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 来源于人诱导多能干细胞(HiPSCs)的胰岛β细胞具有 极大地改变了1型糖尿病(T1D)的治疗。为了实现这些药物的治疗应用 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 提出了一种通过激活CRISPR将HiPSC直接编程到β细胞中的新范式 转录因子(TF)。这些CRISPR激活效应器将使用modRNAs传递到HiPSCs中， 避免通过以前的病毒载体方法进行基因组整合。此外，调查人员还将使用 基因编辑在产生将用于β细胞的通用供体HiPSC克隆方面的最新进展 编程。这些通用的供体β细胞可以用于无免疫的同种异体移植。 拒绝。 首先，研究人员将研究在HiPSCs中激活胰腺转录因子的最佳msgRNAs。接下来，他们 将使用多路msgRNA表达系统表达所有msgRNAs以激活大量msgRNAs 与dCas9激活子modRNA结合，研究β细胞编程。在《目标3》中，调查人员将 通过编程通用捐赠者的HiPSCs来产生免疫特权的β细胞。全能供体hPSCs 将通过去除B2M来抑制T细胞介导的排斥反应以及通过过表达HLA-E三聚体来产生 以避免自然杀伤(NK)细胞介导的移植细胞裂解。然后，研究人员将在体外使用 并在体内进行测试，以表征程序化的β细胞。对于体外实验，研究人员将 进行葡萄糖刺激的胰岛素分泌试验。对于活体检测，研究人员将使用NRG Akita 以测试程序化的β细胞是否能在体内逆转糖尿病。调查员还将使用 人源化NSG小鼠研究来自通用捐赠者HiPSCs的编程β细胞是否表现出免疫力 与正常的HiPSCs产生的细胞相比，它具有逃避特性。