Optimizing Strategies to Overcome Kidney Xenograft Rejection

现在有超过10万名患者在等待肾脏移植，很明显， 缺乏可用的捐赠器官。异种移植是一种很有前途的解决办法。猪是 作为最佳的非人类器官来源，人类对猪器官的免疫反应的效力 阻碍了猪到人的肾移植的临床应用。在本申请中，我们提出 使用尖端的基因工程方法，结合使用下一代的 靶向关键T细胞共刺激通路以降低体液和细胞免疫的药物 接受肾移植的非人灵长类动物（NHP）作为临床前模型的反应， 未来的人体试验第一个也是最主要的障碍是灵长类动物对猪的强大的体液免疫应答 肾脏尽管逐渐引入了一些转基因猪供体，包括敲 α-1，3-gal外，存在预形成的天然抗体以及从头形成的 异种抗原特异性抗体继续导致早期排斥。在这里，我们将使用强大的 CRISPR/Cas9系统的基因组编辑，以消除新的碳水化合物异种抗原产生的 β 4GAL-NT 2酶，并询问该基因缺失的影响（层压在GAL-/-hCD 55转基因上）。 动物）对移植到NHP受体中的猪肾存活的影响。超越体液屏障， 异种移植，对猪器官的强大细胞免疫应答对异种移植提出了第二个挑战。 异种移植的临床应用。免疫抑制策略的发展基于 阻断T细胞活化所需的关键共刺激信号提供了一种潜在的策略， 更有效地控制细胞免疫应答，与标准品相比毒性更低 免疫抑制方案。然而，目前针对两种主要共刺激通路的策略， 使用CTLA 4-IG和抗CD 154 mAb的CD 28和CD 154与疗效问题相关， 安全为代价的重要的是，我们的工作已经证明了新的“下一代”药物的上级功效和安全性。 在小鼠和NHP模型中，与目前的共刺激阻断剂相比，“共刺激阻断剂”。因此我们 接下来将询问这些下一代试剂或域抗体的能力，以更好地控制异种- 反应性免疫和延长异种肾移植存活。最后，我们将采用其他策略， 与T细胞共刺激的阻断协同作用以巩固移植物接受：1）基因组编辑 删除供体组织上的SLA I类/II类的策略，从而减轻通过直接免疫刺激引起的T细胞应答。 2）阻断细胞粘附分子VLA-4和细胞因子 通过CD 122信号传导作为辅助免疫策略来控制异种反应性共刺激， 独立T细胞反应异种移植。总之，这个项目使用尖端技术来开发 降低体液和细胞免疫屏障并延长异种移植存活的新策略。

With over 100,000 patients now on the waiting list for a kidney transplant it is obvious that there is a critical shortage of available donor organs. Xenotransplantation represents a promising solution. While pigs are viewed as the optimal non-human source of organs, the potency of the human immune response to pig organs has prevented the clinical application of pig-to-human kidney transplantation. In this application we propose using cutting-edge genetic engineering approaches in combination with the use of the next generation of agents targeting critical T cell costimulatory pathways to reduce both the humoral and cellular immune response of nonhuman primates (NHP) undergoing kidney transplantation as a preclinical model to inform future human trials. The first and dominant obstacle is the robust humoral immune response of primates to pig kidneys. Despite the gradual introduction of a number of genetic modification to pig donors including knocking out α-1,3-gal, the presence of pre-formed, naturally occurring antibodies as well as the de novo formation of xenoantigen-specific antibodies continues to result in early rejection. Here, we will use the powerful CRISPR/Cas9 system of genome editing to eliminate novel carbohydrate xenoantigens generated by the β4GAL-NT2 enzyme , and interrogate the impact of this gene deletion (layered onto GAL-/- hCD55 transgenic animals) on the survival of pig kidneys transplanted into NHP recipients. Beyond the humoral barrier to xenotransplantation, the robust cellular immune response to pig organs poses a second challenge to the clinical application of xenotransplantation. The development of immunosuppressive strategies based on the blockade of critical costimulatory signals that are required for T cell activation offers a potential strategy to more effectively control the cellular immune response with less toxicity in comparison to standard immunosuppressive regimens. However, current strategies targeting the two dominant costimulatory pathways, CD28 and CD154 using CTLA4-Ig and anti-CD154 mAb have been associated with issues of efficacy and safety. Importantly, our work has demonstrated the superior efficacy and safety of novel “next-generation costimulation blockers” compared to current costimulation blockers in both mouse and NHP models. Thus we will next interrogate the ability of these next generation reagents, or domain antibodies, to better control xeno- reactive immunity and prolong kidney xenograft survival. Finally, we will employ additional strategies that are synergistic with the blockade of T cell costimulation to consolidate graft acceptance: 1) genome-editing strategies to delete SLA class I/ class II on donor tissue and thereby mitigate T cell responses elicited via direct presentation following xenotransplantation, and 2) blockade of the cell adhesion molecule VLA-4 and cytokine signaling through CD122 as adjunct immunotherapeutic strategies to control the xeno-reactive, costimulation- independent T cell response xenotransplantation. In sum, this project uses cutting-edge technology to develop novel strategies to reduce both the humoral and cellular immunity barriers and prolong xenotransplant survival.