Cell-permeable peptides (CPPs) promote the transduction of nonpermissive cells by recombinant adenovirus (rAd) to improve the therapeutic efficacy of rAd. In this study, branched oligomerization of CPPs significantly enhanced the transduction of human mesenchymal stem cells (MSCs) by rAd in a CPP type-independent manner. In particular, tetrameric CPPs increased transduction efficiency at 3000-5000-fold lower concentrations than did monomeric CPPs. Although branched oligomerization of CPPs also increases cytotoxicity, optimal concentrations of tetrameric CPPs required for maximum transduction are at least 300-1000-fold lower than those causing 50% cytotoxicity. Furthermore, although only B60% of MSCs were maximally transduced at 500 mM of monomeric CPPs, 495% of MSCs were transduced with 0.1 mM of tetrameric CPPs. Tetrameric CPPs also significantly increased the formation and net surface charge of CPP/rAd complexes, as well as the binding of rAd to cell membranes at a greater degree than did monomeric CPPs, followed by rapid internalization into MSCs. In a critical-size calvarial defect model, the inclusion of tetrameric CPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs that were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tetrameric CPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tetrameric CPPs to provide an innovative tool for MSC-based gene therapy and for in vitro gene delivery to MSCs.
The viral haemorrhagic septicaemia virus (VHSV) causes high mortality in many marine and freshwater fish species, resulting in heavy economic losses in fish farming. Previously, cholera toxin B subunit (CTB)-fused recombinant viral haemorrhagic septicaemia virus glycoproteins (rec-VHSV-GPs) have been successfully expressed in tobacco, Nicotiana benthamiana. Here, we evaluated the potential of rec-VHSV-GPs as an oral vaccine against a live viral challenge. After immunisation of mice and fish (olive flounder, Paralichthys olivaceous) with those antigenic proteins in a feed additive form, the antibody titres were increased statistically, especially in the primed groups (P < 0.0001) in both the mouse and fish. After the viral challenge under low water temperature culture conditions (below 18 °C), the immunised fish were protected successfully against the challenge, showing a significantly lower mortality rate (P < 0.05). This result suggests that this plant-based immunisation system could induce an effective immune response. It could be used as a candidate to develop an oral vaccine for fish.
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