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

目前造血干细胞(HSC)基因治疗研究中的一个重大障碍是 实现基因修饰细胞的持续高水平植入，提供长期治疗效果。 即使在完全清髓的条件下，亚治疗性植入也会发生，很可能会变得均匀 更大的问题是非清髓性和非遗传毒性的条件反射。唯一的纠正措施是 亚治疗矫正水平是异基因造血干细胞移植，它使患者面临移植的风险- 抗宿主疾病。在这里，我们提出一种替代的、毒性最小的方法来增加植入率。 基因修饰细胞进入治疗范围，避免了与 清髓性预适应和异基因造血干细胞移植。我们的方法将应用于镰状细胞 这两种疾病是世界上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，但在许多情况下也是如此 亚治疗性基因校正水平。在这项应用中，我们提出了一种创新的策略来丰富基因组- 在减少的、非清髓性的背景下编辑和治疗相关的移植后HSC 条件反射。这一战略依赖于三个协调一致的目标，共同解决当前的HSC基因 治疗/基因组编辑移植。特定目标1将优化新型基地的安全性和有效性 编辑/初始编辑，允许同时修饰治疗性珠蛋白靶标和选择基因。 特殊目标2将应用这些新的编辑工具来修改来自健康或 镰状细胞患者，以支持植入和药物后高水平的治疗性珠蛋白生产 小鼠异种移植模型的选择。具体目标3将以这些发现为基础来评估植入物 和在临床相关猕猴自体移植模型中编辑的HSC的选择方案 降低强度条件反射的设置。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调查人员(刘博士)， 血红蛋白病(Weiss博士)和造血干细胞生物学与移植(Kiem博士)。我们的发现应该是 适用于其他疾病，在这些疾病中，经过基因修正的细胞没有天然的选择优势， 对于其他低强度、非遗传毒性的调节方案也是如此。总的来说，拟议的研究将 确定更安全和有效的造血干细胞移植方案，作为临床试验的基础 患有血红蛋白疾病和其他遗传病的患者。

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.