In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

中文翻译：

---

驱动谱系特异性 BCL11A 敲低的新型慢病毒载体用于镰状细胞基因治疗的临床前评估。


在这项工作中，我们提供了临床前数据来支持启动镰状细胞病（SCD）的首次人体试验，该试验使用了一种依赖于发育胎儿到成人血红蛋白转换逆转的方法。通过慢病毒编码的 microRNA 适应的短发夹 RNA (shRNAmiR) 进行红细胞特异性敲低 BCL11A，导致 γ-珠蛋白基因重新激活，同时减少致病性成体镰刀 β-珠蛋白的表达。我们生成了一种精制的慢病毒载体 (LVV) BCH-BB694，该载体的开发是为了克服在原始研究级 LVV 的生产放大过程中观察到的较差的载体滴度。使用良好生产规范 (GMP) 级 BCH-BB694 LVV 转导的健康或镰状细胞供体 CD34+ 细胞实现了高载体拷贝数 (VCN) >5 和基因标记 >80%，导致 3 至 5 倍的诱导胎儿血红蛋白 (HbF) 与模拟转导细胞相比，不影响基因修饰细胞的体外或体内生长、分化和植入。旨在测量载体介导的遗传毒性的体外永生化测定显示，与模拟转导的细胞相比，永生化没有增加。这些数据共同表明，BCH-BB694 LVV 在临床前研究中无毒且有效，并且可以在 GMP 环境中以临床相关规模以高滴度生成，以支持 SCD 治疗的临床测试。