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

项目摘要 酒精性肝病是一个主要的临床挑战，因为它与严重的并发症有关，如肝脏 肝硬化和酒精性肝炎，死亡率很高。我们对潜在机制的理解 酒精性肝病仍在发展;然而，以前的研究已经确定了与酒精性肝病密切相关的细胞。 肝脏对酒精诱导的损伤的反应，包括肌成纤维细胞，巨噬细胞亚群和卵圆细胞， 在人体内被称为小管反应。由于其在酒类中独特而重要的功能， 肝脏疾病，有效和特异性的基因传递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，特别是如果非整合无毒腺相关病毒（AAV） 可以使用矢量。建议的多元化补充条文的研究范围与 亲本R 01 AA 026578，即建立肌成纤维细胞、巨噬细胞 在酒精性肝病小鼠模型中，使用AAV载体观察了肝细胞亚群和卵圆细胞。所述补充剂被 与R 01 AA 026578互补，因为它使用了不同的方法：首先，不是在 如R 01 AA 026578中所做的，在感兴趣的细胞类型中具有未知向性的百万个新产生的衣壳， 该补充剂将在之前在其他环境中成功测试的近200个衣壳中进行鉴定， 包括在肝脏中的器官而不是在细胞水平上，那些使感兴趣的细胞类型稳定的细胞。因此，在本发明中， 所述补充剂通过包括以下步骤使成功靶向感兴趣的细胞类型的可能性最大化 在分析中有希望的现有候选衣壳。二是因为候选人规模较小 衣壳队列，补充将能够采用单细胞RNA测序作为一种有效的方法， 鉴定提供感兴趣的细胞类型的功能性转导的衣壳。使用基本的和 补充剂中的创新技术，包括酒精性肝病的小鼠模型，精确分离 通过流式细胞仪、腺相关病毒生产和单细胞RNA测序相结合对主要肝细胞群进行分析 在测序数据的生物信息学分析的正式培训提供了一个深刻的国家的最先进的研究 培训机会，将作为学员成为医生的职业规划的基础- 科学家作为研究培训的重要组成部分，学员将能够实践和完善他的 新发现的研究技能与经验丰富的年轻科学家密切合作。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培养学员的批判性思维， 创造力，足智多谋和口头和书面表达技能需要成功作为一个独立的 研究人员，并为研究和医学做出有影响力的贡献。

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.