Proteins
The introduction of oligonucleotides into mammalian cells is a powerful tool for gene therapy. Compared with biological (viral vectors) and physical methods, the major advantages of chemical methods (or synthetic vectors) are their simplicity, ease of production, and relatively low toxicity. Protein/peptide-oligonucleotide conjugates offer a unique strategy of delivering genetic material into cells with high efficiencies and cell-specificity. These peptide vectors are able to deliver oligonucleotides into cells by utilizing short sequences of basic amino acid residues that readily cross the plasma membrane. These amino acid sequences, called protein transduction domains or cell-penetrating peptides, are generally divided into two classes: (1) lysine-rich peptides, such as the amphipathic MPG peptide and transportan, and (2) arginine-rich peptides, such as the homeodomain of antennapedia (Antp) and trans-activating transcriptional activators (TAT).
Figure 1. Summary of the common receptors that mediate gene delivery via targeting peptides.
- MPG Peptide
The MPG peptide is a synthetic compound containing a hydrophobic N-terminal region derived from the fusion sequence of HIV gp41 and a hydrophilic region derived from the NLS of the SV40 large T antigen. Complexation of MPG with oligonucleotides was shown to proceed via electrostatic interaction between the basic residues of the NLS region and the phosphonate backbone of the oligonucleotide. Multiple peptides interact with a single oligonucleotide strand to form particle aggregates of oligonucleotide sequences and peptides. The analyses based on circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy have shown that when MPG peptide is complexed with oligonucleotides, the hydrophobic region partially folds into a β-sheet structure. This β-sheet structure promotes cellular uptake by inserting into the plasma membrane and forming a transmembrane pore-like structure.
- Transportan Peptide
The investigation into the mechanism of transportan-protein cellular uptake has suggested alternative routes of translocation. Only a small portion of internalized transportan-protein complexes was detected in the cytoplasm close to the plasma membrane, suggesting that direct passage across the plasma membrane by inverted micelles is not the dominant form of translocation. It has also been determined that clathrin-mediated endocytosis does not contribute significantly to transportan-protein cellular uptake since the treatment of both Hela and Bowes' cell lines with transportan-protein and transferrin, a marker for clathrin-mediated endocytosis, did not result in significant co-internalization. Caveolae-mediated endocytosis was ruled out as a significant contributing factor to transportan-protein cellular uptake due to the lack of a significant accumulation of vesicles at the Golgi apparatus, a typical destination for caveolae-mediated cellular uptake.
- Tat-Based Peptide
Tat protein is an 86-102 amino acid sequence organized into three domains: (1) cationic regions involved in controlling the rate of gene expression, (2) cysteine-rich regions involved in DNA binding, and (3) basic amino acid regions involved in promoting the crossing of the cell membrane. One of the earliest strategies for gene therapy using Tat-peptides involved the inhibition of P-glycoprotein expression. This transmembrane protein functions as a pump to remove drugs that have been internalized by cells, and overexpression of this protein in certain cancer cells often leads to ineffective chemotherapy treatments. By covalently attaching the Tat-peptide with anti-MDR antisense oligonucleotide, it was shown that in vitro P-glycoprotein expression could be significantly inhibited, particularly in the presence of serum, which could potentially lead to a 2- to 3-fold increase in drug uptake.
- Antennapedia Homeodomain Peptide
The antennapedia homeodomain is a 60-amino acid polypeptide corresponding to the Drosophila melanogaster antennapedia homeobox sequence. Cellular uptake of Antp proceeds by a nonendocytic pathway: internalization occurs even at 4°C. Additionally, the translocation of the reverse helix of Antp and a helix composed of D-enantiomers indicates that the internalization of Antp is not receptor-mediated. The replacement of several amino acids with proline disrupted the R-helical structure of Antp but did not hinder cellular uptake, suggesting that the R-helical conformation is not required. Based on this data, scientists have suggested that the internalization of Antp proceeds via electrostatic interactions between the basic amino acid residues of the peptide and the cell membrane, which destabilizes the membrane and leads to the formation of inverted micelles that deliver the peptide across the membrane and into the cytoplasm.
Reference
- Mintzer, M. A.; et al. (2008). Nonviral vectors for gene delivery. Chemical reviews. 109(2): 259-302.
