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Utility of cell-permeable peptides for enhancement of virus-mediated gene transfer to human tumor cells

2006; Future Science Ltd; Volume: 40; Issue: 5 Linguagem: Inglês

10.2144/000112152

1940-9818

Saara Lehmusvaara, Outi Rautsi, Tanja Hakkarainen, Jarmo Wahlfors,

Bacteriophages and microbial interactions

BioTechniquesVol. 40, No. 5 BenchmarksOpen AccessUtility of cell-permeable peptides for enhancement of virus-mediated gene transfer to human tumor cellsSaara Lehmusvaara, Outi Rautsi, Tanja Hakkarainen & Jarmo WahlforsSaara LehmusvaaraA.I. Virtanen Institute, University of Kuopio, Kuopio, Finland, Outi RautsiA.I. Virtanen Institute, University of Kuopio, Kuopio, Finland, Tanja HakkarainenA.I. Virtanen Institute, University of Kuopio, Kuopio, Finland & Jarmo Wahlfors*Address correspondence to Jarmo Wahlfors, Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Neulaniementie 2, P.O. Box 1627, FI-70211 Kuopio, Finland. e-mail: E-mail Address: jarmo.wahlfors@uku.fiA.I. Virtanen Institute, University of Kuopio, Kuopio, FinlandPublished Online:21 May 2018https://doi.org/10.2144/000112152AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Cell-permeable peptides can penetrate the cell membrane and become internalized either alone or coupled to other molecules. Their value has been recognized especially in vaccination and gene therapy studies (for a review, see Reference 1). Gratton et al. reported recently about the use of these peptides in enhancement of virus-mediated gene delivery in vitro and in vivo (2). They showed that polybasic peptides derived from Drosophila Antennapedia homeodomain (Antp) type 1 (HIV-1) transactivator protein (TAT) improved adenoviral and retroviral transduction in cultured monkey COS-7 cells, bovine aortic endothelial cells, and human umbilical vascular endothelial cells, as well as in vivo when precomplexed with viral vector particles. Based on their results, Gratton et al. (2) suggested that highly positively charged peptides can enhance the transduction by concentrating viral particles to the cell surface and by improving receptor-dependent uptake mechanisms.Insufficient transduction efficiency is still considered the major problem in gene therapy research. Because high gene transfer rate is particularly important in most cancer gene therapy approaches and Gratton et al. (2) did not test their concept in human tumor cells, we conducted a series of experiments to verify the utility of cell-permeable peptide-complexed virus vectors in cancer gene therapy. In addition to the Antp peptide (RQIKIWFQNRRMKWKK), we used two versions of the HIV-1 TAT peptide: TAT1 (YGRKKRRQRRR, used in most earlier studies) (1) and TAT2 (GRKKRRQRRRPPQ, presumably used by Gratton et al.) (2). Furthermore, two polycationic compounds, polybrene (hexadimethrine bromide) and protamine sulfate, were used to identify the contribution of a plain electrostatic effect (i.e., the reduction of the electrostatic repulsion between negatively charged viral particles and cell membranes). Polybrene has been known to enhance retroviral infection since the late 1960s (3). It is nowadays a commonly used enhancer of retroviral transduction, and its mechanism of action (4,5) and positive effect to adenoviral gene transfer (6) have been elucidated. Protamine sulfate has been designated as a more clinically relevant alternative for polybrene in retroviral gene therapy (7), and its utility in adenoviral gene transfer has been acknowledged (8).The cell-penetrating peptides were incubated with a serotype 5, E1/E3-deleted adenovirus vector AdTK-GFP (9) and a second generation VSV-G pseudotyped lentivirus vector WOX-TK-GFP (10) as described in the original report (2). Polybrene (8 µg/ mL) and protamine sulfate (5 µg/mL) were added to virus dilutions, and the resulting complexes were then used for transduction of one monkey kidney fibroblast cell line (COS-7) and four different human cancer cell lines representing ovarian carcinoma (SKOV3. ipl, HEY), prostate carcinoma (PC-3), and osteosarcoma (MG-63). The human tumor cell lines were selected due to their known features as targets for viral gene transfer. All of these cell lines were moderate or poor targets for lentiviral and/or adenoviral vectors (9,11), and transduction of these cells would apparently benefit from peptide-mediated enhancement. The results, indicated as proportion of green fluorescent protein (GFP) positive cells, were determined by flow cytometry, and a one-way analysis of variance with Dunnett's post hoc test for multiple comparisons was used for statistical analysis.To verify the results obtained by Gratton and coworkers (2), we examined the peptide- and polycation-mediated enhancement of viral gene transfer efficiency in COS-7 cells (Figure 1). Analysis of TK-GFP positive cells by flow cytometry 2 days (adeno-virus) or 4 days (lentiviras) posttransduction confirmed that the antp peptide can significantly improve adenoviral and lentiviral gene transfer (p 95%; Tallinn, Estonia], The peptides (0.5 mM) were completed with lentivirus and adenovirus vectors as described by Gratton et al. (2). The polycations used were polybrene (8 µg/mL; Sigma-Aldrich Chemie GmbH, Munich, Germany) and protamine sulfate (5 µg/mL; Sigma-Aldrich Chemie). Multiplicity of infection 1 of both vector types was used in transductions, and the results [percent of green fluorescent protein (GFP) positive cells] were obtained with flow cytometry (FACSCalibur™; Becton Dickinson, San Jose, CA, USA) 2–4 days later, The bars are means of 3 different experiments±sem. One-way analysis of variance with Dunnett's post hoc test for multiple comparisons (GraphPad Prism 3.0; GraphPad Software, San Diego, CA, USA) was used for statistical analysis. ***, PTo test the utility of peptide-mediated enhancement in human tumor cell lines, SKOV3.ip1, HEY, PC-3, and MG-63 cells were transduced identically as COS-7 cells. As shown in Figure 2, all the peptides and polycations were able to boost viral gene delivery into human tumor cells similarly as shown with COS-7 monkey fibroblasts. The results regarding the tumor cell lines can be summarized as follows; (i) peptides enhanced significantly both lentiviral and adenoviral gene transfer (P TAT2; (ii) peptides could not significantly improve the poor lentiviral transduction of MG-63 cells; (iii) polycations were efficient transduction enhancers with both vector types (P < 0.001), except in two cases (lentivirus complexed with protamine sulfate, in PC-3 and MG-63 cells); and (iv) polybrene turned out to be a better enhancer than any of the peptides, and in most cases the protamine sulfate effect was also comparable to that of the best peptides.Figure 2. Transduction rates of four human cancer cell lines using peptide- or polycation-assisted lentiviral (A) or adenoviral (B) gene transfer.Identical reagents and experimental conditions as indicated in the legend to the Figure 1 were used. *** P <0.001, and *, P <0.05 as compared with the control group (transduction with no additive compounds). GFP, green fluorescent protein; TAT1 and TAT2, two human immunodeficiency virus type 1 (HIV-1) TAT-derived peptides; Antp, Drosophila Antennapedia homeodomain-derived peptide.Taken together, our results demonstrate that the transduction rate of adenovirus and lentivirus vectors can be significantly boosted with TAT and Antp cell permeable peptides, but similar or better results can be obtained with commonly used polycations. The effect of each peptide or polycation turned out to be surprisingly similar in all the studied cell lines, suggesting that the effect was due to simple electrostatic interactions and was not dependent on, for example, the protein composition of the target cell plasma membrane. However, the enhancing effect appeared to be dependent on the peptide sequence; TAT1 and TAT2 peptides displayed different degrees of enhancement, especially with adenovirus vector, although their net charges were identical. In our studies, we used only one peptide concentration (0.5 mM) that was also chosen by the authors of the original paper (2). It can be speculated that this peptide concentration was already toxic, and lower concentrations could have yielded better results. This was not the case however, since we observed practically no enhancement of gene transfer with 0.1 mM peptide concentration (results not shown). Furthermore, 0.5 mM peptides did not induce any notable cytotoxicity in any of the cell lines (as judged by microscopical examination 48 h posttransduction, results not shown). Higher than 0.5 mM concentrations could theoretically improve the effect, but these concentrations would become very expensive, especially when compared with the polycations. The cost per transduction with the peptides is approximately 500 times higher than with polybrene and protamine (as determined on the basis of prices in Finland in 2005). Even though polybrene yielded better enhancement than protamine sulfate, one has to bear in mind that polybrene may have toxic side effects, and it is not a clinically approved molecule. Thus, considering the in vitro results presented in this report, the clinical utility of the tested molecules and their costs, it is apparent that protamine sulfate has the best risk/benefit ratio compared with any of the peptides or polybrene.AcknowledgmentsThis work was supported by The Academy of Finland grant no. 48590 to J. W. Inbio Ltd. (Tallinn, Estonia) is gratefully acknowledged for their kind cooperation regarding the accelerated peptide delivery.Competing Interests StatementThe authors declare no competing interests.References1. Leifert, J.A. and J.L. Whitton. 2003. 'Translocatory proteins' and 'protein transduction domains': a critical analysis of their biological effects and the underlying mechanisms. Mol. Ther. 8:13–20.Crossref, Medline, CAS, Google Scholar2. Gratton, J.P., J. Yu, J.W. Griffith, R.W. Babbitt, R.S. Scotland, R. Hickey, F.J. Giordano, and W.C. Sessa. 2003. Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo. Nat. Med. 9:357–362.Crossref, Medline, CAS, Google Scholar3. Toyoshima, K. and P.K. Vogt. 1969. Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 38:414–426.Crossref, Medline, CAS, Google Scholar4. Landazuri, N. and J.M. Le Doux. 2004. Complexation of retroviruses with charged polymers enhances gene transfer by increasing the rate that viruses are delivered to cells. J. Gene Med. 6:1304–1319.Crossref, Medline, CAS, Google Scholar5. Davis, H.E., J.R. Morgan, and M.L. Yarmush. 2002. Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys. Chem. 97:159–172.Crossref, Medline, CAS, Google Scholar6. Arcasoy, S.M., J.D. Latoche, M. Gondor, B.R. Pitt, and J.M. Pilewski. 1997. Polycations increase the efficiency of adenovirus-mediated gene- transfer: to epithelial and endothelial-cells in-vitro. Gene Ther. 4:32–38.Crossref, Medline, CAS, Google Scholar7. Cornetta, K. and W.F. Anderson. 1989. Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy. J. Virol. Methods 23:87–194.Crossref, Google Scholar8. Lanuti, M., C. El Kouri, S.D. Force, M.Y. Chang, K. Amin, K. Xu, LA. Blair, L.R. Kaiser, and S.M. Albelda. 1999. Use of protamine to augment adenovirus-mediated cancer gene therapy. Gene Ther. 6:1600–1610.Crossref, Medline, CAS, Google Scholar9. Pellinen, R., T. Hakkarainen, T. Wahlfors, K. Tuilimaki, A. Ketola, A. Tenhunen, T. Salonen, and J. Wahlfors. 2004. Cancer cells as targets for lentivirus-mediated gene transfer and gene therapy. Int. J. Oncol. 25:1753–1762.Medline, CAS, Google Scholar10. Merilainen, O., T. Hakkarainen, T. Wahlfors, R. 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W. Inbio Ltd. (Tallinn, Estonia) is gratefully acknowledged for their kind cooperation regarding the accelerated peptide delivery.Competing Interests StatementThe authors declare no competing interests.PDF download