ABBS 2005,37(09): Targeting Gene-Virotherapy for Cancer

Received:
June 7, 2005

Accepted:
July 13, 2005

*Corresponding
author: Tel, 86-21-54921127 or 86-571-86843181; Fax, 86-21-54921126 or
86-571-86843185; E-mail, [email protected]

Abstract        Gene therapy
and viral therapy for cancer have therapeutic effects, but there has been no
significant breakthrough in these two forms of therapy. Therefore, a new
strategy called “targeting gene-virotherapy”, which combines the advantages of
gene therapy and viral therapy, has been formulated. This new therapy has
stronger antitumor effects than either gene therapy or viral therapy. A
tumor-specific replicative adenovirus vector ZD55 (E1B55KD deleted Adv.) was
constructed and various single therapeutic genes were inserted into ZD55 to
form ZD55-gene. These are the targeting gene-virotherapy genes. But experiments
showed that a single gene was not effective in eliminating the tumor mass, and
therefore two genes were separately inserted into ZD55. This strategy is called
“targeting dual gene-virotherapy” (with PCT patent). Better results were
obtained with this strategy, and all the xenograft tumor masses were completely
eliminated in all mice when two suitable genes producing a synergetic or
compensative effect were chosen. Twenty-six papers on these strategies have
been published by researchers in our laboratory. Furthermore, an adenoviral
vector with two targeting promoters harboring two antitumor genes has been
constructed for cancer therapy. Promising results have been obtained with this
adenoviral vector and another patent has been applied for. This antitumor
strategy can be used to kill tumor cells completely with minimum damage to
normal cells.

Key words       
cancer; therapy; gene; targeting viral vector; targeting gene-virus

Malignant tumors are
becoming more and more common­ and they pose a significant threat to human
lives. There are conventional­ means to treat malignant tumors, such as
surgery, chemotherapy­ and radiotherapy. Unfortunately, these therapies are
ineffective for patients suffering from tumors­ in an advanced stage, so it is
necessary to develop a new strategy, such as gene therapy, for the prevention
and treatment­ of cancer. Gene therapy is a method of delivering­ therapeutic
genes into patients. In 1990,  

Construction of Tumor-conditional
Replicative Viral Vector

Viral vectors are
divided into two types. One type is able to integrate into the chromosome, such
as the retrovirus, adeno-associated virus (AAV) and lentivirus. The other type
of viral vector, such as the adenovirus, EB virus and HSV, is unable to
integrate into the chromosome­ and stays outside the DNA genome. We have been
conducting­ research into the adenovirus for years, as well as the AAV.

There are many ways to
construct tumor-specific replicative­ viral vectors. One way is to delete the
genes necessary for viral replication in normal cells, but which are
unnecessary for tumor cells, such as ONYX-015 [1,6]. E1B55KD is an early
protein of the adenovirus, which binds to p53 and inhibits its function [7].
p53 is the main anti-adenovirus protein in the host cells. p53 is immediately­
activated after adenovirus infection and then the infected cells are induced to
undergo apoptosis [8], which prevents­ the virus from spreading to neighboring
tissues. However, E1B55KD is not required for virus replication in
p53-deficient­ tumor cells as there is no p53 to be activated. Thus, the
E1B55KD-depleted adenovirus can not replicate in normal cells, but replicate in
large amounts in p53-deficient­ tumor cells. The replicated adenoviruse lyses
the tumor cells, releases more viruses to infect other tumor­ cells, and
finally eradicates the tumor [6]. We constructed the modified adenoviral vector
pZD55, with E1B55KD deleted, but EAnother way to construct
tumor-specific replicative viral vectors is to introduce a tumor-specific
promoter to control­ the essential elements of viral replication, such as ERecent studies have
shown that telomerase plays an important role in cell transformation,
tumorgenesis, eternalization and development [15,16]. Human telomerase reverse
transcriptase (hTERT) is the core component of telomerase and is the most
extensively used tumor marker as well. hTERT is not physiologically active in
normal cells, but is highly active in 85%–90% of human tumor cells. Moreover, the activity of hTERT
is correlated with malignancy, which implies that hTERT is a very good target­
for cancer therapy. hTERT expression is highly correlated­ with telomerase
activity and is the critical factor regulating­ telomerase activity in cells [17,18]. hTRER expression­
is mainly regulated at the transcription level and is up-regulated­ in 85%–90% of cancer cells, but not transcribed in
resting­ cells [19]. The hTERT promoter has been cloned by several­ researchers
[19,20] and was utilized to drive exogenous gene expression [21,22]. It can
also restrain the exogenous­ gene expression in cancer cells. We replaced the E 

Targeting Gene-Virotherapy

Targeting
gene-virotherapy has two advantages. The first advantage is that the viral
vector can replicate specifically in tumor cells and kill them accordingly. After
inserting the antitumor genes into ZD55 to form ZD55-genes, these genes will
specifically replicate and kill tumor cells at a rate 600–1000 folds higher than that in normal cells. The
second advantage is that exogenous gene expression increases­ several hundred-
to several thousand folds in parallel with viral replication. Our data revealed
that gene expression is time-dependent and increases with infection.

Different
gene-virotherapy genes can be created using ZD55 by inserting different
antitumor genes. The following­ antitumor genes are available: (1) the tumor
necrosis factor­-related apoptosis-inducing ligand (Trail) pro-apoptotic gene,
which induces apoptosis in tumor cells but not in normal cells; (2) Smac (the
second mitochondria-derived activator­ of caspase) gene, which blocks the
effect of IAP, inhibitor of apoptosis protein; (3) tumor suppressor genes, such
as p53, Rb, PTEN, LFIRE, LPTS, etc.; (4) immune regulator genes, such as IL-2,
IL-12 and IL-24; (5) angiogenesis inhibitory genes, such as angiostatin, k5,
endostatin, VEGI and sFlt1; (6) suicide genes, such as CD and TK; and (7)
anti-free radical genes, such as MnSOD, etc. (Table 2). We have inserted
many of the above antitumor­ genes into ZD55 to form ZD55-gene, respectively.
The therapeutic effect of this targeting gene-virotherapy is much higher than
ONYX-015 or ZD55 alone. The results of our research have been published in many
journals [25,27,30].

Our data on ZD55-IL-24 [Fig.
2(A,B)] have been published­ in Human Gene Therapy [30] and the
pharmacological and toxicilogical study of ZD55-IL-24 will start soon. The
reason of choosing this antitumor drug for clinical use is that in the  

Targeting Dual Gene-Virotherapy

Targeting
gene-virotherapy has been shown to successfully eradicate the transplanted
tumor in animal models in some individual cases, but can not eliminate all
tumors in vivo. In order to improve this targeting gene-virotherapy
strategy, we proposed another new strategy, targeting dual gene-virotherapy. In
this strategy, two genes are inserted into ZD55 or Ad TERT. The two genes have
a synergetic or complementary antitumor effect. It has been found that the
application of ZD55-Trail and Ad-k5 together completely­ eradicates all
transplanted colorectal cancers induced by SW620. The antitumor effect of Trail
is a result of apoptosis and that of k5 is a result of the anti-angiogenesis­
of tumor blood vessels, so these two proteins­ inhibit and kill tumor cells
through different mechanisms and have an synergistic effect. The research
results have been published­ in Molecular Therapy  [Fig. 3(A,B)] [26]. 

Recently, the antitumor
effect of ZD55-MnSOD was found to be about 1000 folds higher than that of
Ad-MnSOD. A new function of Trail was discovered whereby it induces the
expression of MnSOD by the induction of ZD55-Trail. Accordingly, by the
combined use of ZD55-Trail and ZD55-MnSOD, all colorectal xenograft tumors were
completely eliminated in a previous study [Fig. 4(B)] [31]. Moreover, a
new mechanism of H2O2 produced by MnSOD has been found to be
mediated by caspase 8 [Fig. 4(C)]. 

IAP is an inhibitor of
apoptosis. When the IAP level is high, cells will become cancerous easily.
Chemotherapy and Trail treatment are ineffective against high-IAP cancers.
BEL-7404 is a hepatic carcinoma cell line with a high level of IAP. This cell
line and its xenograft cancer are also unaffected by chemotherapy or Trail. But
Smac abolishes IAP activity. By using a combination of ZD55-Trail and
ZD55-Smac, it has been shown that all the BEL-7404 cells can be killed in
vitro, and all the transplanted liver cancers can be completely eradicated in
vivo (Fig. 5). As Smac can block the action of IAP and Trail can
induce Smac, they have a synergetic effect. This strategy can also be used as a
basis for the treatment of cancers with a high IAP level [28].

In summary, targeting
dual gene-virotherapy is a very good strategy for tumor biotherapy and has won
the Debiopharm-CCRF award in  

Double-Targeting Virus
Double-Gene Therapy

An ideal tumor-targeting
vector should be able to specifically­ target all tumor cells with minimum
toxicity to normal cells. ZD55 and Ad hTERT (E 

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