In VitroInhibition of Hepatitis C Virus by Antisense Oligonucleotides in PBMC Compared to Hepatoma Cells

Abstract

Aim. To assess the efficiency of phosphorothioate antisense oligodeoxynucleotide 1 (S-ODN1) on HCV translation inhibition in PBMC compared to hepatoma cells in vitro for the first time. Materials and Methods. The study included 34 treatment naive HCV patients. IRES domain III and IV sequence variations were tested in 45 clones from 9 HCV patients. PBMC of HCV positive patients were subjected to S-ODN in vitro. Concomitantly HepG2 cells infected by the same patient's serum were also treated with S-ODN1 for 24 and 48 hours. Cellular RNA was tested for HCV plus and minus strands by reverse transcription polymerase chain reaction (RT-PCR). Results. Sequence variations were seen in HCV IRES domain III only while domain IV was conserved among all the tested patient's clones. S-ODN1 successfully inhibited HCV translation in HepG2 cells, while in PBMC inhibition was partial. Conclusion. HCV IRES domain IV is more conserved than domain IIId in genotype 4 HCV patients. S-ODN against HCV IRES domain IV was not efficient to inhibit HCV translation in PBMC under the study conditions. Further studies testing other S-ODN targeting other HCV IRES domains in PBMC should be done.

Silencing HCV Replication in Its Reservoir

“…In the current study, 60 genotypes 4 chronic HCV infected patients were enrolled. They are naïve for HCV treatment, HCV RNA was detected in PBMCs of 19 patients out of 60 (about 31.7%), and the remainder show no HCV in its PBMCs, similarly this finding complies with our result that was published 2014 [ 14 ] that represent presence of HCV intra-PBMC in 10 out of 25 HCV infected patients that represent 40% of total study population.…”

“…Our preliminary data showed that siT efficiently suppresses HCV replication in PBMC in vitro. The result of this study overcomes our result of another study previously published in 2014; antisense SODN1 for inhibition of HCV in both PBMC and hepatoma cells because of failure of antisense to eradicate HCV within PBMCs compared to hepatoma cells [ 14 ]. Besides over-coming IFN+RBV too making it promising molecule for either non-responders or relapses’ patients beside those non-suitable for current therapies.…”

“…Cells were harvested, washed 4 times with PBS, and subjected to total cellular RNA extraction. Total RNA extracted from cultured PBMCs was reverse transcribed and amplified using the same assay described previously for both qualitative and quantitative measurement [ 14 ].…”

In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes

“…Genotype 4 is the most prevalent genotype in Egypt. The objective of these experiments was to determine whether our in silico -designed siRNAs could inhibit authentic viruses [ 1 , 63 ]. Naïve Huh-7 cells were inoculated with serum from viremic patients according to Awady et al .…”

“…The viral inoculation was performed as previously described [ 63 , 64 ]. Briefly, Huh-7 cells were grown in a 75 cm 2 culture flask for 48 h to semi-confluence.…”