الفهرس 
1. INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Hepatitis C virus (HCV) infection is a global blood borne disease that
affects almost 3% of world population with a morbidity and mortality that are
second to HIV among the emerging infections. Egypt has reported the highest
prevalence of HCV worldwide, ranging from 6% to more than 40% among region
and demographic groups with 11-14% of the population chronically infected with
the virus. The infection may progress to cirrhosis with the subsequent
development of complications including hepatocellular carcinoma. A significant
increase in HCV is recognized as leading indication for liver transplantation.
Hepatitis C virus is a spherical, enveloped, single stranded, positive-sense
RNA virus classified as the sole member of Hepacivirus genus in the Flavivirdae
family. The HCV RNA genome is approximately 9.6 kb in length with an open
reading frame (ORF) encoding a large viral polyprotein of about 3010 amino
acids. Viral translation is mediated through an internal ribosome entry site (IRES)
found within the 5/UTR. The sequence of 5/UTR ~341 bp in length is highly
conserved even between different HCV isolates. 5/UTR does not encode for
functional protein and contains IRES that initiate translation of the viral
polyprotein in a cap-independent manner. The IRES has a key role in translational
events as it binds independently to the 40S ribosomal subunit and directs the
ribosome to the initiation codon of the HCV mRNA in order to facilitate
translation in a cap-independent manner. It contains four highly structured stemlopped
domains (domain I-IV) that facilitate the translation of HCV RNA.
Domain I is not required for IRES activity but essential for HCV replication, IRES
in Domain II-IV mediates the cap independent translation of viral genes. Domain
III contains subdomains which are essential for the binding of 40S ribosomal
subunit.
HCV mainly is a hepatotropic virus but peripheral blood mononuclear
cells (PBMC) represent alternative extrahepatic site of HCV replication and
proposed as a source of recurrent HCV infection after liver transplantation. The
main goal of therapy in hepatitis C virus (HCV) infection is to achieve a sustained
virological response (SVR), currently defined as undetectable HCV-RNA in
peripheral blood determined 24 weeks after the end of treatment (ETR).
Nonresponders to therapy are sometimes refractory to retreatment and do not
necessarily benefit from escalating the dose. In the currently recommended doses
for the treatment of chronic hepatitis C, interferon causes side effects that are
generally mild and well tolerated. With prolonged therapy, the occurrence of late
and severe side effects should lead to the discontinuation of interferon.
The currently recommended treatment of HCV infected patients is the
combination of PEG-IFN-α , Ribavirin and direct acting antivirus (DAAs) and the
main goal of therapy is to achieve a sustained virological response (SVR), Due to
the limited efficiency of current combination therapy against HCV infection
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Summary and Conclusion
specially for those who aren’t suitable for treatment, non-responders and relapsers,
moreover it is costly, prolonged and have side effect and contraindicated in many
patients, so alternative options are desperately needed out of which the recently
discovered RNAi represent a powerful silencing approach for molecular
therapeutics through a sequence-specific RNA degradation process to silence
virus infection or replication. HCV translation is mediated by a highly conserved
internal ribosome entry site (IRES) within the 5’UTR region making it a relevant
target for new drug development.
In the present study, the efficacy of specially synthesized siRNA molecule
that target 5/UTR of domain IIIC within IRES of HCV RNA (nt 59–79 from the
5’UTR and nt 109–129 from the core area) to eradicate HCV intra-PBMC in-vitro
was tested and compared with traditional IFN/RBV in vitro by using qRT-PCR.
The alignment of HCV sequences typed by TRGUENE (accession
numbers AY661552, AY673080–AY673111, AY624961-AY624986, AY902780-
AY902787) using CLUSTAL analysis in the Bioedit program generated
consensus sequence of the all patients (5’UTR) used for generation of HCV
specific siRNA.also siRNA interaction with HCV was confirmed on HCV
sequence database using software for alignment on this database.
In our study, 40 genotype 4 chronic HCV infected patients who are naïve
for any HCV treatment were enrolled and tested for presence of HCV inta-PBMC
using both qualitative and quantitative techniques of PCR and the result showed
that 19 patients (47.5%) showed the presence of HCV inside PBMCs while the
other had no HCV inside PBMCs. Another four normal samples were recruited in
the study to test the effect of siRNA on non-infected PBMCs cells.
PBMCs were isolated by using Ficoll-hypaque technique and were
cultured with 100 μM Z5 siRNA or a combination of 110 IU/ml PEG-IFN and
210 μM ribavirin with positive GAPDH and negative non-targeting pool controls
for 72 hours. After that HCV load was determined by q-PCR and cell viability
was measured by trypan blue assay. The result show that siRNA was more potent
than tradition combination therapy as siRNA decreased viral load by more than
98% with no effect on gene expression nor cell vitality that reach 95% using
trypan blue exclusion method. While traditional IFN/RBV decreased the viral load by 67% it had obvious cytostatic effect on cells. Using positive GAPDH siRNA together with Z5 siRNA to test effect of siRNA on housekeeping genes and specificity to target desired sequence, iRNA show no off-target interaction with housekeeping genes and high specificity to target its complementary sequence without side-interaction nor cell death.
Western blot analysis was carried out to assess effect of siRNA on viral
protein synthesis and the result showed that not only siRNA suppressed viral
summary and Conclusionprotein ynthesis, but also blocked the replication of sub-genomic viral RNA this expression was reported at Day 1 post Z5 siRNA and assured at day 3 after
transfection.Finally, we can conclude that the advantage of using siRNA as a therapy against HCV infection – as it show good efficacy in silencing HCV.