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

complex into the cytosol from the endosomes (Zuidam and
Barenholz, 1997).
C. Tissue targets using cationic liposomes
in vivo
Although cationic lipids have been used widely for the
delivery of genes very few studies have used systemic i.v.
injection of cationic liposome-plasmid complexes because
of the toxicity of the lipid component and certainly in
animal models, not humans. Administration by i.v.
injection of two types of cationic lipids of similar
structure, DOTMA and DOTAP, has shown that the
transfection efficiency was determined mainly by the
structure of the cationic lipid and the ratio of cationic lipid
to DNA; the luciferase and GFP gene expression in
different organs was transient, with a peak level between 4
and 24 hr, dropping to less than 1% of the peak level by
day 4 (Song et al, 1997).
Figure 5 shows the effect of cationic lipid:DNA ratio
on transfection efficiency after i.v. tail injection.
Luciferase activity was detected in all organs examined
with the highest level in lung. In the absence of neutral
lipid both DOTMA and DOTAP promoted a linear
increase in luciferase activity in the lung with increasing
lipid:DNA from 12:1 to 36:1 nmol lipid: µg of DNA.
DOTMA was 10 times more efficient than DOTAP (10 6
versus 10 7 relative luciferase units (RLU) per mg protein.
Cholesterol (Chol) mixed with DOTMA (1:1 molar ratio)
decreased the level of gene expression in the lung whereas
cholesterol did not affect the transfection efficiency of
DOTAP liposomes. Inclusion of DOPE into either
DOTAP or DOTMA liposomes significantly decreased the
transfection efficiency by 100-fold in the lung.
When a group of four cationic lipids with identical
head group but of different fatty acyl chains were tested
for their transfection efficiencies (Figure 6); these
included DOTAP, DMTAP, DPTAP, and DSTAP. The
C 14 acyl chain-lipid DMTAP had a similar transfection
efficiency as DOTAP which has 18 carbon atoms in the
acyl chain and one double bond (C 18Δ 9); on the contrary,
the transfection efficiencies of DPTAP (C 16) was 10-100
fold lower and that of DSTAP (C 18) was 100 to 1000 fold
lower.
Confocal microscopy of lung tissue after injection of
25 µg pCMV-GFP plasmid DNA complexed with
Gene Therapy and Molecular Biology Vol 1, page 21
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DOTMA liposomes to mice (Figure 7) has shown that the
type of cells that express the transgene are the endothelial
cells that have typical characteristics of neighboring
multiple air-sac structures (Figure 7D).
A number of different organs in vivo can be targeted
after liposomal delivery of genes or oligonucleotides.
Intravenous injection of cationic liposome-plasmid
complexes by tail vein in mice targeted mainly the lung
and to a smaller extend the liver, spleen, heart, kidney and
other organs (Zhu et al, 1993). Intraperitoneal injection of
a plasmid-liposome complex expressing antisense K-ras
RNA in nude mice inoculated i.p. with AsPC-1 pancreatic
cancer cells harboring K-ras point mutations and PCR
analysis indicated that the injected DNA was delivered to
various organs except brain (Aoki et al, 1995).
A number of factors for DOTAP:cholesterol/DNA
complex preparation including the DNA:liposome ratio,
mild sonication, heating, and extrusion were found to be
crucial for improved systemic delivery; maximal gene
expression was obtained when a homogeneous population
of DNA:liposome complexes between 200 to 450 nm in
size were used. Cryo-electron microscopy showed that the
DNA was condensed on the interior of invaginated
liposomes between two lipid bilayers in these
formulations, a factor that was thought to be responsible
for the high transfection efficiency in vivo and for the
broad tissue distribution (Templeton et al, 1997).
Steps to improve for successful liposome-mediated
gene delivery to somatic cells include persistence of the
plasmid in blood circulation, port of entry and transport
across the cell membrane, release from endosomal
compartments into the cytoplasm, nuclear import by
docking through the pore complexes of the nuclear
envelope, expression driven by the appropriate
promoter/enhancer control elements, and persistence of
the plasmid in the nucleus for long periods. A number of
strategies for liposomal delivery and for enhancing the
efficiency of uptake by the cells and release from
endosomal compartments of plasmid or oligonucleotide
DNA are reviewed in the following article (Martin and
Boulikas, 1998).