Enhanced specificity of HPV16 E6E7 siRNA by RNA-DNA chimera modification

• Published in: Cancer gene therapy Vol. 18; no. 8; pp. 587 - 597
• Main Authors: Yamato, K, Egawa, N, Endo, S, Ui-Tei, K, Yamada, T, Saigo, K, Hyodo, I, Kiyono, T, Nakagawa, I
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2011; Nature Publishing Group
• Subjects: 631/1647/1513/1967; 692/699/67/1517/1371; Biomedical and Life Sciences; Biomedicine; Care and treatment; Cell Line, Tumor; Cervical cancer; Cervix; Chimera - genetics; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA, Viral - genetics; Gene Expression; Gene silencing; Gene Therapy; Genetic aspects; HeLa Cells; Human papillomavirus; Human papillomavirus 16; Human papillomavirus 16 - genetics; Humans; Keratinocytes; Nucleotides; Oncogene Proteins, Viral - genetics; Oncogene Proteins, Viral - metabolism; original-article; Papillomavirus E7 Proteins - genetics; Papillomavirus E7 Proteins - metabolism; Papillomavirus infections; Prevention; Repressor Proteins - genetics; Repressor Proteins - metabolism; RNA Interference; RNA modification; RNA, Small Interfering - genetics; RNA, Viral - genetics; siRNA; Transfection; Japan; off-target effect; cervical cancer; siRNA therapy; HPV16
• ISSN: 0929-1903; 1476-5500
• DOI: 10.1038/cgt.2011.28

Although efforts have been made to develop new drugs for infectious and neoplastic diseases utilizing synthetic small interfering RNA(siRNAs), those intrinsically have undesirable effects, including silencing of unintended genes (off-target effect) and nonspecific cytotoxicity. Off-target effects can be avoided by DNA substitution in the guide strand (GS) seed region of nucleotide positions 1–8 and its complementary part of the passenger strand plus the 3′ overhang, which is designated as a double-strand RNA–DNA chimera (dsRDC). In this study, we found that the specificity of potent siRNAs targeting human papillomavirus 16 (HPV16) E6 and E7 oncogenes, which we previously reported, could be enhanced by short dsRDC modification (first six nucleotides from the 5′ end of the GS and its complementary nucleotides of the passenger strand). Such dsRDC modification reduced nonspecific cytotoxicity in two of three siRNAs (497 and 752), although not in the other (573), which correlated with their off-target effects. In addition, silencing activity was marginally impaired in two dsRDCs (497 and 573) and moderately in one (752). Finally, dsRDC-497 induced E6E7-specific growth suppression of cervical cancer cells as well as E6E7-immortalized human keratinocytes. Our results show that dsRDC modification enhances the specificity of E6E7 siRNA, which is required for use in in vivo settings.