Analyzing the mechanism of exosome mediated DNA Methyltransferase activity during sepsis

项目摘要 这项建议是为乔恩·威斯勒博士制定的一项为期五年的研究计划，他是麻省理工学院 创伤、重症监护和烧伤外科。这项建议旨在研究外切体介导的效应。 在约翰·克里斯曼博士的指导下，败血症期间发生的表观遗传调节。克里斯曼医生是 在单核/巨噬细胞生物学和表观遗传学领域的高效研究人员，他有很长的 指导和工作效率的跟踪。该提案的研究和职业发展部分侧重于 技术拓展和专业发展。这包括一个精炼的、合乎逻辑的计划，以及可衡量的短期 和长期的里程碑。利用他的指导团队的丰富经验，这项提议将培训乔恩 是尖端技术，包括CRISPR-基因编辑、活细胞成像、纳米RNA包装和 和外显体生物学，极大地提高了他的机械理解和研究能力。 脓毒症患者表现出严重的免疫抑制，随后感染水平较高。 并发症和长期死亡率增加。我们的初步数据显示DNA显著增加 脓毒症患者外周血中甲基转移酶(DNMT)mRNAs的表达 从这些外显体转移到幼稚的单核细胞。这种转移导致表观遗传事件增加 (启动子甲基化)和基因沉默。我们这项申请的目的是要阐明 Exosome摄取，并在脓毒症的体内模型中靶向这些表观遗传事件。我们假设预防 外切体介导的DNA甲基化允许在脓毒症期间维持宿主的免疫反应。这个 总体目标是确定外显子介导的表观遗传学控制的机制基础。 控制脓毒症相关免疫抑制的事件，并确定靶向的潜在翻译价值 DNMT具有治疗败血症相关并发症的功能。融入新奇、尖端的培训 技术将极大地提高乔恩的科学专业知识，并允许从被指导到 独立的外科医生兼科学家。 目的1：阐明细胞摄取外切体的机制并确定外切体的作用 DNMT作为促进败血症后免疫抑制的表观遗传信使 目的2：建立靶向外切体介导的抗DNMT传递的安全性和有效性，以预防 脓毒症小鼠模型的免疫抑制作用

Project Summary This proposal is for a five-year research program for Dr. Jon Wisler, an Assistant Professor in the Division of Trauma, Critical Care, and Burn Surgery. This proposal aims to study the effects of exosome-mediated epigenetic regulation that occurs during sepsis, under the mentorship of Dr. John Christman. Dr. Christman is a highly productive researcher in the fields of monocyte/macrophage biology and epigenetics who has a long track of mentorship and productivity. The research and career development portions of the proposal focus on technique expansion and professional development. This includes a refined, logical plan with measurable short and long-term milestones. Utilizing the extensive experience of his mentorship team, this proposal will train Jon is cutting-edge technologies including CRISPR-gene editing, live-cell imaging, nanoparticle RNA packaging and delivery, and exosome biology to greatly improve his mechanistic understanding and investigatory capabilities. Patients with sepsis exhibit a profound degree of immunosuppression with higher levels of subsequent infectious complications and increased long-term mortality. Our preliminary data identifies significant increases in DNA Methyltransferases (DNMT) mRNAs in circulating exosomes of patients with sepsis, and that this DNMT mRNA is transferred from these exosomes to naïve monocytes. This transfer results in increased epigenetic events (promoter methylation) and gene silencing. Our intent for this application is to elucidate the mechanisms of exosome uptake, and target these epigenetic events in an in vivo model of sepsis. We hypothesize prevention of exosome-mediated DNA methylation allows for maintenance of the host immune response during sepsis. The overall objective is to identify the mechanistic base that underlies exosome-mediated control of the epigenetic events that govern sepsis-related immunosuppression, and identify the potential translational value of targeting DNMT function to treat complications associated with sepsis. Incorporating the training of novel, cutting-edge techniques will greatly improve the Jon's scientific expertise, and allow for the progression from mentored to independent surgeon-scientist. Aim 1: Elucidate the mechanisms that underlie the cellular uptake of exosomes and define the role of DNMT as an epigenetic messenger that contributes to post-sepsis mediated immunosuppression Aim 2: Establish the safety and efficacy of targeting exosome-mediated delivery of anti-DNMT to prevent immunosuppression during sepsis in mouse models