Intracellular delivery of messenger RNA by recombinant PP7 virus-like particles carrying low molecular weight protamine
Molecules such as mRNAs have been used for the prevention and treatment of various diseases [18–21], including cancer [18–20]. However, there are several obstacles in the in vivo application of mRNAs, including the low in vivo delivery efficiency of mRNA, their lack of cell-type specificity, and instability. In this study, we introduced a delivery system based on PP7 VLPs and a cell-penetrating peptide as a solution to the above limitations of mRNA-targeted delivery.
In order to improve the delivery efficiency of mRNA, the PP7 VLPs were decorated with a non-toxic cell-penetrating peptide, LMWP. This peptide, similar to the peptide TAT, enhanced the intracellular delivery of the linked particles without any dependence on receptors, temperature conditions, or energy use and without causing any alterations to the cell membrane [22–24]. The cell-penetrating assay showed that the LMWP peptide was displayed on the surface of PP7 VLPs.
Previous studies have shown that MS2 capsids specifically encapsidate specific mRNAs that are linked to the MS2 pac site [25–27], and our results showed PP7 capsids could also do this. As was expected, we found that PP7 VLPs carrying the cell-penetrating peptide LMWP packaged a specific mRNA linked with the pac site and protected the RNA from rapid extracellular degradation by RNase. Furthermore, the PP7 VLPs carrying the cell-penetrating peptide LMWP and GFP mRNA efficiently penetrated the cell membrane. To date, the greatest length of RNA packaged using one wild-type pac site in MS2 VLPs fused with a peptide was 970 bases [28]; the length of the RNA using the PP7 VLPs was 720 bases. However, it has not been determined whether more than one kind of RNA was packaged by the PP7 VLPs during their expression in E. coli. The size and characteristics of the foreign RNA that were packaged by the peptide fused to the PP7 coat protein will therefore need to be studied further.
In addition, the mRNA packaged by the PP7 VLPs showed a high level of protein expression in mammalian cells within 24 h. This result was not in accordance with that observed for the naked mRNA vaccine, which showed maximum expression within 12–18 h [29, 30]. This finding underscores the high stability of PP7 VLPs carrying the LMWP peptide.
In 2011, Caldeira and Peabody proved that PP7 VLPs with a peptide insertion were generally stable up to about 70 °C [15]. In this study, the PP7 VLPs carrying the cell-penetrating peptide LMWP and the GFP mRNA showed similar thermal stability up to about 60 °C as those carrying other peptides and, in addition, were more stable than the MS2 VLPs with peptide insertion.
In summary, our current study demonstrates that PP7 VLPs carrying a cell-penetrating peptide and mRNA can be expressed in E. coli with high efficiency, are easily purified, and are nuclease-resistant, non-replicative, nontoxic, and noninfectious.