The Expression And Application Of Bacteriophage MS2 Virus-like Particles Packaging Different Types Of RNA

Avian influenza A (H7N9) is a new subtype of influenza virus that was first found in Shanghai and Anhui provinces of China in March 2013. Due to the high mortality rates in H7N9 infected individuals, accurate diagnosis of the virus is the key to saving lives of patients.As an RNA virus, clinical detection of H7N9 involves nucleic acid extraction, reverse transcription, and PCR. Improper execution of any one of these processes could lead to false positives or negatives, and mislead clinical diagnosis, resulting in even death of the patient. An external quality assessment (EQA) program to detect H7N9 virus was implemented, by the National Center for Clinical Laboratories of China, in June 2013 to evaluate the ability of participating laboratories to detect H7N9 and provide guidance to improve their detection accuracy. Bacteriophage MS2 virus-like particles (MS2 VLPs) that packaged full-length RNA sequences of Hemagglutinin, Neuraminidase, Matrix protein, and Nucleoprotein genes from H7N9 were constructed. The EQA panel, which consisted of six samples that contained different concentrations of RNAs positive for H7N9 and four H7N9-negative samples (including one sample positive for the MP gene of H7N9), was distributed to 79 clinical laboratories that perform molecular diagnosis for H7N9. The performances were classified according to the results of both H7 and N9 genes. Consequently,80 data sets were received, which were generated using commercial (n=60) or in-house (n=17) real-time RT-PCR kits, coupled with a commercial assay employing an isothermal amplification method (n=3). The results show that majority (82.5%) of the data sets correctly identified H7N9; while 17.5% of the data sets required improvement in their diagnostic capability. These "improvable" data sets were mostly derived from false-negative results for N9 at relatively low concentrations. The false-negative rate was 5.6%, whereas the false-positive rate was 0.6%. In addition, we observed variation in the capabilities of different commercially available kits and in-house developed assays. Overall, most of the participating laboratories met the criteria for accurate detection of H7N9.Not only could the exogenous mRNA be packaged into the coat-protein shell, pre-miRNAs could also be packaged, and served as miRNA carriers. In this part, we obtained novel VLPs with a cell-penetrating peptide-HIV-1 TAT displayed on the surface and pre-miR-122 in the coat-protein shell (TAT-MS2-miR122 VLPs). pre-miR-122 was delivered into Hep3B cells by TAT-MS2-miR122 VLPs and then processed into mature miR-122, resulting in the overexpression of miR-122. Target protein suppression was verified and tumor growth was inhibited. Specifically, genetic fusion of two copies of MS2 coat protein formed a single-chain dimeric version of the protein; the HIV-1 TAT peptide sequence and a linker were inserted at the N-terminus of the dimer. This sequence was inserted downstream of the GAL 10 promoter of pESC-URA, and then the pre-miR-122 gene was inserted downstream of the GAL 1 promoter. The vector was transformed into Saccharomyces cerevisiae to express the TAT-MS2-miR122 VLPs. A dose-dependent change in miR-122 level was documented in three different cell lines, and the expression level was stable for about 120 h. After the transfection of TAT-MS2-miR122 VLPs into Hep3B, an increase in miR-122 was observed, and the expression of the insulin-like growth factor 1 receptor (IGFIR), a disintegrin and metalloproteinase 10 (ADAM10) were suppressed. As IGF1R and ADAM10 play important roles in the development of hepatocellular carcinoma, the decrease in IGF1R and ADAM10 inhibited the growth of the hepatocellular carcinoma cells. Moreover, cell proliferation and apoptosis assay demonstrated that, compared to the negative control, TAT-MS2-miR122 VLPs exhibited robust antitumor activity. Taken together, our results show that the TAT-MS2-miR122 VLPs-based miRNA delivery system is effective, stable, cost-effective and has potential clinical applicability. However, the antitumor effects of TAT-MS2-miR122 VLPs in vivo require further study.