Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 169

large T antigen (Pro-Lys-Ser-Lys-Arg-Lys-Val). MPG interacts with DNA via elec-
trostatic interaction between the basic residues of the NLS region and the phos-
phonate backbone of the ODN. Deshayes et al., using circular dichroism (CD) and
Fourier transform infrared (FTIR) spectroscopy, demonstrated that during complex-
ation with ODNs, the hydrophobic region partially folds into a -sheet structure
that facilitates cellular uptake by inserting into the plasma membrane and forming a
transmembrane porelike structure [262] .
MPG vector complexed with pDNA at high charge ratios demonstrates superior
uptake as compared to Lipofectamine TM . In addition, it does not exhibit cytotoxic-
ity and remained stable in the presence of serum. A single-point mutation of the NLS
region of MPG (Lys to Ser) significantly inhibits gene transfer prior to cellular mitosis
due to inhibition in binding of the mutated MPG derivative with importin R, the nuclear
protein involved in recognizing and binding to the NLS of a peptide [263] . However,
the mutated MPG-siRNA complex displayed enhanced gene knockdown compared to
MPG-siRNA because of increased siRNA release into the cytoplasm [264] .
In the late 1990s, 27-amino acid peptide galparan was synthesized, which con-
tains the first 13 amino acids from the N-terminus of galanin (Gly-Trp-Thr-Leu-Asn-
Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro) that is specific for cell surface galanin receptors
[265] . The second sequence of 14 amino acids is mastoparan (Ile-Asn-Leu-Lys-Ala-
Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu), a wasp venom peptide toxin that is believed
to form pores within the plasma membrane to promote cellular uptake. Based on sev-
eral experiments, it was concluded that the mechanism was neither endocytosis nor
receptor mediated [266] . Later, the same group proposed macropinocytosis as the
most probable mode of cellular uptake for transportan-protein complexes [267] .
Only a few studies have been conducted using transportan as a vector for deliv-
ering both siRNA and pDNA into cells. Transportan with an N-terminal cytsteine
residue was covalently attached to siRNA via disulfide linkages. This results in sig-
nificantly higher gene knockdown by transportan-siRNA complexes when compared
to Lipofectamine TM 2000 [268] . In another study with the objective of reducing
cytotoxicity, transportan 10 (TP10), an analogue of transportan, was synthesized by
removing part of the sequence. TP10 was attached to linear PEI (60 kDa) via a suc-
cinimidyl trans -4-(maleimidylmethyl) cyclohexane-1-carboxylate (SMCC) cross-
linker. TP10-PEI-DNA complex displayed twofold improvement in gene transfer
efficiency as compared to PEI (60 kDa) complexes in three different cell lines [269] .
4.5.2 Arginine-Rich Peptides
TAT protein is an 86-102 amino acid sequence consisting of three parts:
1. cationic regions controlling the rate of gene expression,
2. cysteine-rich regions involved in DNA binding, and
3. basic amino acid regions involved in promoting passage through the cell membrane [270] .
However, shorter amino acid sequences incorporating basic amino acid regions
of Tat protein are responsible for cellular uptake. Transduction is possible only with
47-57 residues (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) [271] . Although