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

摘要 在本提案中，我们应用体细胞基因编辑策略来增强胰腺β细胞替代 1型糖尿病（T1 D）的治疗。我们组建了一个团队，结合了β细胞生物学的专业知识， 合成和系统生物学，以及胰岛移植免疫学，以解决有效 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。大部分 移植的胰岛在血管再生发生之前死亡，这限制了治疗的功效。我们有 在局部缺血期间显示缺氧和营养剥夺独立地和协同地杀死移植的胰岛 细胞该提案的目的1提出了这样的假设，即通过删除 β细胞存活的负调节因子或正调节因子的过表达。我们会把这两个目标 以及测试候选调节剂和鉴定人胰岛存活的新调节剂的无偏方法。 我们的团队已经在原代人类胰岛中使用RNAi进行了高通量筛选， 移植存活率作为读数。我们已经准备好将我们的专业知识应用于CRISPRi和cDNA筛选， 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 胰岛移植后胰岛素非依赖率高。免疫系统会部署多个 多余的机制来消除移植的外来组织。这一点，加上脆弱的 移植的胰岛和T1 D受体的免疫功能增强，形成了强大的免疫屏障 贝塔细胞替代疗法我们假设，减少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和阻断细胞浸润将保护胰岛免受 免疫排斥而不需要全身性免疫抑制。在Aim 2中，我们将通过以下方式来验证这一假设： 基因编辑人类胰岛以消除多态性人类白细胞抗原的表达。我们将测试显性 阻断先天性炎性细胞因子TNF、IL-1和1型和2型干扰素的策略。我们还将 通过阻断其运输、激活和效应器功能靶向适应性免疫细胞。成功 我们的假设的确认将提供原则性数据证明，以支持临床翻译的努力， 步我们设想这些策略可以应用于原代人类胰岛、干细胞衍生的β细胞， 甚至异种胰岛。虽然这些CRISPR模式是筛选和证明的强大研究工具， 在实验室中的概念实验中，基础编辑和/或主要编辑可以是优选的实施例。 临床设置。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， 致力于临床翻译

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.