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

• 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
• 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

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.

项目摘要 源自人诱导多能干细胞（hiPSC）的胰腺β细胞具有以下潜力： 1型糖尿病（T1 D）的治疗方法。为了实现这些治疗应用， 然而，我们需要开发一种强大而快速的方法来产生β细胞。在这里，调查人员 提出了通过CRISPR激活hiPSC直接编程为β细胞的新范例， 转录因子（TF）。这些CRISPR激活效应物将使用modRNA递送到hiPSC中， 避免了先前病毒载体方法的基因组整合。此外，研究人员还将使用 基因编辑的最新进展，以产生通用供体hiPSC克隆，其将用于β细胞 编程.这些通用供体β细胞可用于同种异体移植，而无需免疫 拒绝。 首先，研究人员将研究用于在hiPSC中激活胰腺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dCas 9激活剂modRNA偶联以研究β细胞编程。在目标3中，研究人员将 通过通用供体hiPSC的编程产生免疫特权β细胞。通用供体hiPSC 将通过去除B2 M以抑制T细胞介导的排斥反应以及通过HLA-E三聚体的过表达来产生 以避免自然杀伤（NK）细胞介导的移植细胞溶解。研究人员将在体外使用 和体内测定以表征程序化β细胞。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。对于体内试验，研究者将使用NRG秋田 用小鼠测试程序化β细胞是否可以在体内逆转糖尿病。研究人员还将使用 人源化NSG小鼠研究来自通用供体hiPSC的程序化β细胞是否表现出免疫原性， 与由正常hiPSC产生的细胞相比，具有逃避性质。