Intracellular delivery of messenger RNA by recombinant PP7 virus-like particles carrying low molecular weight protamine

Abstract

BackgroundCell-penetrating peptides (CPPs) have been widely used as carriers to transport different molecules into living cells, whereas messenger RNAs (mRNAs) have been utilized as target molecules for the prevention and treatment of various diseases. However, the instability of CPPs and mRNAs has limited their application. Bacteriophage PP7 virus-like particles (VLPs) may protect peptides and RNAs from degradation through displaying foreign peptides on their surface and encapsidating RNA linked with the pac site.ResultsIn this study, the cDNA of the PP7 coat protein single-chain dimer carrying low molecular weight protamine (LMWP) and the cDNA of green fluorescent protein (GFP) were inserted into two multiple cloning sites of pETDuet-1, respectively. PP7 VLPs carrying the LMWP peptide and GFP mRNA were subsequently expressed in Escherichia coli BL21 (DE3) with high yield and thermal stability, and were easily purified. The VLPs were also non-replicative, non-infectious, and non-toxic. Moreover, they penetrated the mouse prostate cancer cells RM-1 after 24 h incubation. Last, PP7 VLPs carrying the LMWP could encapsidate the GFP mRNA, which was translated into mature protein in mammalian cells.ConclusionsRecombinant PP7 VLPs can be used simultaneously as a targeted delivery vector for both peptides and mRNA due to their abilities to package RNA and display peptides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0274-9) contains supplementary material, which is available to authorized users.

The Utilization of Cell-Penetrating Peptides in the Intracellular Delivery of Viral Nanoparticles

“…The genetic modification of viral proteins with the Tat peptide has been described for VPs derived from Ad [55,56,57,58], baculovirus [59] and the PP7 [60,61], MS2 [62], and lambda [63] bacteriophages (Table 3).…”

“…This phenomenon has been demonstrated recently for VPs derived from turnip yellow mosaic virus (a plant-attacking virus) [74] covalently conjugated with Tat: while no native VPs entered the mammalian cells, Tat-assisted transfection was found to be more efficient than lipofection. The modification of VPs derived from the PP7 bacteriophage with CPPs has also been experimentally addressed; unlike unmodified VPs, VPs derived from PP7 with protamine CPP incorporated in a coat protein have been found to be able to enter mouse prostate cancer cells (RM-1), deliver mRNA for a GFP protein, and ensure its expression [61]. Another study has concluded that PP7 VPs modified with Tat peptide successfully packed microRNA precursors (pre-miRNA) which were then protected from nuclease digestion.…”

“…Since some cancer cells have been shown to exhibit limited permissivity due to the low expression of CAR [78], Ad provides an example of a viral vector that may benefit from such modifications with CPPs. A number of studies [44,45,47,50,52,55,56,57,58,59,60,61,62,63,65,66,68,69,70,71,73] have demonstrated that CPPs are able to greatly enhance the transduction of tumor cells derived from a broad range of tissues.…”

Nanoscale platforms for messenger RNA delivery

“…Coat proteins derived from viruses were also used for mRNA delivery (Jekhmane et al, 2017;Li et al, 2014;Sun, Sun, Zhao, & Gao, 2016). Li et al (2014) assembled the recombinant coat protein of bacteriophage MS2 with mRNA encoding granulocyte macrophage colony-stimulating factor.…”

“…The formulated virus‐like particles induced strong antigen‐specific cytotoxic T‐lymphocyte and balanced Th1/Th2 responses, protecting mice against prostate cancer (Li et al, ). Sun et al () developed bacteriophage PP7 coat protein containing low molecular weight protamine, which complexed with GFP mRNA and expressed GFP protein in a mouse cell line. Jekhmane et al designed C‐S10‐K12, an artificial viral coat protein, consisting of three parts: a hydrophilic random coil polypeptide (a C domain contains 400 amino acids), a oligopeptide (GAGAGAGQ)10 (S10), and an oligolysine (K12) (Jekhmane et al, ).…”