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

人类胰岛的基因组编辑以抵抗缺血性损伤并促进免疫逃避

基本信息

批准号：
10504937
负责人：
Gregory Michael Ku
金额：
$ 68.64万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10504937
关键词：
Address Beta Cell Blood Vessels CD28 gene CRISPR interference Cell Death Cell Survival Cells Cellular Stress Cellular biology Cessation of life Chronic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Data Engineering Engraftment Environment Genes Genetic Engineering Glucose Goals HLA Antigens Histocompatibility Antigens Class I Human Hypoxia IFNAR1 gene Immune Immune Evasion Immune response Immune system Immunocompetent Immunologics Immunosuppression Inbred NOD Mice Individual Inflammation Inflammatory Insulin Insulin-Dependent Diabetes Mellitus Interferons Interleukin-1 Intervention Ischemia Islet Cell Islets of Langerhans Islets of Langerhans Transplantation Knowledge Laboratories Lead Leucocytic infiltrate Life Mediating Metabolic Modality Natural regeneration Pancreas Pathway interactions RNA Interference RNA interference screen Research Risk Screening procedure Secondary to Stress Structure of beta Cell of islet Systems Biology TNF gene TNFRSF1A gene Testing Tissues Toxic effect Transplantation Transplantation Immunology Treatment Efficacy Vascular blood supply Vision Work antagonist arm base editing beta cell replacement cell replacement therapy clinical translation curative treatments cytokine efficacy testing experience experimental study genome editing high throughput screening humanized mouse immune function immunogenic immunogenicity improved in vivo intercellular cell adhesion molecule ischemic injury islet islet stem cells mouse model novel nutrient deprivation overexpression prime editing somatic cell gene editing synthetic biology trafficking vascular bed

项目摘要

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.

抽象的在这项提案中，我们应用体细胞基因编辑策略来增强胰腺β细胞的替代 1 型糖尿病 (T1D) 的治疗方法。我们组建了一支结合了 β 细胞生物学专业知识的团队，合成生物学和系统生物学以及胰岛移植免疫学，以解决高效的关键障碍无免疫抑制的胰岛移植。我们提出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。大部分的移植的胰岛在血运重建之前就死亡，这限制了治疗的效果。我们有显示缺血期间的缺氧和营养缺乏独立和协同地杀死移植的胰岛细胞。该提案的目标 1 提出了这样的假设：通过删除可以减轻围移植期死亡 β细胞存活的负调节因子或通过正调节因子的过度表达。我们将有针对性地采取和公正的方法来测试候选调节剂并确定人类胰岛存活的新调节剂。我们的团队已经在体内使用 RNAi 在原代人类胰岛中进行了高通量筛选移植存活率作为读数。我们已准备好将我们的专业知识应用于初级基因的 CRISPRi 和 cDNA 筛选人类的胰岛。既往临床胰岛移植经验表明更强的免疫抑制与之相关胰岛移植后胰岛素依赖率较高。免疫系统会部署多种消除移植的外来组织的冗余机制。这与脆弱性相结合 T1D 受体移植的胰岛和增强的免疫功能形成了强大的免疫屏障 β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并阻止细胞浸润将保护胰岛免受无需全身免疫抑制即可产生免疫排斥反应。在 Aim2 中，我们将通过以下方式检验这个假设：基因编辑人类胰岛以消除多态性人类白细胞抗原的表达。我们将测试主导阻断先天炎症细胞因子 TNF、IL-1 以及 1 型和 2 型干扰素的策略。我们还将通过阻断适应性免疫细胞的运输、激活和效应功能来靶向适应性免疫细胞。成功的我们的假设的确认将为下一步的临床转化工作提供原理验证数据步骤。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 模式是用于筛选和证明的强大研究工具- 实验室中的概念实验、碱基编辑和/或引物编辑可能是优选的实施方案临床环境。我们的最终目标是制定改变游戏规则的策略来解决这些关键障碍，具有临床转化的愿景。