Zero Footprint Induction of Human Hemogenesis to Study Fanconi Anemia

项目摘要 我们无法培养造血干细胞(HSCs)，也无法研究生理性和病理性造血 在体外，仍然是造血生物学中的重大问题。最近的研究集中在重新编程上 多能干细胞(PSCs)或体细胞向造血干细胞和祖细胞(HSPC)转化。这些 然而，研究依赖于整合到宿主基因组中的基因传递系统。这拉大了差距。 通过阻碍这些体外产生的血液产品的应用来实现 移植和药物测试平台。一种在不破坏基因组的情况下从头产生HSPC的方法，以及 将这些研究用于疾病建模，对于理解病理背后的机制至关重要 在Fanconi贫血(FA)等多发性造血疾病中遇到的造血功能。在这种混乱中， 在相关的骨髓衰竭(BMF)之前，CD34+造血细胞显著减少 子宫内的祖细胞。使用最小的一组转录因子(TF)，我们已经证明了我们有能力诱导 小鼠成纤维细胞中的造血程序，随着时间的推移产生HSC样细胞，其表型和 在功能上类似于HSC。将这些方法转化为人真皮成纤维细胞后取得了令人满意的结果 (HDFS)可以提供一种新的FA和其他血液病的体外模型系统，用于药物检测和 基因编辑平台，目标是治疗发现和最终针对患者的造血干细胞移植。 这份F31应用提案的目的是：1)研究非整合性方法诱导血液病 在人成纤维细胞中的程序和2)利用血源性重编程研究病理性造血 这比FA疾病州的BMF更早。产生零足迹造血细胞从头开始的能力 (因此没有插入突变和致癌的风险)具有高度的临床相关性， 在应用这一新的重新编程后，任何关于FA造血缺陷的发现 策略。为了实现这些目标，我将首先生成携带我们的TF鸡尾酒的多顺反子盒式磁带 然后将其转移到一个自我复制的RNA(SrRNA)系统，该系统强大地 在不破坏基因组的情况下表达这些因子。然后将使用这些构造对HDFS进行重新编程，以 确保有效的血液诱导。我还将在患者特定的FA HDFS中诱导造血，以确定 FA通路缺陷在新生造血中的影响。我将严谨地分析全球基因 我们重编程细胞中的表达谱(RNAseq)和候选途径的转铁蛋白结合(ChIP-PCR) 目的：明确FA途径在明确的造血过程中的潜在机制。它的优势在于 建议在于创新的重新编程策略，该策略概括了盘子中的最终造血过程，以及 将这项技术应用于血液病模型后的潜在发现。该项目旨在 将这项研究置于临床环境中，并为未来的内科科学家提供专门培训。

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.