An Enterovirus-Like RNA Construct for Colon Cancer Suicide Gene Therapy.

BACKGROUND: In gene therapy, the use of RNA molecules as therapeutic agents has shown advantages over plasmid DNA, including higher levels of safety. However, transient nature of RNA has been a major obstacle in application of RNA in gene therapy. METHODS: Here, we used the internal ribosomal entry site of encephalomyocarditis virus and the 3' non-translated region of Poliovirus to design an enterovirus-like RNA for the expression of a reporter gene (enhanced green fluorescent protein) and a suicide gene (thymidine kinase of herpes simplex virus). The expression of these genes was evaluated by flow cytometry and cytotoxicity assay in human colorectal adenocarcinoma cell line (SW480). We then armed RNA molecules with a target sequence for hsa-miR-143 to regulate their expression by microRNA (miRNA) mimics. RESULTS: The results showed effective expression of both genes by Entrovirus-like RNA constructs. The data also showed that the restoration of hsa-miR-143 expression in SW480 leads to a significant translation repression of the introduced reporter and suicide genes. CONCLUSION: Collectively, our data suggest the potential use of Entrovirus-like RNA molecules in suicide gene therapy. Additionally, as a consequence of the possible downregulated miRNA expression in cancerous tissues, a decreased expression of gene therapy constructs armed with target sequences for such miRNA in cancer tissue is expected.

Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery.

BACKGROUND: The rapid increase of HIV-1 strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker. METHODS: A PCR-amplified gfp gene (gfp) was cloned into pmzNL4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL4-3/GFP plasmid. GFP-mzNL4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK293T cells co-transfected with pmzNL4-3/GFP and the helper plasmids pSPAX2 and pMD2G, which respectively encode HIV-1 Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p24 antigen assays, and electron and fluorescence microscopy. RESULTS: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions. CONCLUSIONS: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay.

Application of the 3'-noncoding region of poliovirus RNA for cell-based regulation of mRNA stability: implication for biotechnological applications.

Enrichment of production yield of therapeutic proteins in mammalian cell cultures by modulation of the mRNA stability of the target protein to increase its in vivo half-life is a new strategy in biotechnological applications. The present article describes one of the most novel approaches to modulate mRNA stability by application of 3'-noncoding region (3'NCR) from RNA viral genome in the expression constructs. Our data indicated that although utilizing the 3'NCR sequence form poliovirus (PV-3'NCR) downstream of the target gene might generally stabilize the secondary structure of RNA, it influenced the mRNA stability (and thereby the amount of protein production) in a cell type and time-dependent manner, thus indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest to the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

Prevalence of occult hepatitis B virus in plasma and peripheral blood mononuclear cell compartments of patients with chronic hepatitis C infection in tehran-iran.

BACKGROUND: Occult hepatitis B virus (HBV) infection (OBI) is frequently reported in patients with chronic hepatitis C virus (HCV) infection. An association between OBI and more liver damage, cirrhosis, hepatocellular carcinoma, and reduced response to interferon therapy in patients with HCV infection is suggested. OBJECTIVES: The aim of this study was to determine the prevalence of occult HBV, and evaluate its clinical influence on patients with chronic HCV. PATIENTS AND METHODS: A cohort study including50 patients with positive results for HCV, and negative results for HBsAg tests was performed. The patients were divided into two groups: one group had positive results for both HCV and occult HBV tests (n = 18), and the other had positive results for HCV, but negative findings for occult HBV (n = 32). All were treated with PEG-IFN alpha-2a and Ribavirin. Presence of HCV RNA was followed in these patients. RESULTS: HBV-DNA was detected using nested-PCR in 20% of plasma and 32.6% of peripheral blood mononuclear cell (PBMC) compartments. No significant differences were observed between patients with and without occult HBV for sex, age, duration of HCV infection, histological markers, presence of anti-HBc, HCV viral load, and HCV genotype. The response rate was significantly higher in patients with positive results for HBV-DNA test compared to those with negative findings (100% vs. 71.9 %, P < 0.05). CONCLUSIONS: In conclusion, occult HBV was found in 36% of patients with negative results for HBsAg, but positive results for HCV. Detection of HBV-DNA in both PBMCs and plasma together in comparison with plasma alone provided more true identification of OBI.The SVR rate was significantly higher in coinfected patients than mono-infected ones.

Lamivudine-PEGylated chitosan: a novel effective nanosized antiretroviral agent.

Due to the fact that a definite treatment for AIDS disease has not been discovered so far, antiretroviral drugs are relatively significant in controlling the disease. In this study, Lamivudine- which is an old and effective anti-AIDS drug- was connected to PEGylated chitosan nanoparticle in order to produce a new and greater version of Lamivudine. First, physicochemical studies such as HNMR, FTIR spectroscopy and CHN analysis were performed to ensure the proper synthesis. Following that, Lamivudine-PEGylated Chitosan (LPC) drug was evaluated in terms of its inhibitory capability in producing HIV virions and its cytotoxicity in different doses. HIV virions were created by transfection of psPAX2, PmzNL4-3 and pMD2.G plasmids into HEK293T cell line. Also, assessment of the P24 amounts of cell supernatant was performed using ELISA method. Among the different doses of LPC drug (0.1, 1, 10 and 100µM), it was found that 0.1µM was the most effective and least toxic dose compared to Lamivudine in the same dose. Results of this study indicate that LPC drug has the ability to inhibit HIV virus replication in a more significant way in comparison to the old drug. Besides, the drug has a low cytotoxicity and is effective with a lower dose.

Clinical, virologic and phylogenetic features of hepatitis B infection in Iranian patients.

AIM: To characterize the clinical, serologic and virologic features of hepatitis B virus (HBV) infection in Iranian patients with different stages of liver disease. METHODS: Sixty two patients comprising of 12 inactive carriers, 30 chronic hepatitis patients, 13 patients with liver cirrhosis and 7 patients with hepatocellular carcinoma (HCC) were enrolled in the study. The HBV S, C and basal core promoter (BCP) regions were amplified and sequenced, and the clinical, serologic, phylogenetic and virologic characteristics were investigated. RESULTS: The study group consisted of 16 HBeAg-positive and 46 HBeAg-negative patients. Anti-HBe-positive patients were older and had higher levels of ALT, ASL and bilirubin compared to HBeAg-positive patients. Phylogenetic analysis revealed that all patients were infected with genotype D (mostly ayw2). The G1896A precore (PC) mutant was detected in 58.1% patients. HBeAg-negative patients showed a higher rate of PC mutant compared to HBeAg-positive patients (c2 = 9.682, P = 0.003). The majority of patients with HCC were HBeAg-negative and were infected with PC mutant variants. There was no significant difference in the occurrence of BCP mutation between the two groups, while the rate of BCP plus PC mutants was higher in HBeAg-negative patients (c2 = 4.308, P = 0.04). In the HBV S region, the genetic variability was low, and the marked substitution was P120T/S, with a rate of 9.7% (n = 6). CONCLUSION: In conclusion, HBV/D is the predominant genotype in Iran, and the nucleotide variability in the BCP and PC regions may play a role in HBV disease outcome in HBeAg-negative patients.

The phylogenetic analysis of hepatitis C virus isolates obtained from two Iranian carriers revealed evidence for a new subtype of HCV genotype 3.

Classification of hepatitis C virus is based on phylogenetic analysis of the strains reported world wide. Different strains are classified within 6 major genotypes and several minor groups (subtypes). In addition to epidemiologic value of determining genotype/subtype of this virus, the result may change the therapeutic strategy used for a patient. During a survey on hepatitis C in Iran, we found two cases assigned as 1b genotype by PCR-RFLP on 5' UTR, but three based on core region sequencing. Fragments from 5' UTR, Core and NS5b regions were PCR-amplified and sequenced followed by phylogenetic analysis. Although the 5' UTR of this new strain is very similar to genotypes 1 and 6, analysis of core region classifies it in a separate branch of genotype 3, close to subtypes h and k. Further analysis of NS5b region put this new strain in a separate branch near other subtypes of genotype 3 and 4. These data are suggestive of a new subtype within genotype 3.