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

Curriel and coworkers (1991) have used the transferrin
receptor on the surface of mammalian cells to deliver
plasmid-polylysine-transferrin complexes to cells. These
complexes are taken up by endosomes following receptor
binding, a method which suffers from that the endocytosed
DNA is trapped in the intracellular vesicle and is later
largely destroyed by lysosomes; use of the capacity of the
adenoviruses to disrupt endosomes as part of their entry
mechanism to the cells have augmented over 1000-fold the
efficiency of gene transfer. This method has been further
developed in collaboration with Max Birnstiel; true
chemical coupling rather than simple addition of
replication-defective adenovirus particles has shown a
further increase in transfection efficiency (Cotten et al,
1992; Wagner et al, 1992a,b).
A monoclonal antibody against the CE7 antigen
(chCE7) covalently linked to polylysine in the presence of
chloroquine was able to transfect NB cells as efficiently as
DOTAP, transfectam, TF-X50, or lipofectamine;
furthermore, transfection was not observed in cell lines
negative for the CE7 antigen (Coll et al, 1997).
B. Polyethylenimine (PEI, ExGen500)
Polyethylenimine, H 2N-(CH 2-CH 2-NH) n-H, is an
organic polymer with a potential for high cationic charge.
PEI enhanced transfection efficiency in cell culture
(Boussif et al, 1996). ExGen500 is a linear 22 kDa form of
PEI, which was found to be more efficient than lipofectin,
DOTAP and DOGS in delivering the luciferase reporter
gene in both newborn and adult rabbit lungs (Ferrari et al,
1997). The PEI 800 kDa and PEI 25 kDa branched
polymers have also been used to transfer marker genes to
the newborn and adult mouse brain (Boussif et al, 1995;
Abdallah et al, 1996). Another advantage of PEI is that it
yields high transfection efficiencies with a charge ratio of
DNA:PEI close to neutral; this is an advantage as particles
with a net positive charge (cationic lipid-DNA complexes)
interact with circulating serum proteins or anionic
components of the extracellular matrix in the various
tissues hindering their bio-availability (Schwartz et al,
1995).
The high transfection efficiency of ExGen 500 was
suggested to arise from the “proton sponge” effect which
leads to osmotic swelling of endosomes which have
uptaken the DNA complexes (Ferrari et al, 1997).
Different cationic compositions may result in different
targeting and transfection abilities to specific organs; the
branched, 25-kD polyethylenimine polymer (PEI 25k) was
superior over DOTAP and DOGS (Transfectam) in the
efficiency of transfection of the kidney when complexes of
these cations with luciferase plasmid were injected into the
left renal artery of rats; luciferase activity peaked at 2
days, was still significantly higher than controls at 7 days,
Boulikas: An overview on gene therapy
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but was undetectable at 14 days post-injection (Boletta et
al, 1997).
Scanning force microscopy allowed plasmid DNA
strands to be visualized without drying in incomplete
condensates prepared with varying stoichiometries of
lipospermine or polyethylenimine in physiological
solution; discrete nucleation centers of condensation were
observed often surrounded by folded loops of DNA using
either condensing agent; increasing the amount of
lipospermine or polyethylenimine led to further
aggregation through folding rather than winding of the
DNA (Dunlap et al, 1997).
C. APL PolyCat57 and other polymers
APL PolyCat57 is a synthetic polyamino derivative
(nonpeptide, nonlipid polymer) with a glucose backbone
which was used by Goldman and coworkers (1997) for
gene transfer in vivo and in vitro. A variety of human
carcinoma cell lines were transfected with an efficiency
superior to that of Lipofectamine. The polymer-plasmid
complex was resistant to inhibition by serum allowing for
efficient gene transfer in vivo. The level of the luciferase
and β-galactosidase reporter gene expression after
intrathecal injection, evaluated in animal models bearing
stereotactically implanted D54-MG human glioma cell
xenografts, was comparable to that obtained with an
adenoviral vector.
Liposomes composed of the cationic peptide
amphiphile N,N-dihexadecyl-N α-[6-(trimethyl
ammonio)- hexanoyl]-L-alaninamide bromide comprising
an L-alanine residue interposed between a charged head
group and a double-chain segment were more effective
and less toxic than lipofectin, and DOTAP for the
transfection of COS-7 cells (Kato et al, 1996).
D. Adenovirus-polymer complexes
An adenovirus/DNA complex was constructed by
chemically linking poly-L-lysine to the capsid of the
replication-defective adenovirus dl312; this complex was
then coupled with plasmid DNA via ionic interaction. This
system was used to deliver the tumor suppressor protein
p53 to the p53 - human lung cancer cell line H1299, both in
vitro and in vivo, leading to induction of apoptosis;
injection of the complex carrying the p53 gene to
subcutaneous tumor sites 5 days after tumor cell
implantation resulted in a significant inhibition of
tumorigenicity as measured by the number and size of
tumors that developed 21 days after treatment (Nguyen et
al, 1997a,b).
Complexes of cationic polymers and cationic lipids
with adenovirus increased adenovirus uptake and
transgene expression in cells that were inefficiently