Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates

Abstract

Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the ‘cap-independent’ translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5′ or 3′-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37°C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the ‘IRES-dependent’ translation in vitro. The 5′-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines.

Hepatitis C Virus Translation Inhibitors Targeting the Internal Ribosomal Entry Site

“…Inhibition of HCV translation by oligonucleotide-mediated cleavage of the IRES RNA was investigated using ribozymes, 52–58 DNAzymes, 59, 60 conjugated chemical nucleases 61, 62 as well as siRNA and shRNA. 54, 63–72 The design principle shared in common by these constructs requires an accessible single stranded sequence in the IRES 73 that provide the target for a complementary oligonucleotide ligand which is either associated with or, in the case of siRNA and shRNA, directs a ribonuclease activity.…”

IRESPred: Web Server for Prediction of Cellular and Viral Internal Ribosome Entry Site (IRES)

“…Internal ribosome entry sites (IRES), being potential therapeutic targets 9 , determining their presence in a given nucleotide stretch holds clinical importance. Experimental or computational methods capable of demonstrating the presence of IRES in sequences of interest will certainly help biologists working in the area of translation related diseases.…”

“…IRES element therefore, has been tested as a potential therapeutic target 9 . Although, such interventions are yet to be materialized extensively, demonstrating presence of these elements in 5′UTRs, still holds importance.…”

Inhibition of hepatitis C virus gene expression by adenoviral vectors encoding antisense RNA in vitro and in vivo

“…Previously, we and others demonstrated that antisense ODN, hammerhead ribozymes or siRNA targeting sequences of HCV5 0 NCR could inhibit HCV-translation [5][6][7][8]18]. In this work, we extended these studies by testing the inhibitory potential of an asRNA binding the entire HCV5 0 NCR via adenoviral vectors.…”