Enhanced delivery efficiency of recombinant adenovirus into tumor and mesenchymal stem cells by a novel PTD

Abstract

Protein transduction domains (PTDs) are small peptides that facilitate the transduction of large molecules such as polyproteins, DNA and viruses into a eukaryotic cell. Here, we demonstrated that a novel PTD (HP4) derived from herring protamine appeared to enter C6Bu1 rat glioma cell lines more rapidly than other known PTDs such as Tat, Antp and Hph-1. Moreover, HP4 significantly enhanced in vitro transduction of recombinant adenoviruses (rAds) into various cancer cell lines, mesenchymal stem cells (MSCs) and dendritic cells, which are relatively resistant to rAd infection. Enhancement of rAd delivery into C6Bu1 and MSCs by HP4 is 20 and 7 times higher than that by Tat, respectively. The increase in the expression of rAd encoding IL-12N220L by HP4 is proportional to its antitumor effect in the ex vivo transduced mouse colon cancer model. Thus, these results suggest that HP4 could be utilized to improve the transduction efficiency of rAd, resulting in enhanced efficacy of rAd-mediated gene therapy, especially for ex vivotransduced cell therapy.

Branched oligomerization of cell-permeable peptides markedly enhances the transduction efficiency of adenovirus into mesenchymal stem cells

“…Hence, our findings significantly improved the previous reports that monomeric CPPs such as Tat 48À60 , Antp and HP4 enhanced rAd-mediated gene delivery into nonpermissive cells. 15,16 In particular, higher transduction efficiencies were achieved using concentrations of branched tetramers of CPPs at 3000-5000-fold less than that of monomeric CPPs. It is notable that branched oligomerization could increase not only rAd transduction efficacy but also cytotoxicity of CPPs, but optimal concentrations required for maximum transduction (0.03-0.1 mM) are 300-1000-fold lower than concentrations causing 50% toxicity (10-30 mM), suggesting that tetrameric CPPs could be used for safe ex vivo transduction of rAd into MSCs.…”

“…Using four different CPPs, namely Tat, 20 Hph-1, 21 Antp 22 and HP4, 16 a series of branched CPPs were synthesized to include 2, 4, or 8 CPP moieties by conjugating the C termini of the peptides to lysine linkers. Maximum transduction efficiency and minimal cytotoxicity for these synthesized CPPs were evaluated in human bone marrow-derived MSCs (BM-MSCs) and umbilical cord blood-derived MSCs (UCB-MSCs), which do not express CAR ( Figure 1a).…”

“…15,16 CPPs are small, poly-basic peptides that have been shown to transverse the plasma membrane of several mammalian cell types, and thus have been used to deliver various bioactive substrates, including proteins, antisense oligonucleotides, liposomes, plasmid DNAs and small interfering RNAs into cells. Recently, multivalent CPPs such as tetrameric decaarginine, decalysine, Tat peptides and zinc(II)-coordinated oligotyrosine have been reported to show improved efficiency in internalization and intracellular delivery of peptide and DNA.…”

Cross-talk between BubR1 expression and the commitment to differentiate in adipose-derived mesenchymal stem cells

“…The presence of protein transduction domains (PTDs) enables transduction of rAd into adult stem cells (Youn et al, 2008). As expected, co-treatment with HP4-PTD derived from herring protamine (Youn et al, 2008) and with rAd expressing the green fluorescence protein (rAd-GFP) dramatically enhanced in vitro trans- duction of rAd-GFP into ASCs (Figure 2A). Under these experimental conditions, we transduced either rAd-shLuc or rAd-shBubR1 into ASCs, and thereby significantly depleted endogenous BubR1 with rAd-shBubR1 transduction, but not with rAd-shLuc transduction ( Figures 2B and 2C).…”

“…However, the transduction efficiency of recombinant adenoviruses (rAd) is extremely low in stem cells that express very low levels of the primary rAd receptor (Bergelson et al, 1997). The presence of protein transduction domains (PTDs) enables transduction of rAd into adult stem cells (Youn et al, 2008). As expected, co-treatment with HP4-PTD derived from herring protamine (Youn et al, 2008) and with rAd expressing the green fluorescence protein (rAd-GFP) dramatically enhanced in vitro trans- duction of rAd-GFP into ASCs (Figure 2A).…”