Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Abstract

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Targeting Ribosome assembly on the HCV RNA using a small RNA molecule

“…S5) for delivery into animal liver or cells through reconstituted Sendai viral envelopes (F-virosomes). 18 pSUPER SLRef DNA construct expresses SLRef RNA whereas pSUPER NSP produces nonspecific RNA. Initially, to evaluate the efficiency of gene expression mediated by F-virosomes and to test the ability of pSUPER SLRef to inhibit HCV IRES mediated gene expression, Huh7 cells in culture were transfected with inside the transfected cells.…”

“…Initially, to evaluate the efficiency of gene expression mediated by F-virosomes and to test the ability of pSUPER SLRef to inhibit HCV IRES mediated gene expression, Huh7 cells in culture were transfected with inside the transfected cells. 18,22 Here pSUPERNSP expresses non specific RNA sequence. 18 After 36 h of post transfection cells were treated with cycloheximide and mRNAs were separated by velocity sedimentation on a sucrose gradient.…”

“…18,22 Here pSUPERNSP expresses non specific RNA sequence. 18 After 36 h of post transfection cells were treated with cycloheximide and mRNAs were separated by velocity sedimentation on a sucrose gradient. RNA was isolated from free and polysome fractions ( Fig.…”

“…[16][17][18] However these approaches are sequence specific, which can lead to the exertion of selection pressure on the genome for the generation of escape mutants. Hence, SLRef targets the interaction of a host protein with a conserved structural element in the viral IRES will be more efficacious as mutations in the viral genome are less likely to affect such interactions.…”

“…In the past decade, RNA molecules have been explored as possible candidates for novel HCV therapeutics including siRNAs, ribozymes, antisense RNAs, small RNA, and RNA aptamers, as these acts directly on the genome. [16][17][18] In the present study we have studied the interaction of SLIIIef domain of HCV IRES with 40S subunit and host proteins to design small RNA molecules to inhibit HCV translation. We designed three different RNAs namely SLRe, SLRf and SLRef mimicking SLIIIe, SLIIIf and SLIIIef domains of HCV IRES and investigated their ability to interact with 40S ribosome subunit and host proteins.…”