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

the endosomal vesicles. The nuclear uptake of
oligonucleotides depended on several factors such as
charge of the particle where positively charged complexes
were required for enhanced nuclear uptake; DOTAP
increased over 100 fold the antisense activity of a specific
anti-luciferase oligonucleotide. Physicochemical studies of
oligonucleotide-liposome complexes of different cationic
lipid compositions indicated that either
phosphatidylethanolamine or negative charges on other
lipids in the cell membrane are required for efficient
fusion with cationic liposome-oligonucleotide complexes
to promote entry to the cell (Jaaskelainen et al, 1994).
Similar results were reported by Lappalainen et al
(1997); digoxigenin-labeled oligodeoxynucleotides
(ODNs) complexed with the polycationic DOSPA and the
monocationic DDAB (with DOPE as a helper lipid) were
uptaken by CaSki cells in culture by endocytosis. The
nuclear membrane was found to pose a barrier against
nuclear import of ODNs which accumulated in the
perinuclear area. Although DOSPA/DOPE liposomes
could deliver ODNs into the cytosol, they were unable to
mediate nuclear import of ODNs; on the contrary
oligonucleotide-DDAB/DOPE complexes with a net
positive charge were released from vesicles into the
cytoplasm; it was determined that DDAB/DOPE mediated
nuclear import of the oligonucleotides.
DOPE-heme (ferric protoporphyrin IX) conjugates,
inserted in cationic lipid particles with DOTAP, protected
oligoribonucleotides from degradation in human serum
and increased oligoribonucleotide uptake into 2.2.15
human hepatoma cells; the enhancing effect of heme was
evident only at a net negative charge in the particles
(Takle et al, 1997). Uptake of liposomes labeled with 111 In
and composed of DC-Chol and DOPE was primarily by
liver, with some accumulation in spleen and skin and very
little in the lung after i.v. tail injection; preincubation of
cationic liposomes with phosphorothioate oligonucleotide
induced a dramatic, yet transient, accumulation of the lipid
in lung which gradually redistributed to liver. The
mechanism of lung uptake involved entrapment of large
aggregates of oligonucleotides within pulmonary
capillaries at 15 min post-injection via embolism; labeled
oligonucleotide was localized primarily to phagocytic
vacuoles of Kupffer cells at 24 h post-injection; nuclear
uptake of oligonucleotide in vivo was not observed
(Litzinger et al, 1996).
Phosphorothioate oligonucleotides were found in most
tissues 48 h after i.p. administration with highest
concentrations in kidney and liver; complexation of the
oligonucleotide with DOTMA did not affect neither the
oligonucleotide uptake nor its tissue distribution in normal
mice but increased the oligonucleotide cellular uptake (4-
10 times) in LOX ascites tumors (Saijo et al, 1994).
Triplex-forming ODNs were delivered to cells in
culture using adenovirus-polylysine-ODN complexes
Boulikas: An overview on gene therapy
24
designed to take advantage of the receptor mediated
endocytosis of adenoviruses to transfer the ODNs to the
cell nucleus; nuclear uptake peaked at 4 h and intact ODN
persisted in the nucleus with a half-life of 12 h
(Ebbinghaus et al, 1996).
E. Fusogenic peptides enhance gene
transfer efficiency
Enveloped viruses have evolved efficient mechanisms
to release their genomes from the endosomes into the
cytoplasm of the host cells; specific envelope proteins of
the nucleocapsid are capable of destabilizing the
endosomal membrane. Therefore, inactivated viruses have
been used to enhance the transfer of plasmids. Addition of
adenoviral particles capable of inducing endosome lysis
(Blumenthal et al, 1986), mediated by a conformational
change in the adenovirus penton protein induced at the
lower pH of endosomes (Seth, 1994) can increase
transfection efficiency 100-1000 fold using 10 9 adenoviral
particles/ml and the transferrin receptor (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; reviewed by Ledley, 1995).
Use of fusogenic peptides from influenza virus
hemagglutinin HA-2 enhanced greatly the efficiency of
transferrin-polylysine-DNA complex uptake by cells; in
this case the peptide was linked to polylysine and the
complex was delivered by the transferrin receptormediated
endocytosis (Wagner et al, 1992a; Plank et al,
1994). This peptide had the sequence:
GLFEAIAGFIENGWEGMID GGGYC and was able to
induce the release of the fluorescent dye calcein from
liposomes prepared with egg yolk phosphatidylcholine
which was higher at acidic pH; this peptide was also able
to increase up to 10-fold the anti-HIV potency of antisense
oligonucleotides, at a concentration of 0.1-1 mM, using
CEM-SS lymphocytes in culture (Bongartz et al, 1994).
This peptide changes conformation at the slightly more
acidic environment of the endosome destabilizing and
breaking the endosomal membrane (Murata et al, 1992;
Bullough et al, 1994). Fusogenic peptides have been used
by other investigators (Midoux et al, 1993; Kamata et al,
1994). It is thought that several fusogenic peptides selfassemble
following their conformational change forming a
transmembrane channel (Bongartz et al, 1994).
Sendai virosomes were effective for delivering AAV
neuropeptide Y (NPY) cDNA constructs in vivo.
Injections into brain neocortex of Sendai-virosome
encapsulated rAAV construct expressing NPY increased
NPY-like immunoreactivity in neurons but not glia;
injections into the rat hypothalamic para-ventricular
nucleus increased body weight and food intake for 21 days
(Wu et al, 1996). Tomita et al (1996) have found that
newborn mice can sustain expression of the insulin gene