Zero Footprint Induction of Human Hemogenesis to Study Fanconi Anemia

人体造血零足迹诱导研究范可尼贫血

• 批准号: 9261295
• 负责人: Michael Guillermo Daniel
• 金额: $ 4.39万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9261295
• 关键词: Adult; Autologous; Binding; Biological Models; Biology; Blood; Bypass; CD34 gene; CRISPR/Cas technology; Cell Maintenance; Cell physiology; Cells; Clinic; Clinical; DNA Interstrand Crosslinking; DNA Repair Pathway; Defect; Dependency; Dermal; Development; Disease; Disease model; Endothelium; Engineering; Ensure; Fanconi Anemia pathway; Fanconi' s Anemia; Fibroblasts; Future; Gene Expression; Generations; Genes; Genome; Genomic DNA; Genomics; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Human; Impairment; In Vitro; Insertional Mutagenesis; Mediating; Methodology; Methods; Modeling; Molecular Profiling; Multipotent Stem Cells; Mus; Mutation; Pancytopenia; Pathologic; Pathway interactions; Patients; Phenotype; Physicians; Physiological; Pluripotent Stem Cells; Probability; Process; Proteins; RNA; Research; Risk; Scientist; Signal Pathway; Somatic Cell; Stem cells; Subfamily lentivirinae; System; Technology; Therapeutic; Time; Training; Transcriptional Activation; Transgenes; Translating; Transplantation; Work; bench to bedside; blood product; cell type; clinical application; clinical translation; clinically relevant; design; drug testing; gene delivery system; improved; in utero; in vitro Model; innovation; lentiviral-mediated; new therapeutic target; novel; novel therapeutics; overexpression; prevent; progenitor; programs; self-renewal; stem; tool; transcription factor; transcriptome sequencing; tumorigenesis; vector

Project Summary Our inability to culture hematopoietic stem cells (HSCs), or study physiologic and pathologic hematopoiesis in vitro, remain as significant problems in hematopoietic biology. Recent studies focus on reprogramming pluripotent stem cells (PSCs) or somatic cells to hematopoietic stem and progenitor cells (HSPCs). These studies, however, rely on gene delivery systems that integrate into the host genome. This widens the gap between the bench and the bedside by impeding the application of these in vitro generated blood products for transplants and drug testing platforms. A method to generate HSPCs de novo without genomic disruption, and use of these studies for disease modeling, are critical for understanding the mechanisms behind the pathologic hematopoiesis encountered in multiple hematopoietic disorders such as Fanconi Anemia (FA). In this disorder, the associated bone marrow failure (BMF) is preceded by a significant reduction in CD34+ hematopoietic progenitors in utero. Using a minimal set of transcription factors (TFs) we have shown our ability to induce a hemogenic program in mouse fibroblasts that generate HSC-like cells over time that are phenotypically and functionally similar to HSCs. Promising results after translating these methods to human dermal fibroblasts (HDFs) can provide a novel model system of FA and other hematologic disorders in vitro for drug testing and gene editing platforms with the goal of therapeutic discovery and eventual patient-specific HSC transplants. The aims of this F31 application proposal are to 1) investigate non-integrative methods to induce a hemogenic program in human fibroblasts and 2) utilize hemogenic reprogramming to study the pathologic hematopoiesis that precedes BMF in the FA disease state. The ability to generate zero footprint hematopoietic cells de novo (and therefore without the risk of insertional mutagenesis and oncogenesis) is highly clinically relevant, as are any discoveries made regarding defective hematopoiesis in FA after application of this novel reprogramming strategy. To achieve these goals, I will first generate polycistronic cassettes carrying our TF cocktail for hemogenic induction that will then be transferred to a self-replicating RNA (srRNA) system that robustly expresses these factors without genomic disruption. HDFs will then be reprogrammed with these constructs to ensure efficient hemogenic induction. I will also induce hemogenesis in patient-specific FA HDFs to determine the impact of the FA pathway defect in emerging hematopoiesis. I will rigorously analyze global gene expression profiles (RNAseq) and TF binding for candidate pathways (ChIP-PCR) in our reprogrammed cells to identify potential mechanisms behind the FA pathway in definitive hematopoiesis. The strengths of this proposal lie in the innovative reprogramming strategy that recapitulates definitive hematopoiesis in a dish, and the potential findings after applying this technology to hematologic disease models. This project is designed to frame the research in a clinical context and provide specialized training of a future physician scientist.

项目总结