Generation and validation of structurally defined antibody-siRNA conjugates
Generation and validation of structurally defined antibody-siRNA conjugates
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DOI：
10.1093/nar/gkaa286
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发表时间：
2020-06-04
影响因子：
14.9
通讯作者：
Rader, Christoph
中科院分区：
文献类型：
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作者：
Nanna, Alex R.;Kel'in, Alexander, V;Rader, Christoph
通过RNA干扰（RNAi）进行基因沉默已经成为一种强大的治疗策略，可以治疗多种潜在疾病。定制siRNA以沉默对癌细胞生长和功能至关重要的基因可能是一种有效的治疗方法，但必须克服几个挑战才能使其用作治疗方式，其中有效和选择性地递送至癌细胞仍然是最重要的。已经报道了使用抗体进行siRNA递送的尝试，但是这些策略使用导致混合物的非特异性缀合，或需要多个步骤、引入突变或使用酶的位点特异性方法。在这里，我们报告了一种方法，以产生抗体-siRNA（1：2）共轭物（ARC）的结构定义和易于组装。该ARC平台基于工程化的双可变结构域（DVD）抗体，其含有天然的独特反应性赖氨酸残基，用于与β-内酰胺接头官能化的siRNA位点特异性缀合。缀合是有效的，不损害亲本抗体的亲和力，并且利用化学稳定的siRNA。为了验证概念，我们产生了靶向多发性骨髓瘤细胞上的各种细胞表面抗原的DVD-ARC，用于选择性递送靶向β-连环蛋白（CTNNB 1）的siRNA。一组浓度低至10 nM的靶向BCMA的DVD-ARC显示出显著的CTNNB 1 mRNA和蛋白质敲低。
Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to beta-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting beta-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.