Genome editing of human pancreatic islets to withstand ischemic injuries and promote immune evasion

摘要 在这项建议中，我们应用体细胞基因编辑策略来促进胰岛β细胞的替代 1型糖尿病的治疗(T1D)。我们组建了一个团队，结合了贝塔细胞生物学方面的专业知识， 合成和系统生物学，以及胰岛移植免疫学，以解决高效 无免疫抑制的胰岛移植。我们提出了两个正交但互补的 旨在解决胰岛移植中的两个关键挑战-胰岛存活和免疫排斥。大多数 移植的胰岛在血管重建发生之前就死亡了，这限制了治疗的效果。我们有 显示缺血时的低氧和营养剥夺独立和协同杀死移植的胰岛 细胞。这项提案的目标1解决了这样一种假设，即通过删除 β细胞存活的负性调节因子或正调节因子的过度表达。我们会把这两个目标 以及不偏不倚的方法来测试候选调节器，并确定人类胰岛生存的新调节器。 我们的团队已经使用RNAi在原生人类胰岛上进行了高通量筛查，使用的是体内 移植存活作为一种读数。我们准备将我们的专业知识应用于初级CRISPRi和cDNA筛查 人类的小岛。以往的临床胰岛移植经验表明，较强的免疫抑制与 胰岛移植后胰岛素独立率较高。免疫系统部署了多个 消除移植异体组织的多余机制。这一点，再加上美国经济的脆弱性 移植胰岛和提高T1D受者的免疫功能，形成了强大的免疫屏障 到贝塔细胞替代疗法。我们假设，最小化胰岛细胞的多管齐下的方法 免疫原性，中和移植物中的炎症，并阻止细胞渗透将保护胰岛免受 免疫排斥反应，不需要全身免疫抑制。在AIM2中，我们将通过以下方式验证这一假设 对人类胰岛进行基因编辑，以消除人类白细胞多态抗原的表达。我们将测试支配性 阻断先天炎性细胞因子肿瘤坏死因子、白介素1和1型和2型干扰素的策略。我们还将 通过阻止适应性免疫细胞的运输、激活和效应器功能来靶向它们。成功 我们的假设的确认将为下一步的临床翻译工作提供原则证明数据 台阶。我们设想这些策略可以应用于原代人类胰岛，干细胞来源的β细胞， 甚至是异种胰岛。虽然这些CRISPR模式是用于筛选和验证的强大研究工具- 实验室中的概念实验、基础编辑和/或主要编辑可以是 临床环境。我们的最终目标是制定改变游戏规则的战略，以解决这些关键障碍， 着眼于临床翻译。

ABSTRACT In this proposal we apply somatic cell gene editing strategies to enhance pancreatic beta cell replacement therapies for type 1 diabetes (T1D). We have formed a team that combines expertise in beta cell biology, synthetic and systems biology, and islet transplant immunology to address key impediments for efficient immunosuppression-free transplantation of pancreatic islets. We propose two orthogonal yet complementary aims to address two critical challenges in islet transplantation - islet survival and immune rejection. Most of the transplanted islets die before revascularization can occur, which limits the efficacy of the therapy. We have shown hypoxia and nutrient deprivation during ischemia independently and synergistically kill transplanted islet cells. Aim 1 of this proposal addresses the hypothesis that peri-transplant death can be alleviated by deleting negative regulators of beta cell survival or by over-expression of positive regulators. We will take both targeted and unbiased approaches to test candidate regulators and to identify novel regulators of human islet survival. Our team has already performed high-throughput screens using RNAi in primary human islets using in vivo transplant survival as a readout. We are ready to apply our expertise to CRISPRi and cDNA screens of primary human islets. Previous clinical islet transplant experiences show that stronger immunosuppression is associated with higher rate of insulin independence after islet transplantation. The immune system deploys multiple redundant mechanisms to eliminate transplanted foreign tissue. This, combined with the fragility of the transplanted islets and heightened immune functions in T1D recipients, forms a formidable immunological barrier to beta cell replacement therapy. We hypothesize that multipronged approach of minimizing islet cell immunogenicity, neutralizing inflammation in the graft, and blocking cellular infiltrate will shield the islets from immune rejection without the need for systemic immunosuppression. In Aim2, we will test this hypothesis by gene edit human islets to ablate the expression of polymorphic human leukocyte antigens. We will test dominant strategies that block innate inflammatory cytokines TNF, IL-1 and type 1 and type 2 interferons. We will also target adaptive immune cells by blocking their trafficking, activation and effector function. Successful confirmation of our hypotheses will provide proof-of-principle data to support efforts of clinical translation as next steps. We envision that these strategies may be applied to primary human islets, stem cell-derived beta cells, and even xenogeneic islets. While these CRISPR modalities are powerful research tools for screens and proof- of-concept experiments in the laboratory, base editing and/or prime editing may be preferred embodiments in the clinical setting. Our end goal is to generate game-changing strategies to address these key impediments, with a vision towards clinical translation.