Chronic hepatitis C virus (HCV) infection affects ~3% of the world’s population and represents one of the major public health concerns. Being a leading cause of liver cirrhosis and hepatocellular carcinoma chronic HCV infection is now one of the key indications for liver transplantation in developed countries. HCV belongs to the genus Hepacivirus in the family Flaviviridae. The viral genome is a single-stranded RNA of positive polarity ~9500 nt in length. Specific binding of antisense oligonucleotides (ASOs) to a target RNA can inactivate its replication or translation thus interfering with viral life cycle. ASOs act through steric blockade of the translation machinery and degradation of target RNA by ribonuclease H (RNase H). HCV genomic RNA is potential but difficult target for antisense technology-based compounds (ASOs) due to the presence of intensive RNA secondary structures. Thus far, mainly ASOs targeting 5’ non-coding region of HCV RNA, which contains the internal ribosome entry site, have been shown to efficiently inhibit HCV replication.
Aim of current research was to validate ASO-based technology targeting the coding region of HCV: 1) To develop a method of selection of antisense target; 2) To demonstrate the impact of 8-oxo-2’-deoxyguanosine (8-oxo-dG) on the ASO properties and antiviral activities.
GeneCode AS has developed a novel technology by modifying nucleobases in order to improve their binding properties to complementary bases. 8-oxo-dG contains a minimally modified nucleobase, which binds to normal cytosine. The effects of incorporation of such nucleobases into ASOs targeting genome of hepatitis C virus have been analyzed.
In a set of in vitro tests we demonstrated that:
1) 8-oxo-dG supports RNase H mediated degradation of targeted RNA;
2) 8-oxo-dG residues allow for more efficient inhibition of HCV replication in cell culture;
3) 8-oxo-dG modification significantly increases stability of DNA and locked nucleic acid (LNA)/DNA gapmer ASOs in human serum. Positive effects of 8-oxo-dG modification were confirmed in in vivo experiments. Plasmid expressing targeted Rluc and non-targeted Fluc markers was co-transfected with 1500 pmol of control all-DNA ASO, or with increasing amounts of modified LNA/DNA gapmer ASOs. These experiments demonstrated that GeneCode modified ASOs efficiently inhibit expression of targeted marker gene in mouse liver.
Competitive advantages of GeneCode modernized antisense technology: Over years the HCV therapy was based on use of injectable interferon alpha – oral ribavirin combination. This approach has been gradually replaced by using modern oral directly acting antivirals as sofosbuvir. However, these therapies remain expensive. Significant improvement of injectable interferon-alpha based therapy may make it competitive, if the regimen can be delivered with lower frequency (for example, once or twice in month as opposed to daily use of oral drugs), has increased efficiency and can be used against genotypes of HCV with high resistance to oral drugs (such as genotype 3). GeneCode ASOs are injectable compounds and can be combined with other ASOs having different mechanism of action such as miR-122 inhibitors. LNA based ASOs have long serum half-lives and have been shown promising results in clinical trials; GeneCode modification further improves stability of ASO, the most important competitive advantages of this class of compounds. Furthermore 1) GeneCode modified ASOs can be used to target heavily structured RNAs such as coding region of HCV RNA genome; 2) 8-oxo-dG residues reduce the Tm of ASO:RNA duplexes and by doing this may reduce off-target side effects; 3) 8-oxo-dG residues facilitate the cleavage of ASO:RNA duplexes by RNase H at multiple positions within the target region; 4) Incorporation of 8-oxo-dG residues increases stability of ASOs in serum; 5) Combination of these properties resulted in highly active ASOs functional both in in vitro system and in in vivo model.
GENECODE PLATFORM TECHNOLOGIES PATENT:
US Patent no., US 7,786, 292 B2, Date of Patent Aug. 31, 2010, European Patent No EP2013044, Aug. 29, 2012
“ANTISENSE AGENTS COMBINING STRONGLY BOUNDING BASE-MODIFIED OLIGONUCLEOTIDE AND ARTIFICIAL NUCLEASE”
Mutso et al., 2015 (doi: 10.1371/journal.pone.0128686)