Controlled Intracellular Trafficking and Gene Silencing by Oligonucleotide-Signal Peptide Conjugates

Abstract

Oligonucleotide conjugates have been receiving increasing attention as therapeutic agents. It is well known that oligonucleotide conjugates have some improved pharmacological properties such as enhanced resistance against nucleases, extended retentivity in blood, improved cell membrane permeability, specific cell targeting delivery, and so on. The structure of oligonucleotide conjugates can be found in some nucleic acid therapeutics approved to date such as pegaptanib, heplisav-B, and givosiran. Evaluation of biological properties of oligonucleotide-peptide conjugates indicated that (1) the conjugates bound to target RNA with increased affinity, (2) the conjugates were resistant to cellular nuclease degradation, (3) the hybrid duplex of DNA-peptide conjugate with RNA could activate RNase H as effectively as native oligonucleotides, (4) the conjugates with fusion peptides and cationic peptides could penetrate cellular membrane without any transfection reagents, (5) the conjugates with nuclear localization signal (NLS) peptides were localized in cellular nucleus, and (6) the conjugates with nuclear export signal (NES) peptides were localized in the cytoplasm. It was also demonstrated that antisense phosphorothioate oligonucleotide (sASO)-NLS conjugates could be taken up into cells without any transfection reagents and inhibited 99% of human telomerase activity in human leukemia cell line Jurkat and that siRNA-NES conjugates suppressed 95% of BCR/ABL chimeric gene expression in human chronic myelogenous leukemia cell line K562. The results indicated that controlled intracellular localization of therapeutic oligonucleotides significantly enhanced their silencing activities and that oligonucleotide-signal peptide conjugates have a great potential for therapeutic applications.