Rna Vaccine Based On Recombinant MS2Virus-like Particles Against Prostate Cancer

Recently, RNA vaccines show important value in the prevention and therapy of cancer, not only because it induces strong humoral and cellular immune responses, but also because, compared to DNA vaccine, protein vaccine and peptide vaccine, it has several advantages as follows:compared with DNA vaccine, RNA vaccine is safer, and does not have the possibility of integrating into host genome; compared with protein vaccine, RNA vaccine induces stronger cellular immune response; compared with peptide vaccine, RNA vaccine has stronger immunogenicity.However, the instability of naked RNA limits its potential application in the prevention and therapy of disease. Even though there are several approaches to improve intracellular stability and translational efficacy of RNA, such as the modification of RNA skeleton, liposome-or gold particles-packaged RNA, these approaches do not fundamentally solve the problem. Furthermore, lots of RNA used in these approaches is prepared by transcription or synthesis, which is performed under stringent conditions with high cost. Adenovirus and replicative RNA are also used as the delivery vector for RNA, but the diversity of their structural proteins and the potential to recombinant into new virus obstruct their direct use in practice. The most effective delivery approach in clinical research and application is RNA transfected dendritic cells, but their preparation is with high cost and cockamamie manipulation, and the source of autologous dendritic cells is limited. Besides, the inefficiency of microRNA in cell membrane binding and cell entry also hinders its research and application. In view of the disadvantages of current RNA delivery approaches, a safe and effective delivery approach is required, which is prepared easily in mass production, with good cell-penetrability, RNA-stability, and low cost.Considering that the poor stability of RNA and the internalization inefficiency of microRNA, our present study took MS2virus-like particles (VLPs) as the delivery vector of RNA. First, we prepared MS2VLPs containing prostatic acid phosphatase (PAP) mRNA or microRNA by DNA recombinant technology, and investigated their cytotoxicity, the abilities of assembly, packaging RNA, cell penetration and translation. Furthermore, we explored the preventive and therapeutic of these MS2VLPs in vivo and in vitro, and revealed the potential immune mechanism of their anti-tumor activity. First, based on the ability of MS2capsid packaging mRNA, we prepared a stable and effective mRNA vaccine for the prevention and therapy of prostate cancer. In the pESC-URA vector, the gene of MS2coat protein was inserted into the downstream of GAL10promoter, and the fusion genes of human or mouse PAP and GM-CSF were inserted into the downstream of GAL1promoter, then obtaining two vectors pMS2-hPAP-GM-CSF and pMS2-mPAP-GM-CSF, which were transformed into saccharomyces cerevisiae. hPAP-GM-CSF VLPs and mPAP-GM-CSF VLPs were separated from the ultrasonic supernatant of saccharomyces cerevisiae, and were purified by gel filtration affinity. By the same means, MS2VLPs, GM-CSF VLPs, hPAP VLPs and mPAP VLPs were used as the controls.The VLPs mentioned above were identified by SDS-PAGE, transmission electron microscopy (TEM), nuclease-resistant assay and RT-PCR. The results showed that, six VLPs were all expressed in saccharomyces cerevisiae with the diameter of30nm, the molecular weight of14kDa and high yield (2-3mg/L), and the yield was negatively correlated with the length of inserted target gene; VLPs could package target mRNAs with different lengths, from429nt to1652nt, and could protect these mRNA from the degradation of RNase. Additionally, the cytotoxicity assay using cell counting kit-8(CCK-8) showed hPAP-GM-CSF VLPs (0～2000μg/mL) have no cytotoxicity to CHO cells. Moreover, in vitro study revealed MS2VLPs could be phagocytosed by murine macrophagocytes, and the mRNA packaged by MS2capsid could be translated into target protein with activity in mammalian cells, whose translational efficiency was closely related with the incubation time and the proportion of VLPs and cells.Also, we evaluated the immune responses and anti-tumor effects induced by PAP-GM-CSF VLPs. In this study, hPAP-GM-CSF VLPs were injected into C57BL/6mice, which were chosen as the animal model for prostate cancer, then serum IL-12and TNF-a were tested by ELISA. The ELISA results showed serum IL-12and TNF-a were in high levels at4h and24h, respectively, and their levels were correlated with the dose of hPAP-GM-CSF VLPs. Immunization with hPAP-GM-CSF VLPs activated dendritic cells, promoted them into mature DCs, which presented antigen, further activated IgG and T cell immune responses, especially Thl and CTL cell immune responses, but not CD4+regulatory T cells. The results of tumor challenge indicated that, after inoculation with6VLPs for three times, the immune responses induced by hPAP-GM-CSF VLPs protected C57BL/6mice from prostate cancer, but the mice in the other groups all died of cancer. After that,6VLPs were used to treat prostate cancer, and the results demonstrated hPAP-GM-CSF VLPs led to a growth delay, whereas treatment of tumors with the other VLPs was not effective.Next, based on the findings above and the ability of peptide display of MS2capsid, to improve the internalization efficiency of microRNA, cell penetrating peptide—TAT was inserted into MS2coat protein, obtaining a novel VLPs carrying TAT and specific RNA. The expression vectors of the VLPs were based on pESC-URA. In this vector, the gene of two tandem MS2coat protein was inserted into the downstream of GAL10promoter, and the TAT gene was inserted into the second MS2coat protein gene by point mutation, enzyme digestion and ligation. After that, pre-microRNA-23a, pre-microRNA-23b, GFP or GM-CSF genes were inserted into the downstream of GAL1promoter, obtaining four vectors p2MS2-microRNA-23a, p2MS2-microRNA-23b, p2MS2-GFP and p2MS2-GM-CSF. At the same time, we designed a sequence as the control of microRNA-23a, which were transformed into saccharomyces cerevisiae. All these VLPs were separated from the ultrasonic supernatant of saccharomyces cerevisiae, and were purified by gel filtration affinity. The molecular weight of MS2coat protein was identified by SDS-PAGE, approximately72kDa, and the yield of these VLPs decreased compared to the VLPs without TAT. The TEM result showed MS2coat protein carrying TAT could assemble into the whole VLPs with the diameter of30nm in saccharomyces cerevisiae. Additionally, nuclease-resistant assay verified the MS2capsid also protected RNA from degradation, and the identification results of RT-PCR revealed that these VLPs could package specific RNAs (mRNA and microRNA) with the largest fragment of772nt. Cell penetrating assay proved that TAT displayed on the surface of MS2VLPs retained its cell penetrating activity, and the cytotoxicity assay using CCK-8showed TAT-microRNA-23a VLPs (0～1000μg/mL) have no cytotoxicity to CHO cells. Moreover, in vitro study revealed not only mRNA but also microRNA packaged by MS2capsid could be translated into target protein or mature microRNA with higher efficiency in mammalian cells. Finally, we evaluated the anti-tumor activity of TAT-microRNA-23a VLPs. The result indicated that, compared with the positive control TAT-microRNA-23b VLPs, TAT-microRNA-23a VLPs had a stronger antitumor activity (10μg/mL:11.67±1.69vs.9.06±1.50, P<0.001;8.3μg/mL:3.53±0.44vs.1.92±0.12, P<0.01),but TAT-microRNA-con VLPs did not.In conclusion, based on the abilities of MS2VLPs packaging specific RNA and displaying peptide, we prepare two RNA (mRNA and microRNA) vaccines, which are safe, stable, effective, prepared easily in mass production, and with good cell-penetrability, RNA-stability and low cost. MS2VLP-based RNA vaccine strategies we have discussed here have the following advantages over current RNA vaccine approaches, which make them promising cancer vaccines (1) can be prepared easily by recombinant protein technology, and the quality is easy to control;(2) shows high yield in a yeast expression system;(3) has higher stability when incubated with a nuclease;(4) is suitable for phagocytosis and proper protein expression, which can overcome self-tolerance, activate DCs, and induce efficient immune responses;(5) is safe, without obvious adverse effects when administrated intravenously;(6) simultaneously induces humoral and cellular immune responses;(7) generates CD4+T helper cells, especially a Thl-biased response, which is required for the production of effector and memory CD8+T cells;(8) maintains a balance between CD4+T cells and Treg cells;(9) produces a CTL response;(10) shows strong tumor inhibition effects; and (11) MS2capsid can be modified by inserting peptide, which makes it targeted for specific cells. Hence, our study solve the problems of the instability and the internalization inefficiency of RNA successfully, which provides new thinking and innovative solutions for tumor RNA vaccine, and lays the theoretical and practical foundation for the application of these vaccine of MS2VLP-based RNA vaccines in the prevention and therapy of other diseases.