Chemotherapy-free cure of hemoglobin disorders through base editing

项目总结 镰状细胞病(SCD)和输血依赖型b-地中海贫血(TDT)是严重的、普遍的血液疾病。 对于其中的胎儿血红蛋白(HBF)的诱导可以绕过基本的血红蛋白缺陷和造血 干细胞/祖细胞(HSPC)移植(HSCT)具有治疗的潜力。同种异体和自体反式 然而，植物方法可以成功地解决遗传毒性烷基化化学药物的短期和长期毒性问题。 以治疗为基础的调节方案仍然是广泛应用治疗性造血干细胞移植的主要障碍。 适用于SCD和TDT。针对HSPC抗原的免疫治疗已被认为是一种更安全的调节策略。 然而，这些药物的药代动力学目前阻碍了它们的临床疗效。长期目标 我们建议的是通过开发一种有效的、新颖的移植策略来解决这一未得到满足的医疗需求- 自体基因修饰的HSPC在SCD和TDT中的克隆和非基因偶联进行性浓缩 以毒性免疫治疗为基础的表位工程骨髓清除术。我们的中心假设是 HSPC内HBF决定簇和靶表位的多重碱基编辑可以赋予造血功能 对单抗或CAR-T细胞既有HBF诱导能力又有选择性抗性的谱系 影响蛋白质功能或调节的(所谓的隐形状态)。我们已经确定了定义的最低氨基酸 细胞因子受体试剂盒的胞外区(ECD)内的变化，Flt3和IL3RA各自表达 在长期重新填充HSCs中，这在保留生理功能的同时消除了治疗性抗体的识别 用它们各自的配体对刺激的反应。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：1)产生“隐形”的g-珠蛋白 通过多重CRISPR-CAS碱基编辑来解压HPC；ii)验证该方法的有效性 B型血红蛋白病的临床前模型，以及iii)进一步优化和规模化生产工艺 生产适合临床应用的高效、精密的细胞工程产品。我们的目标是：1) 优化多重基础编辑方法，同时降低HBF和工程隐身HSPC 将产生免疫治疗耐药的造血干细胞的表位能够改善镰状 和珠蛋白链在SCD和TDT患者红系细胞中的失衡；2)最大限度地植入编辑的 优化免疫治疗方案，通过模型参数丰富多重编辑的造血干细胞 用于对编辑的HSPC进行治疗选择，从而获得概念验证的免化疗植入物- 编辑的患者HSPC的采集和选择；以及3)确定产生有效的目标基础的条件 在临床规模上没有可测量的偏离目标的编辑。该项目将从根本上促进 用于HSCT的新的非化疗基因疗法治疗本应额外增加的血红蛋白疾病 广泛适用于其他血液病。

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.