Analyzing the mechanism of exosome mediated DNA Methyltransferase activity during sepsis

项目摘要 该提案是为博士乔恩Wisler，在该部门的助理教授五年的研究计划。 创伤、重症监护和烧伤外科。该提案旨在研究外泌体介导的 在John Christman博士的指导下，研究败血症期间发生的表观遗传调节。克里斯曼医生是 他是单核细胞/巨噬细胞生物学和表观遗传学领域的高产研究人员， 指导和生产力的轨迹。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。这包括一个完善的，合乎逻辑的计划， 和长期里程碑。利用他的导师团队的丰富经验，该提案将培训乔恩 是尖端技术，包括CRISPR基因编辑，活细胞成像，纳米颗粒RNA包装和 递送和外泌体生物学，以极大地提高他的机械理解和解释能力。 脓毒症患者表现出严重的免疫抑制， 并发症和长期死亡率增加。我们的初步数据表明， 甲基转移酶（DNMT）mRNA在败血症患者循环外泌体中的表达， 从这些外泌体转移到幼稚单核细胞。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。本申请的目的是阐明 外泌体摄取，并在体内脓毒症模型中靶向这些表观遗传事件。我们假设预防 外泌体介导的DNA甲基化允许在脓毒症期间维持宿主免疫应答。的 总体目标是确定外泌体介导的表观遗传控制的机制基础， 控制脓毒症相关免疫抑制的事件，并确定靶向的潜在转化价值 DNMT的功能是治疗脓毒症相关并发症。培养新颖、前沿、 技术将大大提高乔恩的科学专业知识，并允许从指导到 独立外科医生兼科学家 目的1：阐明细胞摄取外泌体的机制，并确定 DNMT作为表观遗传信使参与脓毒症后免疫抑制 目的2：建立靶向外泌体介导的抗DNMT递送的安全性和有效性，以预防DNMT的发生。 脓毒症小鼠模型中的免疫抑制

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