Optimizing Strategies to Overcome Kidney Xenograft Rejection

目前有超过10万名患者在等待肾脏移植，显然有一个关键的 可供捐献的器官短缺。异种移植是一种很有前途的解决方案。而猪则是 被视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 阻碍了猪对人肾移植的临床应用。在本申请中，我们建议 使用尖端的基因工程方法，结合使用下一代 靶向关键T细胞共刺激通路的药物可降低体液免疫和细胞免疫 非人灵长类动物(NHP)接受肾移植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体液免疫反应。 肾脏。尽管对生猪捐赠者逐步引入了一些基因改造，包括敲门 在α-1，3-Gal之外，存在预先形成的自然产生的抗体以及从新形成的 异种抗原特异性抗体继续导致早期排斥反应。在这里，我们将使用强大的 CRISPR/Cas9基因组编辑系统，以消除由 β4GAL-NT2酶，并检测该基因缺失的影响(层叠到Gal-/-hCD55转基因 动物)对移植到NHP接受者体内的猪肾的存活率的影响。超越了幽默的障碍 异种移植对猪器官的强大细胞免疫反应构成了对 异种移植的临床应用。免疫抑制策略的研究进展 阻断T细胞激活所需的关键共刺激信号提供了一种潜在的策略 与标准相比，更有效地控制细胞免疫反应，毒性更小 免疫抑制疗法。然而，目前针对两个主要的共刺激途径的策略， 使用CTLA4-Ig和抗CD154单抗的CD28和CD154与疗效和 安全。重要的是，我们的工作已经证明了新一代药物的卓越疗效和安全性 在小鼠和NHP模型中，共刺激阻滞剂与目前的共刺激阻滞剂进行了比较。因此，我们 接下来将询问这些下一代试剂或区域抗体更好地控制异种病毒的能力。 反应性免疫和延长异种肾移植存活时间。最后，我们将采用其他策略，这些策略 与阻断T细胞共刺激以巩固移植接受性的协同作用：1)基因组编辑 删除供体组织上SLA I/II类从而减轻直接诱导的T细胞反应的策略 异种移植后的表现以及2)细胞黏附分子VLA-4和细胞因子的阻断 通过CD122作为辅助免疫治疗策略来控制异种反应、共刺激- 独立T细胞反应性异种移植。总之，这个项目使用了尖端技术来开发 减少体液和细胞免疫屏障和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.