被克隆到质粒中，以便电穿孔进入杆状细胞。产生最佳救援表型的构建体将被整合到腺相关病毒载体(AAV)中，以实现稳定的泛视网膜基因转导。通过行为学检测和视网膜电图检测AAV载体对小鼠视功能的影响。拟议的研究和培训计划解决了重要的科学问题，同时为首席调查员在这一翻译研究领域的独立调查职业生涯做好准备。