Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia, Philadelphia, PA.
Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; and.
Blood. 2022 Aug 11;140(6):619-629. doi: 10.1182/blood.2022015825.
Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.
同种异体嵌合抗原受体 T 细胞 (CART) 疗法需要进行多次基因编辑才能在临床上可行。大多数同种异体 CART 都是使用诱导 DNA 双链断裂 (DSB) 的基因编辑技术创建的，这会导致意想不到的靶向编辑结果，从而产生潜在的无法预测的后果。胞嘧啶碱基编辑器 (CBE) 在 T 细胞中安装 C•G 到 T•A 点突变，效率在 90%至 99%之间，可在不产生 DSB 的情况下沉默基因表达，与使用成簇规律间隔短回文重复 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 进行多重编辑相比，大大减少或消除了不必要的编辑结果。我们使用 CBE 开发了 7CAR8，这是一种使用 4 个同时碱基编辑创建的靶向 CD7 的同种异体 CART。我们表明，与 CRISPR-Cas9 不同，CBE 不会影响 T 细胞的增殖，不会导致异常的 DNA 损伤反应途径激活，也不会在多重编辑后导致染色体异常。我们在多种体外和体内模型中证明 7CAR8 对 T 细胞急性淋巴细胞白血病 (T-ALL) 具有高度疗效。因此，CBE 是一种有前途的需要多重基因编辑应用的技术，可用于制造四倍编辑的 7CAR8 细胞，具有很高的临床转化潜力，可用于复发和难治性 T-ALL。
