Lunan Wang

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.

Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1.

ABSTRACT The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5′ terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4°C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA.

RNase-resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system.

ABSTRACT RNase-resistant, noninfectious virus-like particles containing exogenous RNA sequences (armored RNA) are good candidates as RNA controls and standards in RNA virus detection. However, the length of RNA packaged in the virus-like particles with high efficiency is usually less than 500 bases. In this study, we describe a method for producing armored L-RNA. Armored L-RNA is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of a two-plasmid coexpression system in which the coat protein and maturase are expressed from one plasmid and the target RNA sequence with modified MS2 stem-loop (pac site) is transcribed from another plasmid. A 3V armored L-RNA of 2,248 bases containing six gene fragments—hepatitis C virus, severe acute respiratory syndrome coronavirus (SARS-CoV1, SARS-CoV2, and SARS-CoV3), avian influenza virus matrix gene (M300), and H5N1 avian influenza virus (HA300)—was successfully expressed by the two-plasmid coexpression system and was demonstrated to have all of the characteristics of armored RNA. We evaluated the 3V armored L-RNA as a calibrator for multiple virus assays. We used the WHO International Standard for HCV RNA (NIBSC 96/790) to calibrate the chimeric armored L-RNA, which was diluted by 10-fold serial dilutions to obtain samples containing 106 to 102 copies. In conclusion, the approach we used for armored L-RNA preparation is practical and could reduce the labor and cost of quality control in multiplex RNA virus assays. Furthermore, we can assign the chimeric armored RNA with an international unit for quantitative detection.

Immuno-PCR: An ultrasensitive immunoassay for biomolecular detection.

Techniques that combine nucleic acid amplification with an antibody-based assay can dramatically increase the sensitivity of conventional immunoassays. This review summarizes the methodology and applications of one such protein detection technique that has been used for the past 23 years-the immuno-polymerase chain reaction (usually referred to as immuno-PCR or IPCR). The key component of an immuno-PCR is a DNA-antibody conjugate that serves as a bridge to link the solid-phase immunoreaction with nucleic acid amplification. The efficiency of immuno-PCR enables a 10- to 10(9)-fold increase in detection sensitivity compared with that of ELISA. Advancements in immuno-PCR have included improvements of production of the DNA-antibody conjugate, assay formats, and readout methods. As an ultrasensitive protein assay, immuno-PCR has a broad range of applications in immunological research and clinical diagnostics.

A Novel Real-Time Multiplex Reverse Transcriptase-Polymerase Chain Reaction for the Detection of HIV-1 RNA by Using Dual-Specific Armored RNA as Internal Control

Objective: Transmission of HIV-1 and diagnosis of infection in hospitals and public health settings remains a worldwide concern. HIV-1 detection is sometimes not possible using current commercial assays, probably due to mismatches between the selected primers and probes. Methods: By screening primers and probes, we developed a dual-specificity probe real-time reverse transcriptase-polymerase chain reaction (DSPrtRT-PCR) assay using dual-specific armored RNA as the internal control. The specificity and sensitivity were compared between the monospecificity probe real-time and DSPrtRT-PCR techniques. Results: The sensitivity of DSPrtRT-PCR improved significantly, with no effect on its specificity. The detection limit was 173 IU/ml. All the HIV-1 group M and group O could be detected. In clinical assays, 1,000 copies/ml of armored RNA was required as internal control. When applied to negative samples, 100% specificity was achieved. Among 60 samples from the tested patients, DSPrtRT-PCR demonstrated high sensitivity, accurately detecting 50 positives and 10 negatives that were confirmed by the COBAS AmpliScreen assay. Conclusion: DSPrtRT-PCR is a more efficient and effective viral assay with high sensitivity and specificity as compared to monospecificity probe PCR. It can be widely applied in blood donor screening and qualitative individual detection of HIV-1 RNA.

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.

Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.

EdU (5‐ethynyl‐2′‐deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [3H]thymidine incorporation and BrdU (5‐bromo‐2′‐deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assays conditions. We found that the number of EdU+ cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10–50 μM, the optimal incubation time 8–12 h and the proper volume of Click volume 100 μl for labeling 1 × 106 lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X‐100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU+ cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, −80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.

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.