Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 168

were reported as vectors for gene transfer, but momentum in research has increased
recently for the following reasons:

l

Synthetic peptides reduce the number of molecular species in the reaction, because the
complex formed is controlled simultaneously by several kinetic processes from assembly
to DNA uncoating.

l

Selective targeting can be accomplished with synthetic ligands for cell-specific plasma
membrane receptors.

l

Peptides can be synthesized rapidly either automatically or manually in a reproducible
manner.

l

Design and synthesis of complicated structures can be executed with ease.

A major advantage of a synthetic peptide-based DNA delivery system is its flexibil-
ity. By changing the design, the composition of the final complex can be easily modi-
fied in response to experimental results in order to take advantage of specific peptide
sequences to overcome extra- and intracellular barriers to gene delivery. For example,
the structure-function relationships for many receptor ligands are known; it was easier
to obtain receptor ligands with high-binding affinities. Recent advancement in synthetic
peptide chemistry and molecular modeling has allowed synthesis of ligand-modified
peptides recognizable by specific cells; dye-modified peptides to trace their locations
in cells and tissues; and other “intelligent” peptides to achieve functional gene delivery.
It is assumed that the relative ease of peptide construction will provide researchers
with a wide range of molecules, as well as important information about the structural
requirements for functional gene delivery. The pH sensitivity of several peptides can
cause pH-selective endosomal lysis. Nuclear localization sequences (NLS) appear
to be an essential requirement for passage of macromolecules through the nuclear
pore [258] . High affinity DNA-binding peptides are ideally suitable for incorporating
these functions into the gene delivery system.
Peptide-ODN conjugates can deliver genetic material into cells with high effi-
ciencies and cell specificity. Short sequences of basic amino acid that aid in cross-
ing plasma membrane are known as cell-penetrating peptides. Broadly, they can be
divided into lysine-rich peptides, such as the amphipathic MPG peptide and trans-
portan, and arginine-rich peptides, such as transactivating transcriptional activators
(TAT) and the homeodomain of antennapedia (Antp) [259] . Peptide-DNA com-
plexes are generally formed by electrostatic interactions similar to those used to form
lipoplexes or polyplexes, whereas peptide-ODN complexes are formed by covalent
attachment. Haralambidis et al. first reported solid-phase synthetic approaches for
synthesizing peptide-ODN constructs [260] . Another method utilized base-labile
protecting groups such as 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde)
groups to avoid the acid deprotection conditions required to remove BOC-protecting
groups in the presence of the ODN [261] .

4.5.1 Lysine-Rich Peptides
MPG peptide is a synthetic peptide consisting of a hydrophobic N-terminal region
derived by union HIV gp41 (Gly-Ala-Leu-Phe-Leu-Gly-Phe-Leu-Gly-Ala-Ala-Gly-
Ser-Thr-Met-Gly-Ala) and a hydrophilic region derived from the NLS of the SV40