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

delivered by Sendai virus-liposome complexes for at least
8 weeks as assayed by reverse transcriptase PCR and
radioimmunoassay, compared to 2 weeks in adult animals.
A 27 residue peptide vector, containing the fusion
sequence of HIV gp41 and the nuclear localization
sequence of SV40 T antigen was used to deliver
oligonucleotides to cell nuclei very rapidly in cell culture
(1h). The complexes formed strongly increased the
stability of the oligonucleotide to nucleases, enhanced
passage through the plasma membrane, and led to
endosomal internalization (Morris et at, 1997).
Certain cationic lipids are endowed with a better
ability to disrupt the endosomal membrane and promote
release of the plasmid to the cytoplasm, a prelude for its
nuclear import. Presentation of plasmid DNA to COS cell
cultures using three different lipid formulations: (i)
vectamidine (3-tetradecylamino-N-tert-butyl-N'tetradecylpropionamidi
ne), (ii) DOTMA:DOPE
(Lipofectin), and (iii) DMRIE-Chol (1:1) resulted in
complex entry via endocytosis for all three cationic lipids
as revealed using transmission electron microscopy.
However, the endosomal membrane in contact with
complexes containing vectamidine or DMRIE-Chol, but
not Lipofectin, often exhibited a disrupted morphology (El
Ouahabi et al, 1997).
F. Plasmid condensation with spermine,
polylysine, protamine, histones enhances the
transfection efficiency
DNA can be presented to cells in culture as a complex
with polycations such as polylysine, or basic proteins such
as protamine, total histones or specific histone fractions
(Fritz et al, 1996), cationized albumin, and others (Smull
and Ludwig, 1962). These molecules increase the
transfection efficiency. In addition to HMG1, also histone
H1 and HMG17 were identified as transfection-enhancing
proteins in cell culture (Zaitsev et al, 1997). Histone H2A
significantly enhanced in vitro DNA transfection whereas
other histones and anionic liposomes did not (Balicki and
Beutler, 1997). Gene transfer through the asialoglycoprotein
receptor-mediated endocytosis pathway was
enhanced with the histones H1, H2a, H2b, H3, and H4
which were galactosylated with the crosslinker agent, 1ethyl-3-(3-dimethylaminopropyl)carbodiimide,
conjugated
to DNA and then used to transfect HepG2 cells, which
display the asialoglycoprotein receptor (Chen et al, 1994).
Plasmid DNA and HMG1 were efficiently coencapsulated
in liposomes by agitation and sonication, and
were co-introduced into cells by hemagglutinating virus of
Japan (HVJ)-mediated membrane fusion; the presence of
HMG1 enhanced 3-fold the transfection efficiency (Kato
et al, 1991).
The interaction of plasmid DNA with protamine
sulfate followed by the addition of DOTAP cationic
Gene Therapy and Molecular Biology Vol 1, page 25
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liposomes offered a better protection of plasmid DNA
against enzymatic digestion and gave consistently higher
gene expression in mice via tail vein injection compared
with DOTAP/DNA complexes; 50 µg of luciferaseplasmid
per mouse gave 20 ng luciferase protein per mg
extracted tissue protein in the lung which was detected as
early as 1 h after injection, peaked at 6 h and declined
thereafter. Intraportal injection of
protamine/DOTAP/DNA led to about a 100-fold decrease
in gene expression in the lung as compared with i.v.
injection; endothelial cells were the primary locus of lacZ
transgene expression (Li and Huang, 1997). Protamine
sulfate enhanced plasmid delivery into several different
types of cells in vitro using the monovalent cationic
liposomal formulations (DC-Chol and lipofectin); this
effect was less pronounced with the multivalent cationic
liposome formulation, lipofectamine (Sorgi et al, 1997).
Spermine has been found to enhance the transfection
efficiency of DNA-cationic liposome complexes in cell
culture and in animal studies; this biogenic polyamine at
high concentrations caused liposome fusion most likely
promoted by the simultaneous interaction of one molecule
of spermine (four positively charged amino groups) with
the polar head groups of two or more molecules of lipids.
At low concentrations (0.03-0.1 mM) it promoted
anchorage of the liposome-DNA complex to the surface of
cells and enhanced significantly transfection efficiency
(Boulikas et al, in preparation).
Because the receptor for ecotropic viruses is a
transporter for basic amino acids, use of a histone as a
facilitator increased the efficiency of retroviral infection
(Singh and Rigby, 1996). Polybrene is the usual agent
employed during retroviral infection. For supernate
infections, concentrations of 5-10 µg/ml of protamine
provided essentially the same infection efficiency as
polybrene; protamine displayed low toxicity on a range of
cell types and increased 7-fold the efficiency of retroviral
infection (Cornetta and Anderson, 1989).
The polycations polybrene, protamine, DEAE-dextran,
and poly-L-lysine significantly increased the efficiency of
adenovirus-mediated gene transfer in cell culture; this was
thought to act by neutralizing the negative charges
presented by membrane glycoproteins which reduce the
efficiency of adenovirus-mediated gene transfer (Arcasoy
et al, 1997).
G. Targeted gene delivery
Targeting a specific cell type or animal tissue is an
important goal of gene therapy. Many different approaches
have been undertaken to achieve targeting. A recombinant
adenovirus encoding an anti-erbB-2 intracellular singlechain
antibody (sFv) displayed a genetic selectivity for the
erbB-2-positive prostate carcinoma cell lines DU145 and