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

inhibitor of thrombin known today is the polypeptide
hirudin, an anticoagulant derived from the medicinal
leech, Hirudo medicinalis; especially important is a local
delivery of hirudin to prevent thrombosis circumventing
the systemic coagulopathy associated with systemic
administration of the purified protein (for references see
Rade et al, 1996).
The pathogenesis of both atherosclerosis and restenosis
involves the migration of medial smooth-muscle cells
across the internal elastic lamina to form a neointima;
inflammatory reactions involving T cells and other
leukocytes maintain smooth-muscle cell migration,
proliferation and matrix deposition. The stenotic response
involves the expression of HA (hyaluronan) receptors on
both the infiltrating white cells and on smooth-muscle cell
populations; exposure of injured arteries to high
concentrations of HA in vivo resulted in significant
inhibition of neointimal formation (Savani and Turley,
1995). Intervening with the HA and receptor genes could
provide potential molecular targets for restenosis.
A number of drugs have been developed to inhibit
neointima formation such as the drug CVT-313, identified
from a purine analog library; CVT-313 is a specific and
potent inhibitor of CDK2 reducing hyperphosphorylation
of RB (Brooks et al, 1997). The angiotensin-converting
enzyme inhibitor, cilazapril, also prevented myointimal
proliferation after vascular injury (Powell et al, 1989).
However, most of these drugs are toxic and need
continuous administration to the artery. Gene therapy
could result in stable transfection of the arterial wall cells
with the gene of a therapeutic protein circumventing these
problems.
Significant progress has been made in the area of
coronary restenosis, particularly in identifying target genes
to reduce neointima formation in vein grafts used in
coronary bypass surgery. Targets of gene therapy include
the prevention of postangioplasty restenosis, postbypass
atherosclerosis, peripheral atherosclerotic vascular disease
and thrombus formation (reviewed by Malosky and
Kolansky, 1996; Ylä-Herttuala, 1996).
B. VEGF gene transfer for restenosis
Thickening of the arterial intima, composed of smooth
muscle cells, is an important area for intervention by gene
transfer to alleviate the syptoms of restenosis following
balloon treatment. Gene therapy for restenosis may be
achieved following transfer of the gene encoding VEGF;
this therapy has been applied to animal models and has
now entered clinical trials (Isner et al, 1996a). Previous
studies using administration of recombinant, 165 amino
acid, VEGF protein to rabbits in vivo has shown a
significant augmentation in collateral vessel development
by angiography after excision of the ipsilateral femoral
artery in the animal to induce severe hind limb ischemia
(Takeshita et al, 1994a). The rationale behind this
Boulikas: An overview on gene therapy
104
approach is that VEGF promotes endothelial cell
proliferation (Leung et al, 1989) to accelerate reendothelialization
of the artery reducing intimal thickening
and thrombogenicity (Asahara et al, 1996); the inner lining
of the blood vessels is made up of endothelial cells which
are important in preventing the formation of blood clots or
atherosclerotic plaques. Animal studies have shown that
endothelial cells require a relatively long time for growth
after injury. "Naked" plasmid encoding the 165 amino acid
VEGF isoform is being delivered using a hydrogel
/polymer-coated balloon angioplasty catheter without use
of liposomes or recombinant viruses to humans (Isner et
al, 1996a).
Ylä-Herttuala and coworkers (Laitinen et al, 1997a)
have examined and compared the efficiency of lacZ
delivery to the rabbit carotid artery (using a collar method)
with (i) plasmid/liposome complexes (Lipofectin), (ii)
replication-deficient Moloney murine leukemia virus
(MMLV)-derived retroviruses, (iii) pseudotyped vesicular
stomatitis virus protein G (VSV-G)-containing
retroviruses and (iv) adenoviruses. Transfer of the gene to
the adventitia took place with all gene transfer systems
tested except MMLV; the adenovirus gave the highest
gene transfer efficiency and up to 10% of the cells
displayed β-galactosidase activity compared with 0.05%
of cells using VSV-G retrovirus, 0.05% with Lipofectin,
and less than 0.01% with MMLV retrovirus (Figure 31).
During the collar application procedure extravascular
gene transfer took place without intravascular
manipulation; the model is suited for gene transfer studies
involving difussible or secreted gene products (such as
VEGF, see Figure 32) that act primarily on the
endothelium; effects on medial smooth muscle cell (SMC)
proliferation and even endothelium can be achieved from
the adventitial side of the artery (Laitinen et al, 1997a).
Transfer of the VEGF gene was performed on rabbits
using a silicone collar inserted around the carotid arteries.
The collar acted as an agent that caused intimal SMC
growth and as a reservoir for the VEGF gene; the model
preserved the integrity of endothelial cells and permitted
extravascular gene transfer without intravascular
manipulation. A plasmid carrying the mouse VEGF 164
gene under control of the CMV promoter in a mixture with
Lipofectin was injected under anesthesia by gently
opening the collar (Laitinen et al, 1997b). As a control,
arteries were injected with the lacZ cDNA; animals after
3, 7, or 14 days from the operation were sacrificed and the
arteries were examined by electron microscopy using
SMC-specific immunostaining (Figure 32A,B). One week
after VEGF transfer with cationic liposomes there was a
significant reduction in intimal thickening (Figure 32B)
compared to control arteries (A). When the nitric oxide
synthase inhibitor L-NAME was administered to the
animals it abolished the therapeutic efficacy of VEGF and
there was no VEGF-induced reduction in intimal
thickening of the arteries (Laitinen et al, 1997b).