Efficient Gene Suppression by DNA/DNA Double-Stranded Oligonucleotide In Vivo

• Published in: Molecular therapy Vol. 29; no. 2; pp. 838 - 847
• Main Authors: Asami, Yutaro, Nagata, Tetsuya, Yoshioka, Kotaro, Kunieda, Taiki, Yoshida-Tanaka, Kie, Bennett, C. Frank, Seth, Punit P., Yokota, Takanori
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.02.2021; American Society of Gene & Cell Therapy
• Subjects: alpha-Tocopherol; antisense oligonucleotide; Cells, Cultured; DNA - administration & dosage; DNA - genetics; double-stranded antisense oligonucleotide; double-stranded DNA; Gene Expression Regulation; Gene Silencing; Gene Transfer Techniques; heteroduplex oligonucleotide; Oligodeoxyribonucleotides - administration & dosage; Oligodeoxyribonucleotides - genetics; Oligonucleotides, Antisense - administration & dosage; Oligonucleotides, Antisense - genetics; Original; alpha-Tocopherol; heteroduplex oligonucleotide; double-stranded antisense oligonucleotide; antisense oligonucleotide; double-stranded DNA
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2020.10.017

We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2′-O-methyl RNA strand. When α-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2′-O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications. [Display omitted] Yokota, Seth, and colleagues have proposed a new concept of DNA/DNA double-stranded oligonucleotide, composed of a DNA-based antisense oligonucleotide and a DNA-based complementary strand. Systemic administration of this duplex conjugated with α-tocopherol significantly increased the target RNA-suppressing effect of the parent single-stranded antisense oligonucleotide following the degradation of complementary DNA in mouse liver.