Hematopoietic stem cell (HSC) genetic and cellular therapies

造血干细胞 (HSC) 遗传和细胞疗法

• 批准号: 8939915
• 负责人: Andre LaRochelle
• 金额: $ 141.69万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939915
• 关键词: Academic Medical Centers; Address; Adult; Agonist; Animals; Aplastic Anemia; Apoptosis; Autologous; Autologous Transplantation; Bacterial Infections; Biological Assay; Birds; Blood Vessels; CD34 gene; Canis familiaris; Categories; Cell Count; Cell Culture Techniques; Cell Line; Cell Maintenance; Cells; Certification; ChIP-seq; Child; Clinical; Clinical Research; Coculture Techniques; Collaborations; Complementary DNA; Culture Media; Cyclic GMP; DNA Repair Pathway; Data; Defect; Derivation procedure; Development; Disease; Dose; Embryo; Embryonic Development; Employee Strikes; Endothelial Cells; Endothelium; Engraftment; Enrollment; Evaluation; Extramural Activities; Family; Fibronectins; Gene Expression; Gene Expression Profile; Gene Mutation; Generations; Genes; Genetic; Goals; Gold; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Homeostasis; Hospitals; Human; Hypoxia; Hypoxia Inducible Factor; Hypoxia Pathway; ITGB2 gene; Immune; Immunoprecipitation; In Vitro; Individual; Infection; Integrins; Interleukin-3; Laboratories; Lead; Leukocyte Adhesion Deficiency; Leukocyte adhesion deficiency - Type 1; Leukocytes; Life; Ligands; Link; Maintenance; Mediator of activation protein; Methylation; Modification; Molecular; Monitor; Mus; Mutation; Notch Signaling Pathway; Osteoblasts; Oxygen; PTPRC gene; Pancytopenia; Pathway interactions; Patients; Pattern; Pennsylvania; Pharmaceutical Preparations; Phase; Phenotype; Population; Production; Protocols documentation; Regulation; Risk; Role; Safety; Signal Transduction; Site; Stem Cell Development; Stem Cell Factor; Stem cell transplant; Stem cells; Stress; Suspension substance; Suspensions; Syndrome; System; Technology; Testing; Thrombopoietin; Time; Transplantation; Transplantation Conditioning; Umbilical Cord Blood; Undifferentiated; United States National Institutes of Health; Universities; Viral Vector; Washington; Work; analog; bHLH-PAS factor HLF; base; clinical application; comparative; cytokine; experience; follow-up; gene therapy; gene therapy clinical trial; gene transfer vector; hypoxia inducible factor 1; improved; in vivo; induced pluripotent stem cell; leukemia; metabolomics; method development; neutrophil; notch protein; novel; novel strategies; preclinical study; progenitor; reconstitution; response; self-renewal; stem; stem cell differentiation; stem cell technology; stemness; therapeutic transgene; transcription factor; transcriptome sequencing; transduction efficiency; tumorigenesis

Ojective 1: Develop approaches for expansion of hematopoietic stem cells (HSCs) Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivoexpanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Protocols that are based on hematopoietic cytokines (e.g. thrombopoietin TPO, stem cell factor SCF) have failed to support reliable amplification of immature stem cells in culture, suggesting that alternative cytokines or additional factors are required and highly desirable. ELTROMBOPAG. Eltrombopag is a novel TPO agonist with an intrinsic ability to expand HSPC in vivo given observed clinical benefits in patients with aplastic anemia. We have cultured human CD34+ cells for up to 21 days in our standard culture medium supplemented with Eltrombopag or with the combination Eltrombopag + TPO using various cellular or non-cellular support: 1) Fibronectin; 2) Osteoblasts; 3) Endothelial cells. We found that Eltrombopag was not superior to TPO for HSPC expansion in vitro in the absence of a cellular support. Results of transplantation of CD34+ cells expanded on osteoblasts or endothelial support into immune-deficient mice, the gold standard for evaluation of the in vivo repopulating potential of these cells, are pending and will determine the clinical utility of this drug for in vitro expansion of HSCs. Ongoing studies in our laboratory indicate that this drug may work by induction of DNA repair pathways with resulting decrease in apoptosis in HSPC. NOTCH PATHWAY AND HYPOXIA. The primary mediators of hypoxic adaptation are hypoxia-inducible factors (HIF), a family of transcription factors composed of two subunits, an oxygen-labile α subunit that rapidly stabilizes in response to low O2 tensions (HIF-1α and HIF-2α), and a β subunit (HIF-1β) that is constitutively expressed. In immunoprecipitation assays, HIF-1α physically interacted with the intracellular domain of Notch, a critical component for the maintenance of undifferentiated stem and progenitor cell populations, providing a striking molecular link between hypoxia and stemness. Given this recent evidence of a convergence of pathways involved in hypoxia sensing and stem cell maintenance, we are investigating the possibility of stem cell expansion under hypoxic conditions by activation of the canonical Notch signaling pathway using Delta-1 ligand. We have shown that culture of human HSPC under hypoxic conditions in the presence of Notch ligand resulted in 5-fold expansion of the most primitive hematopoietic cells with engraftment potential compared to cells cultured under hypoxia in the absence of Notch ligand. Additional confirmatory studies are ongoing and possible clinical applications are considered. NOVEL PATHWAYS FOR HSC EXPANSION. During homeostasis the HSC pool is maintained at a relatively constant level. In contrast, several murine studies have shown that during hematopoietic stress, such as transplantations, HSCs can and will self-renew extensively, suggesting that HSCs are exposed in vivo to specific factors/signals that promote their self-renewal and amplification. We have transplanted human CD34+ cells into immune-deficient mice and showed evidence of extensive self-renewal in vivo. To identify novel factors/pathways involved in HSC expansion, we are comparing gene expression/methylation patterns between HSC at steady state (before transplant) and after transplant, using RNA-Seq, CHIP-Seq and metabolomics approaches. Optimal transplantation conditions have been identified in the past year and comparative global transcriptome analyses are ongoing. These studies will have a significant impact on the global understanding of human HSC self-renewal and could lead to the development of novel approaches for HSC expansion in vitro. development of novel approaches for HSC expansion in vitro. Objective 2: Develop approaches for differentiation of iPSCs into HSCs With the development of induced pluripotent stem cell (iPSC) technologies emerged the concept of generating iPSCs from an individual patient, correcting the genetic defect using gene specific targeting for safe integration of the therapeutic transgenes, and differentiating the disease-free iPSCs into transplantable HSCs. Currently used protocols for iPSC differentiation into HSCs can generally be divided into two main categories: those that co-culture stem cells with stromal layers (e.g. OP9), and those that culture stem cells in suspension to form embryoid bodies (EBs). However, these protocols remain inefficient at producing the quantity and quality of HSCs required for clinical applications. On the basis of recent data demonstrating that definitive HSCs are generated from a unique population of endothelial cells known as hemogenic endothelium (HE), we have established a novel system for de novo generation of transplantable HSCs from iPSCs. Human iPSCs derived from normal individuals are cultured in the presence of a cytokine combination that favors development of an adherent layer of HE in vitro. Over a period of 12-14 days, these cells further differentiate to produce and release cells in suspension. Up to 70% of these cells have a CD45+CD34+ phenotype compared to 10-15% CD45+CD34+ using current co-culture or EB-based protocols. We have characterized these cells further and demonstrated a subpopulation with the most defined HSC phenotype described (CD34+CD38-CD45RA-CD90+CD49f+Rholo). We have demonstrated that these cells can generate hematopoietic progenitors in vitro but they are functionally inapt at establishing hematopoiesis in vivo, suggesting a developmental defect in iPSC-derived HSPC. Various approaches are underway to address this fundamental issue: 1) Comparative global transcriptional analysis of primary and iPSC-derived HSPC; 2) Instructive potential of endothelial cells; 3) Differentiation of iPSC under hypoxic conditions; 4) Optimization of currently used approach for mesodermal differentiation. We have also initiated the derivation and genetic correction of iPSC lines derived from patients with life-threatening bone marrow failure syndromes for eventual clinical applications. Objective 3: Initiate a first-in-human clinical trial of gene therapy for patients with leukocyte adhesion deficiency type 1 (LAD-1) Patients with Leukocyte Adhesion Deficiency Type 1 (LAD-1) suffer life-threatening bacterial infections that stem from the inability of their neutrophils to adhere to blood vessel walls and migrate to sites of infections. Heterogeneous genetic mutations in the leukocyte integrin CD18 subunit have been associated with this abnormal phenotype. HSPC transplantation represents the only curative therapy for LAD-1, but there are well-described limitations with this treatment. Gene therapy represents an alternative, potentially safer, curative option for these patients. Foamy Viral Vectors (FVV) have several advantages over currently used gene transfer vectors. In pre-clinical studies, we demonstrated correction of the LAD-1 phenotype in four dogs with canine leukocyte adhesion deficiency (CLAD), the animal counterpart to LAD-1 in humans. There has been no emergence of clonal dominance or leukemia after several years of follow-up. In this project, we are developing a human gene therapy clinical trial for LAD-1 using FVV. This project is conducted in two consecutive phases: 1) Development of methods for production, certification and testing of transduction efficiency of cGMP-like and cGMP grade lots of ΔΦMSCV-hCD18 FVV for clinical use; 2) Patients with LAD-1 will be enrolled and monitored for safety and efficacy in a non-randomized phase I gene therapy clinical trial.

目标 1：开发造血干细胞 (HSC) 扩增方法 由于无法产生足够的干细胞数量以及起始细胞群的过度分化，改善离体扩增的造血干细胞和祖细胞（HSPC）的造血重建和植入潜力的尝试基本上不成功。基于造血细胞因子（例如血小板生成素 TPO、干细胞因子 SCF）的方案未能支持培养中未成熟干细胞的可靠扩增，这表明需要并且非常需要替代细胞因子或其他因子。 艾尔腾博帕格。艾曲波帕是一种新型 TPO 激动剂，具有在再生障碍性贫血患者中观察到的临床益处，具有体内扩增 HSPC 的内在能力。我们使用各种细胞或非细胞支持物，在添加艾曲波帕或艾曲波帕 + TPO 组合的标准培养基中培养人类 CD34+ 细胞长达 21 天：1) 纤连蛋白； 2）成骨细胞； 3）内皮细胞。我们发现，在没有细胞支持的情况下，艾曲波帕在体外 HSPC 扩增方面并不优于 TPO。将在成骨细胞或内皮支持上扩增的 CD34+ 细胞移植到免疫缺陷小鼠体内的结果（评估这些细胞体内增殖潜力的金标准）正在等待中，并将确定该药物体外扩增 HSC 的临床效用。我们实验室正在进行的研究表明，该药物可能通过诱导 DNA 修复途径发挥作用，从而减少 HSPC 的细胞凋亡。 缺口通路和缺氧。缺氧适应的主要介质是缺氧诱导因子 (HIF)，这是一个由两个亚基组成的转录因子家族，一个氧不稳定 α 亚基，可响应低 O2 张力而快速稳定（HIF-1α 和 HIF-2α），和组成型表达的 β 亚基 (HIF-1β)。在免疫沉淀试验中，HIF-1α 与 Notch 的细胞内结构域发生物理相互作用，Notch 是维持未分化干细胞和祖细胞群的关键成分，在缺氧和干性之间提供了惊人的分子联系。鉴于最近证据表明缺氧传感和干细胞维持涉及的途径趋同，我们正在研究通过使用 Delta-1 配体激活经典 Notch 信号通路来在缺氧条件下进行干细胞扩增的可能性。我们已经证明，与缺乏Notch配体的缺氧条件下培养的细胞相比，在存在Notch配体的缺氧条件下培养人HSPC导致具有植入潜力的最原始造血细胞扩增5倍。其他验证性研究正在进行中，并考虑可能的临床应用。 HSC 扩展的新途径。在体内平衡期间，HSC 池保持在相对恒定的水平。相比之下，一些小鼠研究表明，在造血应激期间，例如移植，HSC 能够并且将会广泛地自我更新，这表明 HSC 在体内暴露于促进其自我更新和扩增的特定因素/信号。我们将人类 CD34+ 细胞移植到免疫缺陷小鼠体内，并显示出体内广泛自我更新的证据。为了确定参与 HSC 扩增的新因子/途径，我们使用 RNA-Seq、CHIP-Seq 和代谢组学方法比较稳态（移植前）和移植后 HSC 之间的基因表达/甲基化模式。去年已经确定了最佳移植条件，并且正在进行比较性全球转录组分析。这些研究将对人类 HSC 自我更新的全球理解产生重大影响，并可能导致 HSC 体外扩增新方法的开发。 开发 HSC 体外扩增新方法。 目标 2：开发 iPSC 分化为 HSC 的方法 随着诱导多能干细胞 (iPSC) 技术的发展，出现了从个体患者生成 iPSC、使用基因特异性靶向纠正遗传缺陷以安全整合治疗性转基因、并将无病 iPSC 分化为可移植 HSC 的概念。目前使用的 iPSC 分化为 HSC 的方案通常可分为两大类：干细胞与基质层（例如 OP9）共培养的方案，以及悬浮培养干细胞形成胚状体（EB）的方案。然而，这些方案在生产临床应用所需的 HSC 数量和质量方面仍然效率低下。 最近的数据表明，最终的 HSC 是由称为造血内皮 (HE) 的独特内皮细胞群产生的，我们建立了一个新的系统，用于从 iPSC 中从头产生可移植的 HSC。源自正常个体的人 iPSC 在细胞因子组合的存在下进行培养，该细胞因子组合有利于体外 HE 粘附层的发育。在 12-14 天的时间内，这些细胞进一步分化，产生并释放悬浮细胞。使用当前共培养或基于 EB 的方案时，这些细胞中高达 70% 具有 CD45+CD34+ 表型，而 10-15% CD45+CD34+ 表型。我们进一步表征了这些细胞，并证明了具有最明确的 HSC 表型的亚群 (CD34+CD38-CD45RA-CD90+CD49f+Rholo)。我们已经证明这些细胞可以在体外产生造血祖细胞，但它们在功能上不适合在体内建立造血功能，这表明 iPSC 衍生的 HSPC 存在发育缺陷。正在采取各种方法来解决这一基本问题：1) 对原代和 iPSC 衍生的 HSPC 进行比较全局转录分析； 2）内皮细胞的指导潜力； 3）低氧条件下iPSC的分化； 4) 优化目前使用的中胚层分化方法。我们还开始对来自患有危及生命的骨髓衰竭综合征的患者的 iPSC 系进行衍生和基因校正，以供最终的临床应用。 目标 3：启动针对 1 型白细胞粘附缺陷 (LAD-1) 患者的基因治疗的首次人体临床试验 1 型白细胞粘附缺陷 (LAD-1) 患者会遭受危及生命的细菌感染，这是由于中性粒细胞无法粘附在血管壁上并迁移到感染部位所致。白细胞整合素 CD18 亚基的异质基因突变与这种异常表型有关。 HSPC 移植是 LAD-1 的唯一治疗方法，但这种治疗方法存在明显的局限性。对于这些患者来说，基因疗法代表了一种可能更安全的替代治疗选择。泡沫病毒载体 (FVV) 比目前使用的基因转移载体有几个优点。在临床前研究中，我们证明了四只患有犬白细胞粘附缺陷（CLAD）的狗的 LAD-1 表型得到了纠正，CLAD 是人类 LAD-1 的动物对应物。经过几年的随访，没有出现克隆优势或白血病。在这个项目中，我们正在利用 FVV 开发 LAD-1 的人类基因治疗临床试验。该项目分两个连续阶段进行：1）开发用于临床使用的cGMP类和cGMP级批次的ΔΦMSCV-hCD18 FVV的生产、认证和转导效率测试方法； 2) LAD-1患者将被纳入非随机I期基因治疗临床试验并监测其安全性和有效性。