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

expressing preferentially HSV-tk into adult liver cells; this
led to an approach for the treatment of hepatocellular
carcinomas (Su et al, 1996). Subcutaneous tumors induced
by injection of RM-1 (mouse prostate cancer) cells in mice
followed by injection of HSV tk in an adenovirus vector
and treatment with ganciclovir for 6 days showed
reduction in tumor volume (16% of control) and higher
apoptotic index in tumor cells (Eastham et al, 1996).
Recombinant adenoviruses carrying the HSV-tk gene
under control of the CMV promoter displayed a significant
cell killing efficiency for the eradication of brain tumors
and leptomeningeal metastases in rats (Vincent et al,
1997).
Pancreatic cancer is the fifth leading cause of cancer
death in the United States. In order to treat peritoneal
dissemination, one of the most common complications of
the malignancies of the digestive system such as gastric or
pancreatic cancers, mice were intraperitoneally (i.p.)
inoculated with the human pancreatic cancer cell line
PSN-1; i.p. transfer of the HSV-tk suicidal gene under
control of the potent hybrid CAG promoter was achieved
with a DNA-lipopolyamine complex given eight days
from the injection of cancer cells; animals were treated
with GCV for 8 days; 8 out of 14 mice treated with HSVtk
and GCV were free of tumors on day 24. The gene
transfer method resulted in the transduction of tumor
nodule cells and not in normal organs as shown by reverse
transcription polymerase chain reaction (RT-PCR)
analysis as well as by transfer of the lacZ gene under
similar conditions and localization of the blue staining;
HSV-tk was expressed in about 10% of tumor cells but not
in the normal pancreas or in the small intestine (Aoki et al,
1997).
A murine pancreatic ductal adenocarcinoma cell line
was used to induce intrahepatic solid tumors into the left
lateral liver lobe; intratumoral injection of an adenovirus
vector carrying the HSV-tk gene under control of the RSV
promoter in combination with intraperitoneal administration
of ganciclovir caused a significant reduction in tumor
volume and necrosis; because pancreatic cancer patients
have an overall low survival since metastases have already
taken place at the time of diagnosis and because surgical
resection of pancreatic cancers does not significantly
change the clinical outcome even in combination with
chemotherapy, gene therapy might offer an effective
approach in the near future (Block et al, 1997).
HSV-tk gene transfer was successfully used to
eradicate adenocarcinoma-derived peritoneal
carcinomatosis, a common clinical situation which, in
most cases cannot be controlled by surgery or
chemotherapy. DHD/K12 colon carcinoma cells stably
expressing the HSV-tk gene were injected
intraperitoneally to rats leading to the development of
peritoneal carcinomatosis within 2-3 weeks from injection
(Figure 25A). Treatment of these animals with GCV
(Figure 25C) resulted in the eradication of the peritoneal
Boulikas: An overview on gene therapy
74
tumor nodes. It ought to be emphasized, however, that the
same spectacular results are not expected when treating
tumors in patients; tumor cells in patients need first to be
transduced with the HSV-tk gene whereas the cells used to
elicit these tumors in animals were already transduced
with the HSV-tk gene in cell culture and most or all cells
were expressing the viral thymidine kinase.
Retrovirus-mediated transfer of HSV-tk was used to
kill proliferating cells in rabbit models of proliferative
vitreoretinopathy (PVR); traction retinal detachment
results from proliferation of retinal pigment epithelial,
glial, macrophages, and fibroblast cells in the vitreous
cavity of the eye forming contractile membranes on both
surfaces of the retina; PVR may ensue after retinal surgery
or trauma and can be induced in rabbit models by surgical
vitrectomy to facilitate cell attachment to the retina.
Injection, into the vitreous cavity, of rabbit dermal
fibroblasts transduced in vitro with retroviral vectors
carrying the HSV-tk gene was used to preferentially kill
proliferating cells for PVR in rabbit models; all eyes
received 0.2 mg GCV on the following day and on day 4;
significant inhibition of PVR was observed thus providing
a novel therapeutic strategy for this disease (Kimura et al,
1996).
E. Expression of cytosine deaminase (CD)
gene from E. coli and treatment with 5fluorocytosine
Another suicide gene approach has been the expression
of the cytosine deaminase (CD) from E. coli; mammalian
cells, unlike certain bacteria and fungi, do not posses this
enzyme. The CD protein normally catalyzes the
conversion of cytosine to uracil but has been exploited for
the conversion of the prodrug 5-fluorocytosine (5FC) into
the toxic 5-fluorouracil (5FU); treatment of cells,
transfected with this construct, with 5FC resulted in the
conversion of the 5FC into the antitumor drug 5FU into
CD-expressing tumor cells (Mullen et al, 1992; Austin and
Huber, 1993; Huber et al, 1993; 1994; Richards et al,
1995).
This approach has been used for the treatment of
primary and metastatic hepatic tumors based on the
overexpression of the suicidal CD gene under control of
the regulatory regions of the tumor marker gene
carcinoembryonic antigen (Richards et al, 1995, see
below).
Szary et al (1997) have developed a model for tumor
radiosensitization using the CD gene/5FC system; when
melanoma cells were transfected with the CD gene,
subsequent treatment with 5FC sensitized the cells to
radiation damage; 5FC did not change the radiosensitivity
of parental, nontransfected cells; increased toxicity to
radiation damage was thought to arise from 5-fluorouracil
generated by CD.