Lee Jeong Min, Zeng Jing, Liu Pengpeng, Nguyen My Anh, Suchenski Loustaunau Diego, Bauer Daniel E, Kurt Yilmaz Nese, Wolfe Scot A, Schiffer Celia A
Department of Biochemistry and Molecular Biotechnology, UMass Chan Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
Division of Hematology/Oncology, Boston Children's Hospital, 1 Blackfan Circle, Boston, MA 02115, USA.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1217.
Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting CE_CBE RNP complexes efficiently target cytosines in TC dinucleotides with minimal off-target or bystander mutations. Delivery of additional uracil glycosylase inhibitor protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation. A single electroporation was sufficient to effectively edit the therapeutically relevant locus BCL11A for sickle cell disease in hematopoietic stem and progenitor cells in a dose-dependent manner without cellular toxicity. Significantly, these CE_CBE RNPs permitted highly efficient editing and engraftment of transplanted cells in mice. Thus, our designed CBE proteins provide promising reagents for RNP-based editing at disease-related sites.
最近，胞嘧啶碱基编辑器（CBEs）已成为一种有前景的治疗工具，可用于特定单核苷酸变体的编辑以及破坏与疾病相关的特定基因。尽管有此前景，但目前可用的CBEs存在脱靶和旁观者编辑活性的重大缺陷，部分原因在于其递送机制，这限制了它们的潜在应用。在本研究中，我们设计了优化的、可溶且稳定的嵌入Cas的CBEs（CE_CBEs），整合了多项最新进展，并将其有效配制为核糖核蛋白（RNP）复合物直接递送至细胞中。我们得到的CE_CBE RNP复合物能够高效靶向TC二核苷酸中的胞嘧啶，脱靶或旁观者突变极少。共转染额外的尿嘧啶糖基化酶抑制蛋白以剂量依赖方式进一步提高了C到T的编辑效率和靶点纯度，同时将插入缺失的形成降至最低。单次电穿孔足以在造血干细胞和祖细胞中以剂量依赖方式有效编辑镰状细胞病的治疗相关位点BCL11A，且无细胞毒性。重要的是，这些CE_CBE RNPs能够使移植细胞在小鼠体内高效编辑并植入。因此，我们设计的CBE蛋白为基于RNP的疾病相关位点编辑提供了有前景的试剂。
