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

Abstract

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 0 -Omethyl RNA strand. When a-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 0 -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.

Single Stranded Fully Modified-Phosphorothioate Oligonucleotides can Induce Structured Nuclear Inclusions, Alter Nuclear Protein Localization and Disturb the Transcriptome In Vitro

“…Consequently, our findings of reduced off-target binding by duplex oligonucleotides suggest that steric blocking AOs utilizing all-phosphorothioate modified bases could be delivered as duplexes to reduce off-target effects associated with the backbone chemistry. Nagata and others ( Asada et al, 2021 ; Asami et al, 2021 ; Nagata et al, 2021 ) designed fully phosphorothioated gapmers annealed to modified RNA and DNA oligonucleotides to form heteroduplexes, conjugated to cholesterol or α-tocopherol at the 5′ end of the RNA strand, and showed delivery to the central nervous system (CNS) after subcutaneous or intravenous administration in mice and rats. In the context of the CNS, the heteroduplex was far more effective in knocking down the target than the single stranded conjugated gapmer ( Nagata et al, 2021 ).…”

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics

“…This may have implications such as premature in vivo release; however, up to 60% of the assembly remained intact after 72 h of incubation in 50% human serum. Moreover, it would be expected that greater degradation would occur intracellularly due to the combined presence of both DNase I and II nucleases. , Some high molecular weight (Mw) bands nonmigrating in the native PAGE were observed after incubating the assembly in serum. These bands could indicate a degree of aggregation possibly due to interaction of serum proteins with the assembly; however, these represent only around 30% of the assembly.…”

Recent Advances in RNA Therapy and Its Carriers to Treat the Single-Gene Neurological Disorders

“…Up to date, three gapmers have been approved for clinical use, Mipomersen, Inotersen, and Volanesorsen. In addition to the single stranded oligonucleotides, double stranded DNA-RNA heteroduplex oligonucleotides (HDO) [ 30 , 31 ], and overhanging heteroduplex oligonucleotides (ODO) [ 32 ] were recently reported to have more potent gene silencing activities.…”