Targeting AAV vectors to cell types involved in alcohol-induced liver injury

将 AAV 载体靶向参与酒精性肝损伤的细胞类型

• 批准号: 10403762
• 负责人: Holger Willenbring
• 金额: $ 9.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10403762
• 关键词: Alcohol-Induced Disorders; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcohols; Anti-Inflammatory Agents; Binding; Bioinformatics; California; Capsid; Cardiac Myocytes; Catheters; Cells; Cirrhosis; Clinical; Clinical Trials; Collaborations; Collagen; Creativeness; Critical Thinking; DNA; DNA Shuffling; Data; Development; Diet; Ethanol; Ethanol toxicity; Exhibits; Foundations; Gene Delivery; Gene Expression; Genome; Goals; Hematopoietic; Hepatocyte; Human; Inflammatory; Inflammatory Infiltrate; Kupffer Cells; Laboratories; Libraries; Life; Liver; Liver Cirrhosis; Liver Failure; Liver Fibrosis; Medicine; Mentors; Methods; MicroRNAs; Mus; Muscle Fibers; Myofibroblast; Oral; Organ; Parents; Patients; Physicians; Population; Predisposition; Production; Publishing; RNA; Reaction; Reporting; Research; Research Personnel; Research Training; Residual state; Scientist; Serotyping; Source; Specificity; Speed; Structure; Technology; Testing; Training; Tropism; Universities; Validation; Vertebral column; Viral; Viral Vector; Virus; alcohol response; base; career; cell type; cellular transduction; cholangiocyte; clinical application; clinical translation; cohort; experience; gene therapy; immunogenicity; in vivo; innovative technologies; insight; interest; liver inflammation; macrophage; mortality; mouse model; next generation sequencing; novel therapeutics; oval cell; prevent; progenitor; promoter; single-cell RNA sequencing; skills; training opportunity; transcription factor; vector; western diet

Project Summary Alcoholic liver disease is a major clinical challenge because it is associated with severe complications like liver cirrhosis and alcoholic hepatitis, which carry a high mortality. Our understanding of the mechanisms underlying alcoholic liver disease is still evolving; however, previous research has identified cells critically involved in the liver's response to alcohol-induced injury, including myofibroblasts, macrophage subsets and oval cells, which are referred to as ductular reaction in humans. Because of their unique and important functions in alcoholic liver disease, efficient and specific gene delivery to these cell types would have many applications in research and therapy of alcoholic liver disease, particularly if nonintegrating nontoxic adenoassociated viral (AAV) vectors could be used. The research scope of the proposed diversity supplement is the same as that of the parent R01 AA026578, that is, to establish efficient and specific transduction of myofibroblasts, macrophage subsets and oval cells with AAV vectors in mouse models of alcoholic liver disease. The supplement is complementary to R01 AA026578 because it uses a different approach: First, instead of selecting over a million newly generated capsids with unknown tropism in the cell types of interest as done in R01 AA026578, the supplement will identify among almost two hundred capsids previously successfully tested in other settings, including in the liver at the organ but not at the cell level, those that transduce the cell types of interest. Thus, the supplement maximizes the likelihood of successful targeting of the cell types of interest by including promising existing candidate capsids in the analysis. Second, because of the smaller scale of the candidate capsid cohort, the supplement will be able to employ single-cell RNA sequencing as an efficient method to identify capsids affording functional transduction of the cell types of interest. The use of essential and innovative technologies in the supplement, including mouse models of alcoholic liver disease, precise isolation of the principal liver cell populations by FACS, AAV production and single-cell RNA sequencing in combination with formal training in bioinformatic analysis of sequencing data provides a profound state-of-the-art research training opportunity that will serve as the foundation for the trainee's career plan of becoming a physician- scientist. As an essential part of the research training, the trainee will be able to practice and refine his newfound research skills in close collaboration with experienced junior scientists. A milestone-based structured mentoring plan developed by the experienced PI will further equip the trainee with the critical thinking, creativity, resourcefulness and oral and written presentations skills needed to succeed as an independent researcher and make impactful contributions to research and medicine.

项目摘要 酒精性肝病是一项重大的临床挑战，因为它与肝脏等严重并发症有关。 肝硬变和酒精性肝炎，死亡率很高。我们对潜在机制的理解 酒精性肝病仍在发展中；然而，先前的研究发现，细胞与酒精性肝病 肝脏对酒精损伤的反应，包括肌成纤维细胞、巨噬细胞亚群和卵圆细胞，这些细胞 在人类中被称为导管反应。因为它们在酒精中具有独特而重要的作用 肝脏疾病，高效和特异地将基因输送到这些细胞类型将在研究中有许多应用 和酒精性肝病的治疗，特别是在非整合非毒性腺相关病毒(AAV)的情况下 可以使用矢量。拟议的多样性补充方案的研究范围与 亲本R01 AA026578，即建立高效、特异的肌成纤维细胞、巨噬细胞转导系统 小鼠酒精性肝病模型中携带AAV载体的细胞亚群和卵圆形细胞。副刊是 与R01 AA026578互补，因为它使用了一种不同的方法：首先，不是在 如在R01AA026578中所做的，在感兴趣的细胞类型中具有未知取向的新产生的衣壳蛋白， 该补充材料将在之前在其他环境中成功测试的近200个衣壳中进行识别， 包括肝脏中的器官，但不是在细胞水平上，那些转导感兴趣的细胞类型的那些。因此， 补充剂最大限度地提高了成功靶向感兴趣细胞类型的可能性，包括 在分析中，有希望的现有候选人衣冠楚楚。其次，由于候选人的规模较小 衣壳序列，补充剂将能够使用单细胞RNA测序作为一种有效的方法 鉴定衣壳，为感兴趣的细胞类型提供功能转导。使用Essential和 补充剂中的创新技术，包括小鼠酒精性肝病模型，精确分离 流式细胞仪、AAV产生和单细胞RNA测序相结合的主要肝细胞群的研究 经过正式的生物信息学培训，测序数据分析提供了一项深入的最先进的研究 培训机会将成为学员成为医生职业计划的基础- 科学家。作为研究培训的一个重要部分，学员将能够练习和完善他的 与经验丰富的初级科学家密切合作，掌握新的研究技能。基于里程碑的结构化 由经验丰富的PI制定的指导计划将进一步装备学员的批判性思维， 创造力、足智多谋和口头和书面陈述技能，需要作为一名独立的 研究人员，并为研究和医学做出有影响力的贡献。