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.

项目总结