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

(responsible for the formation of the extravascular fibrin
gel) through postcapillary venules in tumor cells is
responsible for generating the supporting matrix for
fibroblast migration, angiogenesis, and fibroplasia
(reviewed by Senger et al, 1993). Migration of
macrophages and fibroblasts in the fibrin gel in tumors is
determined by fibrinogen concentration and Factor XIII
crosslinking of α and γ chains. Tumor cells as well as
inflammatory cells in healing wounds produce a higher
concentration of degradative proteases for extracellular
matrix but also protease inhibitors which explains the
resistance toward degradation of the fibrin gel in solid
tumors (reviewed by Senger et al, 1993).
The mechanism of overexpression of VEGF in solid
tumors might involve its induction by hypoxia (Shweiki et
al, 1992); the rapid proliferation of the cells in the center
of the tumor induces an increase in the interstitial pressure
and may lead to closure of capillaries by compression;
inefficient vascular supply, including the compensatory
development of collateral blood vessels in ischaemic
tissues, leads to neovascularization via production of
VEGF. A clustering of capillaries alongside VEGFproducing
cells in a subset of glioblastoma cells
immediately proximal to necrotic foci have been observed
in intracranial brain neoplasms obtained from surgical
specimens; this was thought to be the result of a local
angiogenic response elicited by VEGF (Shweiki et al,
1992). VEGF expression by hypoxia was also induced in
skeletal muscle myoblasts, in the fibroblast mouse L cell
line, and in cells from rat heart muscle (Shweiki et al,
1992).
Targeting of VEGF gene leading to its transcriptional
inactivation (e.g. via triplex-forming oligonucleotides or
antisense vectors) is expected to limit growth in solid
tumors via inhibition in neo-vascularization; the prolonged
sustenance of hypoxia in the center of the tumor is also
expected to induce p53. An important concept to
understand is that tumor angiogenesis results from a
balance between angiogenic and anti-angiogenic factors.
Expression of VEGF 165 in rat C6 glioma cells and
subcutaneous injection of the transduced cells in athymic
mice has shown that tumors from cells expressing VEGF
grew slower than tumors developed from nontransduced
C6 cells, were highly vascularized, and contained varying
degrees of necrosis and eosinophilic infiltrate (Saleh,
1996).
VEGF plays an important role not only in
carcinogenesis but also restenosis (see below). VEGF
promotes endothelial cell proliferation to accelerate reendothelialization
of the artery reducing intimal
thickening; up-regulation of VEGF is the desired effect for
treatment of restenosis (Asahara et al, 1996; Isner et al,
1996a). The transfer of the VEGF gene demonstrates a
special mission of gene therapy: how to treat one human
disease by upregulating the expression of a specific gene
while treating a different disease by downregulating the
Boulikas: An overview on gene therapy
102
expression of the same gene. Targeting is important. Also,
exploring the molecular mechanisms affected by the
transfer and overexpression of the cDNA of a gene, in all
aspects and at their entire spectrum, is essential for a
successful gene therapy application.
F. Transfer of the VEGF gene in ischemia
VEGF gene transfer can improve blood supply to the
ischaemic limb and is a promising approach for the
treatment of acute limb ischemia. In a gene therapy
approach for tissue ischemia, the VEGF 165 cDNA under
the transcriptional control of the HSV immediate-early 4/5
promoter was used to transduce BLK-CL4 fibroblasts
resulting in the secretion of high levels of biologically
active VEGF; when the transduced cells were resuspended
in basement membrane extract (matrigel) and were
injected subcutaneously into syngeneic C57BL/6 mice
they showed a strong angiogenic response (Mesri et al,
1995). Regional angiogenesis was induced in nonischemic
retroperitoneal adipose tissue by adenoviral VEGF gene
transfer supporting a 123% increase in vessel number
compared to control (Magovern et al, 1997).
Treatment of a 71 year-old patient with an ischaemic
leg with 2 mg phVEGF 165 plasmid applied to the hydrogel
polymer coating of an angioplasty balloon and reaching
the distal popliteal artery resulted in an increase in
collateral vessels at the knee, mid-tibial, and ankle levels,
which persisted for 12-weeks (Isner et al, 1996a).
Ischemia, induced in the hindlimb of rats by excision
of the femoral artery, was experimentally treated by
transfer of the VEGF gene; therapeutic angiogenesis
produced morphologically similar, but significantly more
extensive, networks of collateral microvessels (Takeshita
et al, 1997). Direct i.m. injection of naked VEGF 165
plasmid DNA into the ischemic thigh muscles in rabbits
resulted in more angiographically recognizable collateral
vessels at 30 days posttransfection (Tsurumi et al, 1996,
1997).
G. Cancer treatment with angiogenesis
inhibitors
On November 20, 1997 the first exciting data on
clinical trials using the TNP-470, a drug extracted from
fungi which inhibits angiogenesis, were reported in a
speech before the National Institutes of Health by Judah
Folkman (Harvard University). A woman in Texas with
cervical cancer and metastasis to lungs had been tumorfree
for months after treated with TNP-470; and a young
girl with a slow-growing bone tumor in her jaw was
cancer-free after treatment with IFN-α. Both TNP-470 and
IFN-α are relatively weak inhibitors of blood vessel
formation compared with angiostatin (O'Reilly et al, 1994,
1996), endostatin (O'Reilly et al, 1997), and vasculostatin