A novel potent strategy for gene delivery using a single peptide vector as a carrier

Centre de Recherches de Biochimie Macromoleculaire, UPR-1086 CNRS, 1919 Route de Mende, 34293 Montpellier Cedex 5, France.
Nucleic Acids Research (Impact Factor: 9.11). 10/1999; 27(17):3510-7. DOI: 10.1093/nar/27.17.3510
Source: PubMed


We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides into cultured cells. Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells. In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects them from degradation in cell culture media. MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids into several different cell lines in only 1 h. Moreover, MPG enables complete expression of the gene products encoded by the plasmids it delivers into cultured cells. Finally, we have investigated the potential of MPG as an efficient delivery agent for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene. MPG efficiently delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C expression levels and promoted a block to cell cycle progression. Based on our results, we conclude that MPG is a potent delivery agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its contribution to the development of new gene therapy strategies could be of prime interest.

Download full-text


Available from: May C Morris,
  • Source
    • "This suggests that pepM translocates bilayers in a receptor- or transporter-independent way, in tune with the ability of DENV C protein to deliver ssDNA at 4°C (Figures 3B,C). The divergent behavior of pepR and pepM suggests that the cationic domain favors non-covalent nucleic acid binding [26], [27], while the hydrophobic one may favors membrane interaction, perturbation and consequent translocation. It should be noted, however, that these domains cannot be simplistically assigned to the nucleic acid-binding and membrane-binding functionalities of DENV C protein, respectively, since both peptides have the ability to complex ssDNA and to interact with lipids [36], [52]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.
    PLoS ONE 12/2013; 8(12):e81450. DOI:10.1371/journal.pone.0081450 · 3.23 Impact Factor
  • Source
    • "In the case of MPG complexation , no significant difference in silencing effect was seen at all molar ratios (Supplementary Fig. 5). It is possible that unstable complex formation (lanes 3 and 4, Fig. 1a) and degradation by nucleases [52] may have occurred. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The foreign body reaction often interferes with the long-term functionality and performance of implanted biomedical devices through fibrous capsule formation. While many implant modification techniques have been adopted in attempts to control fibrous encapsulation, the outcomes remained sub-optimal. Nanofiber scaffold-mediated RNA interference may serve as an alternative approach through the localized and sustained delivery of siRNA at implant sites. In this study, we investigated the efficacy of siRNA-poly(caprolactone-co-ethylethylene phosphate) nanofibers in controlling fibrous capsule formation through the down-regulation of collagen type I (COL1A1) in vitro and in vivo. By encapsulating complexes of COL1A1 siRNA with a transfection reagent (Transit TKO) or the cell penetrating peptides CADY or MPG within the nanofibers (550-650nm in diameter), a sustained release of siRNA was obtained for at least 28days (loading efficiency ∼60-67%). Scaffold-mediated transfection significantly enhanced cellular uptake of oligonucleotides and prolonged in vitro gene silencing duration by at least 2-3 times as compared to conventional bolus delivery of siRNA (14days vs. 5-7days by bolus delivery). In vivo subcutaneous implantation of siRNA scaffolds revealed a significant decrease in fibrous capsule thickness at weeks 2 and 4 as compared to plain nanofibers (p<0.05). Taken together, the results demonstrated the efficacy of scaffold-mediated siRNA gene-silencing in providing effective long-term control of fibrous capsule formation.
    Acta biomaterialia 10/2012; 9(1). DOI:10.1016/j.actbio.2012.09.029 · 6.03 Impact Factor
  • Source
    • "A series of small protein domains (PTD) have been shown to cross biological membranes efficiently and independently of transporters or specific receptors and to promote the delivery of peptides and proteins into cells (Blanchard et al. 1999). TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics (Morris et al. 1999). Similarly, some specific protein domains and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells (Gupta and Torchilin 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cited By (since 1996):37, Export Date: 23 March 2014, Source: Scopus
Show more