Tingting Jia

Development of a microRNA delivery system based on bacteriophage MS2 virus‐like particles

Recently, microRNA (miRNA)‐mediated RNA interference has been developed as a useful tool in gene function analysis and gene therapy. A major obstacle in miRNA‐mediated RNAi is cellular delivery, which requires an efficient and flexible delivery system. The self‐assembly of the MS2 bacteriophage capsids has been used to develop virus‐like particles (VLPs) for RNA and drug delivery. However, MS2 VLP‐mediated miRNA delivery has not yet been reported. We therefore used an Escherichia coli expression system to produce the pre‐miR 146a contained MS2 VLPs, and then conjugated these particles with HIV‐1 Tat47–57 peptide. The conjugated MS2 VLPs effectively transferred the packaged pre‐miR146a RNA into various cells and tissues, with 0.92–14.76‐fold higher expression of miR‐146a in vitro and about two‐fold higher expression in vivo, and subsequently suppressed its targeting gene. These findings suggest that MS2 VLPs can be used as a novel vehicle in miRNA delivery systems, and may have applications in gene therapy.

MS2 VLP-based delivery of microRNA-146a inhibits autoantibody production in lupus-prone mice

Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies. Recent studies suggest that microRNAs (miRNAs) play an essential role in immunoregulation and may be involved in the pathogenesis of SLE. Therefore, it was of interest to investigate the potential therapeutic application of miRNAs in SLE, a concept that has not been thoroughly investigated thus far. Virus-like particles (VLPs) are a type of recombinant nanoparticle enveloped by certain proteins derived from the outer coat of a virus. Herein, we describe a novel miRNA-delivery approach via bacteriophage MS2 VLPs and investigate the therapeutic effects of miR-146a, a well-studied and SLE-related miRNA, in BXSB lupus-prone mice. Methods VLPs containing miR-146a, and the control VLPs, were prepared using an Escherichia coli expression system and then administered to lupus-prone mice over a 12-day period. We performed an enzyme-linked immunosorbent assay to evaluate the anti-dsDNA antibody, autoantibody to nuclear antigen (ANA), total IgG and total IgM levels in serum. The expression of miR-146a was analyzed by qRT-PCR. SLE-related cytokines as well as some toll-like receptor signaling pathway molecules were also measured. Results Treatment with MS2-miR146a VLP showed profound effects on lupus-prone BXSB mice, including an increased level of mature miR-146a, which led to a significant reduction in the expression of autoantibodies and total IgG. Remarkably, these mice also exhibited reduced levels of proinflammatorycytokines, including IFN-Interferon-α (IFN-α), Interleukin-1β (Il-1β) and Interleukin-6 (Il-6). Moreover, we showed that the toll-like receptor pathway was involved in this regulation. Conclusion Restoring the loss of miR-146a was effective in eliminating the production of autoantibodies and ameliorating SLE progression in lupus-prone mice. Thus, the induction of dysregulated miRNAs by an MS2 VLP-based delivery system may lead to novel therapies.

Messenger RNA vaccine based on recombinant MS2 virus-like particles against prostate cancer.

Prostate cancer (PCa) is the most diagnosed cancer in the western male population with high mortality. Recently, alternative approaches based on immunotherapy including mRNA vaccines for PCa have shown therapeutic promise. However, for mRNA vaccine, several disadvantages such as the instability of mRNA, the high cost of gold particles, the limited production scale for mRNA‐transfected dendritic cells in vitro, limit their development. Herein, recombinant bacteriophage MS2 virus‐like particles (VLPs), which based on the interaction of a 19‐nucleotide RNA aptamer and the coat protein of bacteriophage MS2, successfully addressed these questions, in which target mRNA was packaged by MS2 capsid. MS2 VLP‐based mRNA vaccines were easily prepared by recombinant protein technology, nontoxic and RNase‐resistant. We show the packaged mRNA was translated into protein as early as 12 hr after phagocytosed by macrophages. Moreover, MS2 VLP‐based mRNA vaccines induced strong humoral and cellular immune responses, especially antigen‐specific cytotoxic T‐lymphocyte (CTL) and balanced Th1/Th2 responses without upregulation of CD4+ regulatory T cells, and protected C57BL/6 mice against PCa completely. As a therapeutic vaccine, MS2 VLP‐based mRNA vaccines delayed tumor growth. Our results provide proof of concept on the efficacy and safety of MS2 VLP‐based mRNA vaccine, which provides a new delivery approach for mRNA vaccine and implies important clinical value for the prevention and therapy of PCa.

Armored long non-coding RNA MEG3 targeting EGFR based on recombinant MS2 bacteriophage virus-like particles against hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 (MEG3) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15, but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.

External Quality Assessment for Avian Influenza A (H7N9) Virus Detection Using Armored RNA

ABSTRACT An external quality assessment (EQA) program for the molecular detection of avian influenza A (H7N9) virus was implemented by the National Center for Clinical Laboratories (NCCL) of China in June 2013. Virus-like particles (VLPs) that contained full-length RNA sequences of the hemagglutinin (HA), neuraminidase (NA), matrix protein (MP), and nucleoprotein (NP) genes from the H7N9 virus (armored RNAs) were constructed. The EQA panel, comprising 6 samples with different concentrations of armored RNAs positive for H7N9 viruses and four H7N9-negative samples (including one sample positive for only the MP gene of the H7N9 virus), was distributed to 79 laboratories in China that carry out the molecular detection of H7N9 viruses. The overall performances of the data sets were classified according to the results for the H7 and N9 genes. Consequently, we received 80 data sets (one participating group provided two sets of results) which were generated using commercial (n = 60) or in-house (n = 17) reverse transcription-quantitative PCR (qRT-PCR) kits and a commercial assay that employed isothermal amplification method (n = 3). The results revealed that the majority (82.5%) of the data sets correctly identified the H7N9 virus, while 17.5% of the data sets needed improvements in their diagnostic capabilities. These “improvable” data sets were derived mostly from false-negative results for the N9 gene at relatively low concentrations. The false-negative rate was 5.6%, and the false-positive rate was 0.6%. In addition, we observed varied diagnostic capabilities between the different commercially available kits and the in-house-developed assays, with the assay manufactured by BioPerfectus Technologies (Jiangsu, China) performing better than the others. Overall, the majority of laboratories have reliable diagnostic capacities for the detection of H7N9 virus.

Novel miR-122 delivery system based on MS2 virus like particle surface displaying cell-penetrating peptide TAT for hepatocellular carcinoma

Current treatments for hepatocellular carcinoma (HCC) have shown inadequate. MicroRNA-122 (miR-122) mediated RNA interference brings new prospects. A safe, efficient miRNA delivery system is an indispensable assurance. Previously, we developed an MS2 bacteriophage virus-like particle (VLP)-based microRNA delivery system crosslinked with the HIV TAT peptide, which served as an effective inhibitor in the treatments of systemic lupus erythematosus and osteoporosis. However, defects, such as low crosslinking efficiency, high cost, and potential toxicity of the crosslinking agent, needed to be confronted. Therefore, TAT peptide was designed to display on the surface of MS2 VLPs, instead of being chemically crosslinked, using the platform of phage surface display. The results reflected that MS2 VLPs displaying TAT could effectively penetrate the cytomembrane and deliver miR-122. Additionally, its inhibitory effects on HCC were significant in Hep3B, HepG2, and Huh7 cells and Hep3B related animal models. Thus, we have established a novel miR-122 delivery system based on MS2 VLPs surface displaying TAT peptide, which could effectively perform the function of penetrating cytomembrane and the inhibition of HCC.

Using a novel microRNA delivery system to inhibit osteoclastogenesis.

Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.

Strategies for active TNF-α vaccination in rheumatoid arthritis treatment.

Local overexpression of tumor necrosis factors alpha (TNF-α) is critically involved in the inflammatory response and tissue destruction of rheumatoid arthritis (RA). Currently, the blockade of TNF-α by passive immunotherapy is indeed efficacious in the treatment of RA, but it still present some disadvantages. Induction of high level of anti-TNF-α neutralizing autoantibodies by TNF-α autovaccine has been developed to avoid these shortcomings. This review is to briefly introduce several vaccination approaches that have been used to induce a B cell response, including coupled TNF-α (entire/peptide) with a carrier protein, modified TNF-α with foreign Th cell epitopes, and engineered DNA vaccine. These methods showed remarkable therapeutic efficiency in experimental animals which indicated that active TNF-α immunization would be a promising and cost-effective new treatment option for RA.

External Quality Assessment for the Detection of Measles Virus by Reverse Transcription-PCR Using Armored RNA

In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7%) were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL). In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection of measles virus.

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy

Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses.