Hepatitis C virus (HCV)
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 the 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 a potential but difficult target for antisense technology-based compounds (ASOs) due to the presence of intensive RNA secondary structures. Thus far, mainly ASOs targeting a 5’ non-coding region of HCV RNA, which contains the internal ribosome entry site, have been shown to efficiently inhibit HCV replication.
The aim of the current research is 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-o xo2’- deoxyguanosine (8-o xo-dG ) on the ASO properties and antiviral activities.
Approaching GeneCode AS has developed a novel technology by modifying nucleobases to improve their binding properties to complementary bases. 8-o xo-dG contains a minimally modified nucleobase, which binds to normal cytosine. The effects of incorporation of such nucleobases into ASOs targeting the genome of the hepatitis C virus have been analysed.
In a set of in vitro tests we demonstrated that:
- 8-o xo-dG supports RNase H mediated degradation of targeted RNA];
- 8-o xo-dG residues allowing for more efficient inhibition of HCV replication in cell culture;
- 8-o xo-dG modification significantly increases the stability of DNA and locked nucleic acid (LNA) / DNA gapmer ASOs in human serum. The positive effects of 8-o xo-dG modification were confirmed in vivo experiments. Plasmid expressing targeted Rluc and non-targeted Fluc markers were co-transfected with 1500 pmol of a control all-DNA ASO or with increasing amounts of modified LNA / DNA gapmer ASOs. These experiments demonstrate that GeneCode-modified ASOs efficiently inhibit the expression of the targeted marker gene in mouse liver.
Competitive advantages of GeneCode modernised antisense technology: Over the years, HCV therapy was based on the use of an injectable interferon alpha oral ribavirin combination. This approach has been gradually replaced by using modern oral direct-acting antivirals such 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 a lower frequency (for example, once or twice a month as opposed to the 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 mechanisms of action such as miR-122 inhibitors. LNA-based ASOs have long serum half-lives and have shown promising results in clinical trials. GeneCode modification further improves the stability of an ASO, the most important competitive advantage of this class of compounds. Furthermore, 1. GeneCode modified ASOs can be used to target heavily structured RNAs such as the coding region of HCV RNA genome; 2. 8-o xo-dG residues reduce the Tm of ASO: RNA duplexes and by doing this may reduce off-target side effects ; 3. 8-o xo-dG residues facilitate the cleavage of ASO: RNA duplexes by RNase H at multiple positions within the target region; 4. Incorporation of 8-o xo-dG residues increases the stability of ASOs in serum; 5. A combination of these properties resulted in highly active ASOs, functional both in the in vitro system and the in vivo model.
GeneCode platform technologies patent:
US Patent no. US 7,78 6, 292 B2, Date of Patent: Aug 31, 2010, European Patent No E P 2 013 044, Aug 29, 2012
“ANTISENSE AGENTS COMBINING STRONGLY BOUNDING BASE-MODIFIED OLIGONUCLEOTIDE AND ARTIFICIAL NUCLEASE”
PUBLICATION: Mutso et al., 2015 (doi: 10 .13 7 1 /journal.pone.01 28686)