Liver Xenotransplantation using CRISPR-modified Porcine Organs

项目总结/摘要 由于器官供应不足，肝移植未能充分发挥其拯救生命的潜力。 每年都有数千名有可能挽救生命的患者在等待同种异体移植时死亡，数百名其他患者接受同种异体移植。 “边缘”捐赠器官，通常是在绝望的情况下，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。肝 异种移植为器官短缺提供了一个潜在的解决方案，但临床应用一直受到阻碍 四个主要障碍：1）人畜共患病感染人类的风险，2）强烈的免疫反应， 异种组织，和3）由于蛋白质稀少引起的物种分化而导致的生理不相容性： 蛋白质相互作用，如果这些障碍可以持续克服，4）需要在临床上确定一个 适用的IS方案。 一个具有前所未有潜力的新工具，可以解决这些广泛应用的障碍， 在CRISPR-Cas9等技术中发现了异种移植，这大大增加了基因编辑 特异性和效率。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去除猪基因组中的62个功能性猪内源性逆转录病毒拷贝， （PERV），基本上消除了PERV传播给器官接受者的风险。在目前的提案中， 我们探索了先进的基因编辑技术解决免疫和生理障碍的能力， 当移植到转化狒狒模型中时，肝脏异种移植物的立即移植物功能障碍。 总体目标是预防初始异种移植物功能障碍（IXD），以获得猪肝的长期存活 在狒狒接受者的异种移植中，Aim I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，和2）血小板消耗。心脏病的最新发现 异种移植研究已经揭示了IR损伤对早期心脏异种移植死亡的关键作用。 我们在离体肝脏灌注和人血异种肝脏灌注方面的丰富经验以及 最近报道的无缺血肝移植技术应该对这个问题给出一个明确的答案。 目的II将重点关注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）获得设计成 解决NHP中猪肝异种移植发生的补体和凝血功能失调， 3)减轻先天和细胞介导的免疫和炎症。为了实现这一目标，我们将利用 肝移植，离体肝灌注与人血和体外试验，以及建立在我们的实验室和 一组具有不同基因编辑表达的CRISPR修饰猪。

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.