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

normal cells in the surroundings. Treatment of rats at day
5 after transplantation with ganciclovir (GCV) resulted in
the complete regression of the tumor cell mass; this was
thought to be induced by killing of cells that respond to
signals promoting angiogenesis in the immediate vicinity
of the tumor; vascular endothelial cells in the normal brain
tissue, exhibiting cycling at a low rate, apparently were
not affected. Other proliferating tissues, such as intestinal
epithelium, thymus, and bone marrow, which might also
uptake the retroviral HSV-tk vector and then be destroyed
during GCV treatment were not affected by this approach
over a 30 day period of treatment with GCV (Culver et al,
1992).
A replication-defective, highly purified retroviral
vector at titers of 10 8 colony forming units/mL was used to
treat 9L gliosarcoma cells in rat brain. Animals with
established 9L tumors treated with intralesional injection
of the HSV-tk retrovirus followed by GCV treatment
showed at day 26 that 29% (4/14) had no tumor and 50%
(7/14) of the animals had < 1% tumor volume; substantial
numbers of CD4 +
Gene Therapy and Molecular Biology Vol 1, page 73
and CD8 +
lymphocytes infiltrated the
tumors of animals treated with HSV-tk and GCV; the
former tumor bed in cured animals contained cell debris,
immune cells, and fibroblasts without signs of damage to
the adjacent brain tissue (Kruse et al, 1997).
C. The bystander effect of HSV-tk/GCV
During HSV-tk/GCV treatment of brain tumors
products from the dying cells in the brain tumor killed
nearby non-HSVtk-transduced cancer cells without
affecting normal cells, an effect described as "bystander"
antitumor effect (Culver et al, 1992). The bystander effect
of the HSV-tk plus GCV system appears to be powerful
and significant, circumventing the low efficiency of
transduction in vivo with recombinant retroviruses.
Because of this effect, the low-level percentage of cells
that can be transduced with a retrovirus can cause the
elimination of a much larger percentage of proliferating
cells in their surroundings (Kimura et al, 1996).
In vitro, the “bystander” effect works by transfer of
cytotoxic small molecules between cells via gap junctions.
In order to understand the “bystander effect” mechanism
during which adjacent nontransduced tumor cells are
killed, Yamamoto et al (1997) used Renca cells from a
renal carcinoma cell line transduced with a retroviral
vector bearing the HSV-tk gene to inoculate BALB/c
mice. After complete regression of inoculated tumors with
GCV treatment, the animals were challenged with
nontransduced tumor cells. In these animals, tumorspecific
cytotoxic CD8 +
T cells were efficiently induced
which promoted the rejection or significant growth
inhibition of challenged tumor cells.
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In a similar experiment, set to assess the “bystander
effect” in vivo, mixtures of HSV-tk−transduced and
nontransduced oral squamous carcinoma cells were
implanted subcutaneously in the left flank of nude mice,
and naive HSV tk − cells were implanted subcutaneously in
the right flank. Treatment with GCV eradicated the tumors
in the left flank consistent with a predicted bystander
effect but also resolved or arrested the growth of the naive
tumors in the right flank. The histology of regressing
tumors from the right flank showed an infiltration of
lymphoid cells suggesting that an immune-related
antitumor response accounted for the distant bystander
effect (Bi et al, 1997; see also Ramesh et al, 1998 this
volume).
The induction of higher levels of HSV-tk expression
does not augment the sensitivity to GCV: adenoviral
vectors that expressed HSV-tk at different efficiencies
from CMV versus RSV promoters did not display a
significant difference in antitumor effects; thus, increasing
the HSV-TK enzyme levels per cell above a minimal
threshold level will not be effective in cell killing with
GCV. To enhance the therapeutic responses of the HSVtk/GCV
system one needs to improve other parameters
such as to use higher doses of GCV, to enhance the
"bystander effect," to engineer mutant HSV-tk genes with
higher substrate affinities, or to discover vectors with
increased transduction efficiencies (Elshami et al, 1997).
Suicide gene therapy may be useful not only for shortterm
tumor regression mediated by direct cell killing and
bystander effect, but may also exert a therapeutic
vaccination effect resulting in long-term tumor regression
and prevention of recurrence (Yamamoto et al, 1997).
D. Additional examples of tumor
eradication with HSV-tk/GCV
Chen et al (1996) used a recombinant adenoviral
vector containing the HSV-tk gene for the treatment of
metastatic colon carcinoma in the mouse liver; the HSV-tk
alone exhibited substantial regression, although all treated
animals suffered from subsequent relapses. Delivery of the
HSV-tk + mouse IL-2 genes in adenoviral vectors to the
hepatic tumors induced an effective antitumor immune
response which nevertheless waned with time, and the
treated animals eventually succumbed to hepatic tumor
relapse; however, after combination treatment with HSVtk,
mouse IL-2, and mouse GM-CSF a fraction of the
animals developed long-term antitumor immunity and
survived for more than 4 months without tumor recurrence
(Chen et al, 1996).
Microinjection of the HSV-tk gene, under control of αfetoprotein
enhancer and albumin promoter, in a linear
form flanked by the adeno-associated virus ITRs into
pronuclei of mouse embryos led to transgenic animals