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Cell-Penetrating Peptides as Vectors for Delivery of Nucleic Acids 355
hydrophilic part of Simian virus SV40 large tumor antigen) have been shown to act
as carriers of nucleic acids at physiological and decreased temperatures
(Table 16.1). 55 The ability of this peptide to act as carrier for nucleic acids was
further investigated using antisense technology. Antisense phosphorothioate ODN
covalently attached to the formerly described peptide was able to inhibit L-type
Ca 2+ channel β subunit synthesis in cardiac cell line H9C2.
Another report showed the use of MTS (MPM) derived from the hydrophobic
region of signal peptide sequence of the Kaposi fibroblast growth factor, or of the
above-mentioned MTS in complex with NLS of transcription nuclear factor NFκB. 56
The phosphorothioate ODNs were cross-linked to the carrier peptides by disulphide
bond. These constructs translocated membranes and localized in the nucleus. Unfortunately,
the constructs did not show any antisense effect in vitro. Experiments with
a cell-free luciferase reporter assay were successful as all antisense sequences
decreased luciferase expression by 60 to 80% of basal level at 2 µM. The inhibition
of luciferase expression by control sequences was observed by 10 to 20%. We explain
it by the fact that the authors of this study used high concentrations of construct.
Morris et al. 57 developed a 27-amino acid-long peptide called MPG that contains
a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic
domain derived from the nuclear localization sequence of SV40 T-antigen
(Table 16.1). Binding of both single- and double-stranded phosphorothioate ODNs
to MPG occurs through electrostatic interactions, which involve basic residues of
the peptide vector. MPG peptide translocates fluorescein-labeled phosphorothioate
ODN conjugates into cultured mammalian cells in less than 1 h with high efficiency
(90%, as measured by confocal microscopy). The speed of the uptake indicates that
the mechanism does not follow the endosomal pathway. Experiments with MPGmediated
ON uptake on HS68 fibroblasts at low temperature to block the endosomal
pathway confirm the assumption. At 4°C the oligonucleotides were also rapidly
internalized into cells in the previously described time frame and localized to the
nucleus.
Vidal et al. 58 used MPG peptide for transmembrane delivery of fluorescentlabeled
single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase.
In the presence of the peptide vector, the mRNA was delivered into the
cytoplasm of HS68 human fibroblasts. The same authors used a similar strategy for
gene delivery into cultured cells. 59 MPG promoted delivery and expression of the
pRL-SV40 plasmid encoding the reporter R. reniformis luciferase in HS-68 and NIH
3T3 fibroblasts, C2C12 myoblasts and COS-7 cells, as well as human CEM-SS
lymphoblasts (Table 16.1). The degree of transfection using MPG was at least twoand
sevenfold higher than that obtained with Lipofectamine ® for fibroblasts or COS-
7 and C2C12 myoblasts, respectively. They also demonstrated that MPG-mediated
delivery of a plasmid carrying full-length antisense cdc25C cDNA into mammalian
fibroblasts promotes cell cycle arrest at the G2/M transition.
Zanta et al. 60 demonstrated that NLS peptide (PKKKRKVEDPYC) alone was
sufficient to deliver 3.3 kbp CMV luciferase gene. The efficiency of transfection
was enhanced 10- to 1000-fold as a result of addition of NLS. Whether the enhancement
of transfection is caused by improved membrane penetration or just by