Multiplex base/prime editors for in vivo selection of modified HSPCs in hemoglobinopathy

目前造血干细胞（HSC）基因治疗研究的一个重要障碍是， 实现基因修饰细胞的持续高水平植入以提供长期治疗功效。 即使在完全清髓性预处理下也会发生亚治疗性植入， 非清髓性和非遗传毒性条件作用更大的问题。唯一的纠正措施 亚治疗校正水平是同种异体HSC移植，这使患者面临移植物- 抗宿主病在这里，我们提出了一种替代的，毒性最小的方法，以增加植入 基因修饰细胞进入治疗范围，避免与 清髓性预处理和异基因HSC移植。我们的方法将应用于镰状细胞 疾病（SCD）和β-地中海贫血，其代表全世界最常见的严重单基因疾病。 这些疾病的基因替代疗法已经显示出有希望的早期结果，但在许多情况下， 亚治疗基因校正水平。在这个应用中，我们提出了一个创新的策略来丰富基因组- 在减少的、非清髓性的背景下，移植后编辑和治疗相关的HSC 条件反射这一策略依赖于三个协调的目标，共同解决目前的HSC基因 治疗/基因组编辑移植。具体目标1将优化新型基质的安全性和有效性 编辑器/引物编辑器以允许治疗性珠蛋白靶标和选择基因的同时修饰。 Specific Aim 2将应用这些新的编辑工具来修饰来自健康或健康人的HSC。 镰状细胞患者，以支持移植后高水平的治疗性珠蛋白产生， 在小鼠异种移植模型中的选择。具体目标3将基于这些发现来评价植入 在临床相关的恒河猴自体移植模型中编辑的HSC的选择方案， 设置低强度条件反射。为了实现这些目标，我们组建了一个多学科团队， 其中包括在基础编辑/主要编辑方面具有互补专业知识的研究者（刘博士）， 血红蛋白病（韦斯博士）和HSC生物学和移植（Kiem博士）。我们的发现应该是 适用于遗传校正细胞不具有天然选择优势的其它疾病， 也适用于其他低强度、非遗传毒性的预处理方案。总体而言，拟议的研究将 定义一个更安全有效的HSC移植方案，作为临床试验的基础， 患有血红蛋白病和其他遗传性疾病的患者。

A significant obstacle in current hematopoietic stem cell (HSC) gene therapy studies has been the ability to achieve persisting high-level engraftment of gene-modified cells to provide long-term therapeutic efficacy. Subtherapeutic engraftment occurs even under full myeloablative conditioning and will likely become an even bigger issue with nonmyeloablative and nongenotoxic conditioning. The only corrective measure for subtherapeutic correction levels is an allogeneic HSC transplant, which subjects patients to the risks of graft- versus-host disease. Here we propose an alternative, minimally toxic approach to increase engraftment of gene-modified cells into the therapeutic range that avoids the significant side-effects associated with myeloablative conditioning and allogeneic HSC transplantation. Our approach will be applied to sickle cell disease (SCD) and ß-thalassemia, which represent the most common severe monogenic diseases worldwide. Gene replacement therapies for these disorders have shown promising early results but also in many cases subtherapeutic gene-correction levels. In this application, we propose an innovative strategy to enrich genome- edited and therapeutically relevant HSC post-transplantation in the context of reduced, nonmyeloablative conditioning. This strategy relies on three coordinated aims that collectively address current HSC gene therapy/genome editing transplantation. Specific Aim 1 will optimize safety and efficacy of novel base editors/prime editors to allow simultaneous modification of the therapeutic globin target and of the selection gene. Specific Aim 2 will apply these novel editing tools to the modification of HSCs derived from both healthy or sickle cell patients in order to support high levels of therapeutic globin production upon engraftment and drug selection in the mouse xenograft model. Specific Aim 3 will build upon these findings to evaluate engraftment and selection protocols for edited HSC in the clinically relevant rhesus macaque autologous transplant model in the setting of reduced-intensity conditioning. To accomplish these aims, we assembled a multidisciplinary team which includes investigators with complementary expertise in base editing/prime editing (Dr. Liu), hemoglobinopathies (Dr. Weiss) and HSC biology and transplantation (Dr. Kiem). Our findings should be applicable to other diseases in which genetically corrected cells do not have a natural selective advantage and also to other reduced-intensity, nongenotoxic conditioning regimens. Collectively, the proposed studies will define a safer and effective HSCs transplantation protocol that will serve as a foundation for clinical testing in patients suffering from hemoglobinopathies and from other genetic diseases.