ModRNA-based Direct Programming of Universal Donor hiPSCs into Immune Evasive Beta Cells

基于 ModRNA 的通用供体 hiPSC 直接编程至免疫逃避型 β 细胞

基本信息

批准号：
10774361
负责人：
Xiaojun Lian
金额：
$ 24.05万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10774361
关键词：
Acceleration Affect Beta Cell Biological Assay Blood Glucose C-Peptide CRISPR-mediated transcriptional activation Cell Differentiation process Cell Line Cell Therapy Cell Transplantation Cells Cellular biology Chemicals Clone Cells Cloning Complementary DNA Coupled Cytolysis Defect Development Diabetes Mellitus Electron Microscope Engineering Exhibits Gene Expression Generations Genes Genomics Glucose Goals Growth Factor Guide RNA HSF1 Homologous Transplantation Human Image Immune Immune Evasion Immunologic Tests In Vitro Individual Insulin-Dependent Diabetes Mellitus Islet Cell Measures Mediating Mediator of activation protein Metabolic Diseases Mus Mutagenesis Natural Killer Cells Pancreas Patients Persons Population Property Public Health RNA Research Personnel Speed Structure of beta Cell of islet System T-Lymphocyte Technology Testing Therapeutic Time Time Study Transcription Initiation Site Transfection Transfer RNA Transplantation United States Viral Vector activin A aptamer autoimmune pathogenesis cost delivery vehicle design diabetic differentiation protocol directed differentiation donor stem cell endocrine pancreas development experimental study functional restoration genomic locus humanized mouse in vivo induced pluripotent stem cell innovation insulin secretion mouse model novel novel therapeutics oligodendrocyte progenitor overexpression p65 programs screening single-cell RNA sequencing stem cells transcription factor vector

项目摘要

Project Summary Pancreatic beta cells derived from human induced pluripotent stem cells (hiPSCs) hold the potential to dramatically change the treatment of type 1 diabetes (T1D). To realize the therapeutic application of these cells, however, we need to develop a robust and rapid means for generating beta cells. Here, the investigators propose a novel paradigm for direct programming of hiPSC into beta cells via CRISPR activation of transcription factors (TFs). These CRISPR activation effectors will be delivered into hiPSCs using modRNAs, avoiding genomic integrations by previous viral vector approach. Furthermore, the investigators will also use recent advances in gene editing to generate universal donor hiPSC clones, which will be used for beta cell programming. These universal donor beta cells can be used for allogeneic transplantation without immune rejections. First, the investigators will investigate optimal msgRNAs for activation of pancreatic TFs in hiPSCs. Next, they will use a multiplexed msgRNA expression system to express all msgRNAs to activate numerous msgRNAs coupled with the dCas9 activator modRNA to study beta cell programming. In Aim 3, the investigators will generate immune-privileged beta cells via programming of universal donor hiPSCs. Universal donor hiPSCs will be generated by removing B2M to inhibit T cell-mediated rejection, and by overexpression of HLA-E trimer to avoid the natural killer (NK) cell-mediated lysis of transplanted cells. The investigators will then use in vitro and in vivo assays to characterize programmed beta cells. For in vitro experiments, the investigator will perform glucose stimulated insulin secretion assay. For in vivo assays, the investigator will use the NRG Akita mice to test whether programmed beta cells can reverse diabetes in vivo. The investigator will also use humanized NSG mice to study whether programmed beta cells from universal donor hiPSCs exhibit immune evasive properties as compared to cells generated from normal hiPSCs.

项目概要源自人类诱导多能干细胞 (hiPSC) 的胰腺 β 细胞具有以下潜力：显着改变 1 型糖尿病 (T1D) 的治疗。实现这些技术的治疗应用然而，我们需要开发一种强大而快速的方法来生成β细胞。在这里，调查人员提出了一种通过 CRISPR 激活将 hiPSC 直接编程到 β 细胞的新范例转录因子（TF）。这些 CRISPR 激活效应器将使用 modRNA 传递到 hiPSC 中，避免通过以前的病毒载体方法进行基因组整合。此外，研究人员还将使用基因编辑产生通用供体 hiPSC 克隆的最新进展，该克隆将用于 β 细胞编程。这些通用供体β细胞可用于同种异体移植，无需免疫拒绝。首先，研究人员将研究激活 hiPSC 中胰腺 TF 的最佳 msgRNA。接下来，他们将使用多重 msgRNA 表达系统来表达所有 msgRNA，以激活大量 msgRNA 与 dCas9 激活剂 modRNA 结合来研究 β 细胞编程。在目标 3 中，调查人员将通过通用供体 hiPSC 的编程生成免疫特权的 β 细胞。通用供体 hiPSC 将通过去除 B2M 以抑制 T 细胞介导的排斥反应以及 HLA-E 三聚体的过度表达来产生避免自然杀伤 (NK) 细胞介导的移植细胞裂解。研究人员随后将在体外使用以及表征程序化β细胞的体内测定。对于体外实验，研究者将进行葡萄糖刺激的胰岛素分泌测定。对于体内测定，研究人员将使用 NRG Akita 小鼠测试编程的β细胞是否可以在体内逆转糖尿病。调查员还将使用人源化 NSG 小鼠研究来自通用供体 hiPSC 的编程 β 细胞是否表现出免疫与正常 hiPSC 生成的细胞相比，具有逃避特性。