Zhou Liujuan, Bi Jie, Chang Shenghai, Bai Zhaoshuai, Yu Junqi, Wang Ruru, Li Zhihang, Zhang Xing, Chou James J, Pan Liqiang
Assembly Medicine, LLC, Shanghai, 201203, China.
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
Angew Chem Int Ed Engl. 2025 Jan 15;64(3):e202415272. doi: 10.1002/anie.202415272. Epub 2024 Nov 6.
Antibody-oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO). Here, we have developed a modular AOC (MAOC) approach based on accurate self-assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO-to-antibody ratio were generated by L-DNA scaffold mediated precise self-assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti-TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo-EM structure of the sdAb-TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic human TfR1 mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity-related morbidity or mortality. Collectively, our data suggest that the self-assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi-specific targeting and delivery.
抗体 - 寡核苷酸缀合物（AOC）可实现反义寡核苷酸（ASO）的优先细胞靶向和增强的细胞摄取。在此，我们基于分别制备的抗体和ASO模块的精确自组装开发了一种模块化AOC（MAOC）方法。通过L - DNA支架介导的抗体和ASO的精确自组装产生了具有确定的ASO与抗体比例的均一多聚体AOC。MAOC方法已用于通过转铁蛋白受体（TfR1）介导的内化来递送外显子跳跃ASO。我们发现一种抗TfR1单域抗体（sdAb）可大大增强ASO的核递送。sdAb - TfR1复合物的冷冻电镜结构显示了一个新的表位，该表位与内源性TfR1配体的结合位点不重叠。在转基因人TfR1小鼠模型中，对用于单外显子跳跃的一种ASO和用于双外显子跳跃的两种ASO的MAOC进行体内功能分析表明，MAOC在心脏和骨骼肌中的ASO浓度和外显子跳跃效率均显著高于传统ASO。MAOC治疗在体内耐受性良好，且与任何毒性相关的发病率或死亡率无关。总体而言，我们的数据表明，自组装的MAOC是通过多特异性靶向和递送拓宽ASO治疗应用的可行选择。
