01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

Boulikas: An overview on gene therapy
Figure 1. Role of E1A and E1B19-kDa proteins of adenovirus in apoptosis.
penetration of the adenoviral particles by acid-catalyzed
rupture of the endosomal membrane involving the penton
protein and the integrins and allowing escape to the
cytoplasmic compartment; a decrease in endosome pH
during internalization expose hydrophobic domains of
these adenoviral capsid proteins which permits these
proteins to insert into the vesicle membrane in a fashion
that ultimately disrupts its integrity (Seth et al, 1984). At
the final step the adenoviral particle is attached to the
cytoplasmic side of pore complexes and the DNA is
released to the interior of pore annuli entering the
nucleoplasm.
These highly ordered processes are accompanied by
losses or protease degradation of specific proteins on the
viral particles; the fibers and some of the penton base
complexes on the adenovirus surface are already lost
during the process of endocytosis; a viral protease,
L3/p23, located inside the capsid at 10 copies per virion,
plays a key role in the stepwise dismantling and in the
weakening of the capsid structure culminating with the
release of the adenovirus DNA by degrading of the viral
capsid protein VI (Greber et al, 1996). The mechanism of
disruption of endosomes by the adenoviral particles has
been exploited to augment efficiency of transfection with
transferrin-polylysine-DNA complexes (see fusogenic
peptides and Curiel et al, 1991; Cotten et al, 1992; Wagner
et al, 1992b; Cristiano et al, 1993; Morishita et al, 1993;
Harries et al, 1993; Curiel, 1994).
To overcome one of the major limitations to the
clinical utility of adenoviruses which is the low efficiency
of gene transfer achieved in vivo, Arcasoy et al (1997)
found that the presence of the polycations polybrene,
protamine, DEAE-dextran, and poly-L-lysine significantly
increased the transfection efficiency in cell culture using
the lacZ gene; because the polyanion heparin did not
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significantly alter gene transfer efficiency, but completely
abrogated the effects of polycations it supports the idea
that the negative charges presented by membrane
glycoproteins reduce the efficiency of adenovirusmediated
gene transfer, an obstacle that can be overcome
by polycations.
D. Advantages and drawbacks of
adenoviral vectors in gene delivery
Adenoviruses possess a linear double-stranded genome
which can be manipulated to accommodate up to 7.5 kb of
DNA. Adenoviruses have the advantage of being able to
infect nondividing cells. Other advantages are the rarity of
recombination events between adenoviral vectors and the
host chromosomes, the absence of induction of human
malignancies by adenoviruses, and the relative safety of
their use as vaccines (e.g. Ballay et al, 1985; Haj-Ahmad
and Graham, 1986). For safety, replication-deficient,
infectious adenoviruses are being used in somatic gene
transfer; for example deletion in a portion of the E3 region
of the virus permits encapsidation whereas deletion of a
portion of the E1A coding sequence impairs viral
replication (Gilardi et al, 1990; Rosenfeld et al, 1991).
E. Deletion of adenoviral DNA sequences
for gene delivery
First generation recombinant adenoviruses were
rendered defective by deletion of sequences spanning the
E1A and E1B genes; these adenoviruses expressed low
levels of early and late viral genes responsible for
activating destructive cellular immune responses. Further
deletion of other essential genes and growth in new
packaging cell lines or incorporation of temperature