2'-O-甲氧基乙基硫代磷酸酯剪接转换寡核苷酸的肽缀合物显示内体中的包埋增加,ACS Omega

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2'-O-甲氧基乙基硫代磷酸酯剪接转换寡核苷酸的肽缀合物显示内体中的包埋增加
ACS Omega ( IF 3.7 ) Pub Date : 2023-10-17 , DOI: 10.1021/acsomega.3c05144
Alyssa C Hill ₁ , J Philipp Becker ₁ , Daria Slominski ₁ , François Halloy ₁ , Christoffer Søndergaard ₂ , Jacob Ravn ₂ , Jonathan Hall ₁

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反义寡核苷酸 (ASO) 是改变基因表达的短单链核酸分子。然而，它们转运到适当的细胞区室是其效力的限制因素。在这里，我们合成了先前开发用于治疗罕见疾病红细胞生成性原卟啉症的剪接转换寡核苷酸（SSO）。使用化学连接定量聚合酶链反应 (CL-qPCR)，我们对自由摄取后细胞和亚细胞区室中的 SSO 进行了定量。为了驱动核定位，我们使用硫醇-马来酰亚胺化学将核定位信号（NLS）肽共价结合到2′- O-甲氧基乙基硫代磷酸酯SSO上。缀合物和亲本 SSO 显示出相似的 RNA 靶标结合亲和力。游离摄取后，对细胞和亚细胞区室中的缀合物进行 CL-qPCR 定量，结果显示一种缀合物相对于亲本 SSO 具有更好的核积累。然而，与改变目标前mRNA剪接的母体SSO相比，在体外自由摄取条件下，缀合物在剪接校正时没有活性。通过阳离子脂质介导的转染将缀合物递送到细胞中，或通过用氯喹（一种内体破坏剂）处理已自由吸收缀合物的细胞，可以赋予缀合物剪接转换活性。我们的结果确定了肽-寡核苷酸缀合物活性的主要障碍是内体截留。

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Peptide Conjugates of a 2′-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes

Antisense oligonucleotides (ASOs) are short, single-stranded nucleic acid molecules that alter gene expression. However, their transport into appropriate cellular compartments is a limiting factor in their potency. Here, we synthesized splice-switching oligonucleotides (SSOs) previously developed to treat the rare disease erythropoietic protoporphyria. Using chemical ligation-quantitative polymerase chain reaction (CL-qPCR), we quantified the SSOs in cells and subcellular compartments following free uptake. To drive nuclear localization, we covalently conjugated nuclear localization signal (NLS) peptides to a lead 2′-O-methoxyethyl phosphorothioate SSO using thiol–maleimide chemistry. The conjugates and parent SSO displayed similar RNA target-binding affinities. CL-qPCR quantification of the conjugates in cells and subcellular compartments following free uptake revealed one conjugate with better nuclear accumulation relative to the parent SSO. However, compared to the parent SSO, which altered the splicing of the target pre-mRNA, the conjugates were inactive at splice correction under free uptake conditions in vitro. Splice-switching activity could be conferred on the conjugates by delivering them into cells via cationic lipid-mediated transfection or by treating the cells into which the conjugates had been freely taken up with chloroquine, an endosome-disrupting agent. Our results identify the major barrier to the activity of the peptide–oligonucleotide conjugates as endosomal entrapment.

更新日期：2023-10-17

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