CRISPR-Modified Cardiac Xenograft Transplantation

摘要摘要：CRISPR改良异种心脏移植 在过去的三年里，异种心脏移植取得了两项重大突破。首先， 以前难以克服的迟发性异种移植排斥反应(DXR)在猪到狒狒之间已经被克服了 心脏异种模型。在这个模型中，DXR主要表现为接受者的消耗性凝血障碍(CC) 移植物中的血栓性微血管病(TM)。在巴布亚的异位模型研究中，我们的 ‘Mohiuddin/NIH’组使用转基因GTKO.hCPRP.hTBM心脏和一种治疗方案 包括诱导T和B耗尽，MMF和类固醇-所有临床使用的药物-与一个实验性的 针对CD40的单抗。只要CD40治疗被预防，DXR就会被预防 在一个例子中，异种移植物的存活时间延长了两年以上。 第二个重大进展来自慕尼黑小组，他们报告说原位手术的存活率一直很高。 超过180天的异种心脏移植受体使用我们的免疫调节方案和 GTKO.hCPRP.hTBM心脏。实现原位异种心脏移植的一致存活 由最初的异种移植功能障碍(IxD)预防，这是一种难以由多个 经验丰富的临床移植团队。重要的是，慕尼黑研究小组发现，最大限度地减少移植物缺血 对持续预防IxD来说是必要和充分的。发现缺血最小化足以 克服IxD障碍是一项“突破性”的进步，即使通过 缺血最小化还没有被很好地理解。 在这个项目中，我们假设损伤GTKO.hCPRP.hTBM心脏的IxD机制可能是 通过在Pig 2.0的一个或多个版本中表达的附加的聚焦于异种的遗传修饰来解决， 由我们在eGenesis的合作者使用创新的CRISPR驱动的基因编辑工具创建。Pigg 2.0是 旨在通过12种异种特异性损伤机制的组合来解决多种已知的异种特异性损伤机制 有针对性的基因改造。该项目利用了几个定义良好的版本的可用性 Pigg 2.0，我们在多种体外、体外和在体心脏异种模型方面的丰富经验，以及强大的 在有或没有缺血的情况下，确定猪2.0是否受到心脏IxD保护的机械测试能力 最小化(目标1)。具体来说，我们将使用这些独特的资源来了解a)Pig 2.0的版本 含有凝血级联调节基因盒的IxD保护；b)是否缺血 最小化对于预防易感猪2.0心脏的IxD是必要的和/或充分的；以及c)为了定义 临床可接受的PIG 2.0心脏免疫抑制方案，一贯安全和 有效(目标2)。该项目的结果将产生可能催化其他细胞和器官进展的见解 异种移植，并告知临床异种心脏移植试验设计。

SUMMARY ABSTRACT: CRISPR-Modified Cardiac Xenograft Transplantation The past three years have witnessed two major breakthroughs in heart xenotransplantation. First, the previously intractable barrier of delayed xenograft rejection (DXR) has been overcome in the pig-to-baboon heart xeno model. In this model, DXR is manifest primarily as consumptive coagulopathy (CC) in the recipient and thrombotic microangiopathy (TM) in the graft. Working in the heterotopic model in baboons, our `Mohiuddin/NIH' group used genetically modified GTKO.hCPRP.hTBM hearts and a treatment regimen including “induction” T and B depletion, MMF, and steroids – all clinically used drugs – with an experimental monoclonal antibody directed against CD40. DXR was prevented for as long as CD40 treatment was continued, and in one instance xenograft survival was extended beyond two years. The second major advance came from the Munich group, who reported consistent survival of orthotopic pig heart xenograft recipients beyond 180 days using our immunomodulatory regimen and GTKO.hCPRP.hTBM hearts. Accomplishing consistent survival of orthotopic heart xenografts had previously been prevented by initial xenograft dysfunction (IXD), a phenomenon refractory to concerted efforts by multiple experienced clinical transplant teams. Importantly, the Munich group found that minimizing graft ischemia was necessary and sufficient to consistently prevent IXD. Discovering that ischemia minimization was sufficient to overcome the IXD barrier is a `breakthrough' advance, even as the mechanism of improved graft protection by ischemia minimization is not yet well understood. In this Project, we hypothesize that IXD mechanisms that injure GTKO.hCPRP.hTBM hearts may be addressed by the additional xeno-focused genetic modifications expressed in one or more versions of Pig 2.0, created by our collaborators at eGenesis using innovative CRISPR-driven gene editing tools. Pig 2.0 is designed to address multiple known xeno-specific injury mechanisms through a combination of 12 xeno- targeted genetic modifications. This Project takes advantage of availability of several well-defined versions of Pig 2.0, our deep experience with multiple in vitro, ex vivo, and in vivo heart xeno models, and a robust mechanistic assay capability to determine whether Pig 2.0 is protected from heart IXD, with or without ischemia minimization (Aim 1). Specifically, we will use these unique resources to learn a) whether versions of Pig 2.0 containing a coagulation cascade regulatory gene cassette are protected from IXD; b) whether ischemia minimization is necessary and/or sufficient to prevent IXD in susceptible Pig 2.0 hearts; and c) to define a clinically acceptable immunosuppression regimen for Pig 2.0 hearts in baboons that is consistently safe and effective (Aim 2). Results from this Project will yield insights likely to catalyze progress for other cell and organ xenografts, and inform clinical heart xenotransplantation trial design.