Targeting a Novel Hematopoietic Stem Cell Population in Non-Human Primates for Effective and Sustained Gene Therapy

针对非人类灵长类动物的新型造血干细胞群进行有效和持续的基因治疗

• 批准号: 10287494
• 负责人: HANS-PETER KIEM
• 金额: $ 20.59万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-11-10 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10287494
• 关键词: Affect; Allogenic; Animals; Autologous; Autologous Transplantation; Blood; Blood Cells; Blood Platelets; Bone Marrow; CCR5 gene; CD34 gene; CRISPR/Cas technology; Cells; Clinical; Clonality; Data; Disease; Engraftment; Family; Flow Cytometry; Frequencies; Gene-Modified; Genes; Genetic Diseases; Genetic Transcription; Goals; Grant; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobinopathies; Human; Immune; Immune system; Immunologic Deficiency Syndromes; In Vitro; Lentivirus; Life; Maintenance; Mediating; Outcome; Patients; Phenotype; Population; Property; Recovery; Regimen; Safety; Siblings; Source; Stem cell transplant; Supplementation; Technology; Testing; Time; Translating; Transplantation; Umbilical Cord Blood; Whole-Body Irradiation; base; conditioning; cost; curative treatments; gene therapy; graft vs host disease; hematopoietic stem cell expansion; high throughput screening; improved; in vivo; neutrophil; nonhuman primate; novel; peripheral blood; side effect; small molecule; stem cell gene therapy; stem cell population; stem cells; success; transcriptome sequencing; transplant model; treatment strategy; zinc finger nuclease; zinc finger nuclease technology

Project Summary Autologous hematopoietic stem cell (HSC) gene therapy is one of the most promising strategies for the treatment of genetic diseases affecting the blood and immune system. Currently, the only curative treatment for patients with diseases like hemoglobinopathies or immunodeficiencies is an allogeneic HSC transplant. While outcomes with an HLA-matched sibling donor are very encouraging, most patients will not have an HLA-matched family donor. HSC transplants from unrelated donors or partially matched donors can be associated with life- threatening side effects especially from graft-vs-host disease (GVHD) and related infectious complications. To avoid these complications, efforts have been made to use the patients´ own (autologous) HSCs and correct them with either lentivirus-mediated gene therapy or gene editing. A critical problem and bottleneck, however, has been the inability to identify and purify a “true” HSC population and therefore optimize conditions for such cells that allow for rapid multi-lineage engraftment. A reliable strategy to determine the quality and quantity of such a HSC population would be a major advance for high-throughput screening of ex vivo gene editing and expansion conditions. Using our nonhuman primate (NHP) stem cell transplantation model, we have recently identified an exclusive HSC-enriched population capable of multi-lineage short-term and long-term engraftment that is evolutionary conserved between human and NHPs. This HSC-enriched population accounts for ~3-5% of the entire CD34+ cell population, reducing the number of cells for gene therapy approaches by ~25-fold. Most importantly, flow-cytometric quantification of this HSC-enriched phenotype allowed us to reliably predict engraftment success as well as the onset of neutrophil and platelet recovery after HSC transplantation regardless of the source, gene modification, or ex vivo expansion. Availability of this novel HSC-enriched phenotype in combination with the ability to easily enrich for this subpopulation will help overcome current limitations of autologous HSC gene therapy. We hypothesize that gene editing of this HSC-enriched population will improve targeting, enhance gene editing efficiency, and increase in vivo persistence of gene-modified and corrected blood and immune cells. Furthermore, we hypothesize that this phenotype will serve as a reliable read-out to develop ex vivo culture conditions for the expansion of gene-modified HSCs promoting improved engraftment, which in turn should enable us to reduce the intensity of currently used conditioning regimens for HSC gene therapy. Evolutionary conservation of this HSC-enriched phenotype between human and NHP cells will allow us to rapidly translate these findings to clinical HSC gene therapy studies.

项目摘要 自体造血干细胞（HSC）基因疗法是治疗最有前途的策略之一 影响血液和免疫系统的遗传疾病。目前，对患者的唯一现代治疗 诸如血红蛋白病或免疫缺陷之类的疾病是一种同种异体HSC移植。同时结果 伴随HLA匹配的兄弟姐妹捐赠者非常令人鼓舞，大多数患者将没有HLA匹配的家庭 捐助者。来自无关捐助者或部分匹配的捐助者的HSC移植可以与生活有关 威胁性副作用，尤其是基层宿主疾病（GVHD）和相关感染并发症。到 避免这些并发症，已经努力使用患者自己的（自体）HSC并纠正它们 与慢病毒介导的基因疗法或基因编辑。但是，一个关键的问题和瓶颈有 是无法识别和净化“真实”的HSC种群，因此可以优化此类细胞的条件 这允许快速的多部门植入。确定此类质量和数量的可靠策略 HSC人口将是对离体基因编辑和扩展的高通量筛查的重大进步 状况。使用我们的非人类灵长类动物（NHP）干细胞移植模型，我们最近确定了 富含HSC的人群，能够短期和长期植入多段的人群 人与NHP之间的进化保守。富含HSC的人口约占约3-5％ 整个CD34+细胞群，将基因治疗方法的细胞数量减少了约25倍。最多 重要的是，这种富含HSC的表型的流量量化量化使我们能够可靠地预测 HSC移植后的植入成功以及中性粒细胞和血小板恢复的开始 源，基因修饰或离体扩展。这种新颖的HSC增益表型的可用性 结合轻松丰富此亚群的能力将有助于克服当前的局限 自体HSC基因治疗。我们假设这种富含HSC的人群的基因编辑将改善 靶向，增强基因编辑效率以及增加基因修饰和校正的体内持久性 血液和免疫细胞。此外，我们假设这种表型将作为可靠的读者 开发离体培养条件，以扩大基因修饰的HSC，以促进促进植入的植入 反过来，这应该使我们能够降低HSC基因当前使用的调节方案的强度 治疗。人类和NHP细胞之间这种富含HSC的表型的进化保护将使我们 快速将这些发现转化为临床HSC基因治疗研究。