Liver Xenotransplantation using CRISPR-modified Porcine Organs

使用 CRISPR 修饰的猪器官进行肝脏异种移植

基本信息

批准号：
10333323
负责人：
JAMES FRANCIS MARKMANN
金额：
$ 75.49万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10333323
关键词：
Address Adhesions Allografting Animal Organ Antibodies Binding Blood Blood Coagulation Disorders Blood Coagulation Factor Blood Platelets CRISPR/Cas technology Candidate Disease Gene Cardiac Cellular Immunity Clinical Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Process Complement Consumption Cryopreservation Data Endogenous Retroviruses Engineering Evaluation Family suidae Fright Functional disorder Genes Genetic Genetic Engineering Goals Heart Human Immune response Immunologics Immunosuppression Infection Inflammation Infusion procedures Injury Investigation Ischemia Kidney Knock-in Knock-out Life Liver Modeling Modification Organ Organ Donor Outcome Papio Pathway interactions Patients Perfusion Physiological Primates Production Protein Engineering Proteins Recovery Regimen Reperfusion Injury Reperfusion Therapy Reporting Resources Risk Savings Site Source Specificity System Techniques Technology Testing Time Tissues Transplantation Treatment Protocols Vertebral column Work Xenograft procedure Zoonoses adaptive immune response allotransplant attenuation clinical application clinically relevant design ex vivo perfusion experience genetic payload graft dysfunction heart xenograft improved improved outcome in vitro Assay in vivo industry partner liver transplantation liver xenograft novel pig genome porcine model prevent protein protein interaction success synergism tool transmission process transplant model transplantation therapy vascular bed

项目摘要

PROJECT SUMMARY / ABSTRACT Liver transplantation has failed to reach its full potential for saving lives due to the inadequate organ supply. Every year thousands of potentially salvageable patients die awaiting an allograft, hundreds of others receive a `marginal” donor organ, often in desperate circumstances, and a many times this number of potential recipients are never offered listing for lifesaving organs due to the need to ration this precious but limited resource. Liver xenotransplantation offers a potential solution to the organ shortage but clinical application has been stymied by four principle hurdles: 1) risk of zoonotic infection of humans, 2) the vigorous immune response mounted to xenogeneic tissues, and 3) physiologic incompatibilities due to species divergence arising in a rarity of protein: protein interactions, and if these obstacles can be consistently overcome, the 4) need to identify a clinically applicable IS regimen. A new tool with unprecedented potential to address these barriers to widespread application of xenotransplantation is found in technology such as CRISPR-Cas9 that dramatically increases gene editing specificity and efficiency. A powerful example is found in recent work by our industry partner, eGenesis, who used CRISPR-Cas9 to rid the pig genome of 62 copies of functional porcine endogenous retroviruses (PERVs), essentially eliminating the risk of PERV transmission to an organ recipient. In the current proposal, we explore the ability of advanced gene editing to address immunologic and physiologic barriers that cause immediate graft dysfunction of liver xenografts when transplanted into a translational baboon model. With the general goal preventing initial xenograft dysfunction (IXD) to gain long term survival of pig liver xenografts in baboon recipients, studies in Aim I will focus on two recently identified potential impediments to xenograft survival: 1) ischemia reperfusion injury, and 2) platelet consumption. Recent findings in cardiac xenotransplantation studies have exposed the critical contribution of IR injury to early heart xenograft demise. Our wealth of experience with ex vivo liver perfusion and also xeno liver perfusion with human blood and the recently reported technique for ischemia free liver transplants should yield a definitive answer to this question. Aim II will focus on understanding the impact of gene edits that: 1) eliminate expression of the three major antibody targets of preformed human anti-pig antibodies, 2) gain expression of human proteins designed to address complement and coagulation dysregulation occurring with porcine liver xenotransplants in NHP, and 3) mitigate innate and cell-mediated immunity and inflammation. To accomplish this, we will take advantage of liver transplant, ex vivo liver perfusion with human blood and in vitro assays well established in our lab and a panel of CRISPR modified pigs with varied expression of genetic edits.

项目概要/摘要由于器官供应不足，肝移植未能充分发挥挽救生命的潜力。每年都有数千名可能挽救的患者在等待同种异体移植的过程中死亡，还有数百人接受了移植手术 “边缘”捐赠器官，往往处于绝望的境地，而潜在接受者的数量是这个数字的许多倍由于需要配给这种宝贵但有限的资源，因此从未提供救生器官的清单。异种移植为器官短缺提供了潜在的解决方案，但临床应用却受到阻碍面临四个主要障碍：1）人类感染人畜共患病的风险，2）强烈的免疫反应异种组织，以及 3) 由于稀有蛋白质引起的物种差异而导致的生理不相容性：蛋白质相互作用，如果这些障碍能够持续克服，4）需要确定临床适用的 IS 方案。一种具有前所未有潜力的新工具，可以解决广泛应用的这些障碍 CRISPR-Cas9 等技术可显着增强基因编辑能力，从而实现异种移植我们的行业合作伙伴 eGenesis 最近的工作就是一个有力的例子。使用 CRISPR-Cas9 去除猪基因组中 62 个拷贝的功能性猪内源性逆转录病毒（PERV），从根本上消除了 PERV 传播给器官接受者的风险。我们探索先进基因编辑的能力，以解决导致免疫和生理障碍的问题当移植到狒狒模型中时，肝异种移植物会立即出现移植功能障碍。总体目标是防止初始异种移植功能障碍 (IXD)，以获得猪肝的长期存活为了在狒狒受体中进行异种移植，Aim I 中的研究将重点关注最近发现的两个潜在障碍异种移植存活：1) 缺血再灌注损伤，2) 心脏血小板消耗的最新发现。异种移植研究揭示了IR损伤对心脏异种移植物早期死亡的关键作用。我们在离体肝脏灌注和用人血进行异种肝脏灌注方面拥有丰富的经验，最近报道的无缺血肝移植技术应该可以为这个问题提供明确的答案。目标 II 将重点了解基因编辑的影响：1）消除三个主要基因的表达预先形成的人抗猪抗体的抗体靶标，2) 获得旨在表达的人蛋白解决 NHP 中猪肝异种移植发生的补体和凝血失调问题，以及 3）减轻先天性和细胞介导的免疫和炎症为了实现这一目标，我们将利用这一点。我们的实验室已建立了肝移植、人血离体肝脏灌注和体外测定方法一组经过 CRISPR 修饰的猪，具有不同的基因编辑表达。