Chemotherapy-free cure of hemoglobin disorders through base editing

项目摘要 镰状细胞病（SCD）和输血依赖性b-地中海贫血（TDT）是严重的、流行的血液疾病 胎儿血红蛋白（HbF）诱导可以绕过基本的血红蛋白缺陷和造血缺陷。 干/祖细胞（HSPC）移植（HSCT）提供了治疗潜力。同种异体和自体反式- 然而，植物方法可以成功，遗传毒性烷基化化学的短期和长期毒性， 基于治疗的预处理方案仍然是广泛应用治愈性HSCT的实质性障碍 对于SCD和TDT。已提出靶向HSPC抗原的免疫疗法作为更安全的调节策略， 然而，这些药物的药代动力学目前妨碍了它们的临床功效。远景目标 我们的建议是通过开发一种有效的、新颖的移植策略来解决这种未满足的医疗需求， 在SCD和TDT中，通过偶联非基因修饰的HSPCs， 基于毒性免疫疗法的骨髓消融与表位工程。我们的核心假设是 HSPC内HbF决定簇和靶向表位的多重碱基编辑可以赋予造血功能。 具有HbF诱导能力和对单克隆抗体或CAR-T细胞的选择性抗性的谱系， 影响蛋白质功能或调节（所谓的隐形状态）。我们已经确定了最小的氨基酸 细胞因子受体KIT、FLT 3和IL 3RA的细胞外结构域（ECD）内的变化， 在长期再生HSC中，消除治疗性Ab的识别，同时保持HSC的生理活性， 对各自配体刺激的反应。在这里，我们将利用这些成果，进一步扩大 这些创新的基因工程工具的目标是i）产生“隐形”G-球蛋白 ii）通过多重CRISPR-Cas碱基编辑去阻遏的HSPC; b-血红蛋白病的临床前模型，和iii）进一步优化和扩大制造工艺， 生产有效和精确工程化的细胞产品，适用于临床。我们的目标：1） 优化多重碱基编辑方法，以同时抑制HbF和工程化隐形HSPC 表位，其将产生能够改善镰状化的免疫疗法抗性造血干细胞 2）为了最大限度地植入经编辑的红细胞， 通过优化免疫治疗方案以通过建模参数富集多重编辑的HSPCs的HSC 用于编辑的HSPC的治疗性选择，从而获得概念验证的无化疗的移植物， 编辑的患者HSPC的分割和选择;以及3）鉴定产生有效靶向碱基的条件 在临床规模上没有可测量的脱靶编辑。该项目将提供一个基本的进步， 一种新的无化疗的基因治疗方法，用于血红蛋白病的HSCT， 广泛适用于其他造血系统疾病。

PROJECT SUMMARY Sickle cell disease (SCD) and transfusion-dependent b-thalassemia (TDT) are severe, prevalent blood disorders for which fetal hemoglobin (HbF) induction can bypass the fundamental hemoglobin defects and hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative potential. Allogeneic and autologous trans- plant approaches can succeed, nonetheless, the short- and long-term toxicities of genotoxic alkylating chemo- therapy-based conditioning regimens remain a substantial barrier to the widespread application of curative HSCT for SCD and TDT. Immunotherapies targeting HSPC antigens have been proposed as a safer conditioning strat- egy, however the pharmacokinetics of these agents currently hamper their clinical efficacy. The long-term goal of our proposal is to address this unmet medical need by developing an effective, novel strategy for the engraft- ment and progressive enrichment of autologous gene modified HSPCs in SCD and TDT by coupling non-geno- toxic immunotherapy-based myeloablation with epitope-engineering. Our central hypothesis is that precise multiplexed base editing of HbF determinants and targeted epitopes within HSPCs can endow hematopoietic lineages with both HbF induction capacity and selective resistance to monoclonal Abs or CAR-T cells without affecting protein function or regulation (so-called stealth status). We have identified defined minimal amino-acid changes within the extracellular domains (ECD) of the cytokine receptors KIT, FLT3 and IL3RA, each expressed in long-term repopulating HSCs, that abrogate recognition by therapeutic Abs while preserving physiologic responses to stimulation with their respective ligands. Here, we will capitalize on these results and further expand the reach of these innovative genetic engineering tools with the objectives to i) generate “stealth” g-globin derepressed HSPCs by multiplex CRISPR-Cas base-editing; ii) validate efficacy of this approach on suitable pre-clinical models of b-hemoglobinopathy, and iii) further optimize and scale the manufacturing process for production of efficiently and precisely engineered cellular products suitable to progress to the clinic. We aim: 1) to optimize multiplex base editing approaches to simultaneously derepress HbF and engineer stealth HSPC epitopes that will generate immunotherapy resistant hematopoietic stem cells capable of ameliorating sickling and globin chain imbalance in SCD and TDT patient erythroid cells; 2) to maximize the engraftment of edited HSCs by optimizing immunotherapy regimens to enrich multiplex edited HSPCs through modeling parameters for therapeutic selection of edited HSPCs, and thereby obtaining proof-of-concept chemotherapy-free engraft- ment and selection of edited patient HSPCs; and 3) to identify conditions that produce efficient on-target base edits without measurable off-targets at clinical scale. This project will provide fundamental advancement of a new chemotherapy-free gene therapy approach to HSCT for hemoglobinopathies that should additionally have broad applicability to other hematopoietic disorders.