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

项目摘要 自体造血干细胞基因治疗是最有前途的治疗策略之一 影响血液和免疫系统的遗传性疾病。目前，对患者的唯一治疗方法 血红蛋白病或免疫缺陷等疾病的患者是异基因HSC移植。虽然成果 与HLA匹配的同胞供体是非常令人鼓舞的，大多数患者不会有HLA匹配的家庭 供体来自无关供体或部分匹配供体的HSC移植可能与生命相关， 危险的副作用，特别是来自移植物抗宿主病（GVHD）和相关的感染并发症。到 为了避免这些并发症，已经努力使用患者自己的（自体）HSC并纠正它们， 慢病毒介导的基因治疗或基因编辑。然而，一个关键的问题和瓶颈是， 无法鉴定和纯化“真正的”HSC群体，因此无法优化此类细胞的条件 允许快速的多谱系移植。一个可靠的战略，以确定这种质量和数量的 HSC群体将是离体基因编辑和扩增的高通量筛选的重大进展 条件使用我们的非人灵长类动物（NHP）干细胞移植模型，我们最近发现了一种 能够进行多谱系短期和长期植入的专属HSC富集群体， 在人类和NHP之间进化保守。这种HSC富集的群体占约3%-5%。 整个CD 34+细胞群，减少约25倍的细胞数量的基因治疗方法。最 重要是，流式细胞术定量这种HSC富集表型使我们能够可靠地预测 造血干细胞移植后的移植成功率以及中性粒细胞和血小板恢复的开始， 来源、基因修饰或离体扩增。这种新的HSC富集表型在 结合容易富集该亚群的能力将有助于克服目前的限制， 自体HSC基因治疗。我们假设，这种HSC富集群体的基因编辑将改善 靶向，增强基因编辑效率，并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，这种表型将作为一个可靠的读出， 开发用于扩增基因修饰的HSC的离体培养条件，促进改善的植入， 这反过来又使我们能够减少目前使用的HSC基因预处理方案的强度， 疗法在人类和NHP细胞之间这种HSC富集表型的进化保守性将使我们能够 快速将这些发现转化为临床HSC基因治疗研究。

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.