Antisense oligonucleotides (ASOs) are small, single-stranded molecules that can suppress the expression of targeted genes and thereby achieve therapeutic effects. However, many ASO-based drug candidates accumulate too slowly, show too little potency, or even exert off-target effects. To overcome these difficulties, drug designers have decorated or packaged ASOs in various ways.
One approach, the construction of RNA/RNA heteroduplex oligonucleotides (HDOs), has shown promise. A refinement of this approach, the construction of DNA/DNA HDOs, has been attempted in a new study. It, too, shows promise as a modulator of gene expression. Moreover, it is more stable and easier to manufacture.
Details about the new DNA/DNA HDOs recently appeared in the journal Molecular Therapy, in an article (“Efficient Gene Suppression by DNA/DNA Double-Stranded Oligonucleotide In Vivo”) contributed by scientists from Tokyo Medical and Dental University (TMDU) and Ionis Pharmaceuticals. According to this article, DNA/DNA HDOs consist of two complementary DNA strands, each of which is flanked by different molecular structures. One strand is flanked by locked nucleic acids (LNAs); the other, by 2′-O-methyl nucleic acids.
“Our experiments indicate that the preserved stability of HDO in the serum until uptake by the cells as well as efficient degradation and unwinding of the complementary strand from the ASO strand inside the cells are essential beneficial features of the HDO technology,” the article’s authors wrote. “In addition, phosphodiester RNA is dispensable as the complementary strand of HDO and can be replaced by phosphodiester DNA.
“The latter is less expensive and chemically more stable during synthesis than PO RNA. Our findings, therefore, expand possibilities of the future HDO design and the range of applications of this molecular technology.
The TMDU and Ionis researchers indicated that the current study advances their earlier work, which involved single-stranded RNA ADOs and RNA/DNA HDOs. For example, the researchers had earlier developed an HDO wherein the single-stranded ASO was hybridized to complementary RNA and conjugated with tocopherol. Toc-HDO(coRNA) proved more stable in serum, efficiently deliverable to target cells, and more potent than the parent ASO.
“Cellular uptake was mostly in the intact form, and the parent ASO was released intracellularly,” noted Yutaro Asami, a TDMU researcher and the first author of the current study. “Consequently, we proposed replacing the phosphodiester RNA of the complementary strand with phosphodiester DNA that is more stable and easier to manufacture.”
The researchers bioengineered a DNA/DNA double-stranded oligonucelotide: Toc-HDO(coDNA). The relatively low DNAse in serum would promote stability and the molecule would be activated intracellularly by DNase degradation. The efficacy of this molecular modification was evaluated using murine hepatocyte uptake assay, quantitative real-time PCR assay for RNA levels, and fluorescence-based determination of hepatic ASO concentrations.
“We could establish the efficacy of Toc-HDO(coDNA) on mRNA expression levels in comparison with parent ASOs of varied compositions,” asserted Asami. “Moreover, we also elucidated coDNA strand structure-activity relationships and degradation kinetics in mouse liver cells.”
Many drugs work by modifying specific disease-related proteins. Unfortunately, they may also affect nontargeted proteins causing side effects that downgrade their safety and clinical applicability. Nucleic-acid therapeutics employs an emerging class of drugs including ASOs that target disease at the genetic level by suppressing the expression of pathogenic proteins. By modifying targets hitherto undruggable by conventional pharmacotherapy, they offer potential for treating intractable diseases such as spinal muscular atrophy and Huntington disease, with several candidates in clinical use and more on the horizon.
ASOs are synthetic single-stranded molecules comprising a few dozen base pairs capable of regulating gene expression through binding to the “sense” strand of mRNA targets. Arranging nucleotides, the building blocks of genetic code, in an “antisense” or opposing order can suppress a specific RNA sequence and prevent production of harmful proteins.
“HDO technology promises personalized targeted therapy for several neurodegenerative and other intractable diseases,” declared Takanori Yokota, MD, PhD, a professor of neurology and neurological science at TDMU and the current study’s senior author. “Our innovative molecular structural modifications, by enhancing clinical potency and safety, help enlarge the therapeutic toolkit on this versatile platform.”