NEW ORLEANSA mutant her-pesvirus has been shown, in vitro and
in vivo, to be highly oncolytic against colon carcinoma cells in
liver metastases. On-colysis induced by the replication of this
virus, combined with cyclophosphamide prodrug activation, appears to
hold therapeutic promise in this setting, according to research
presented at the Society of Surgical Oncology Cancer Symposium.
Timothy Pawlik, MD, MPH, a resident at the University of Michigan,
Ann Arbor, presented research he is conducting with Kenneth Tanabe,
MD, of Massachusetts General Hospital, Boston.
A Replicating Vector
Most gene therapy approaches have used viral vectors modified so that
they cannot replicate in humans. But this study used a virus that can
replicate in humans, specifically, herpes simplex virus type 1
(HSV-1) The investigators chose HSV-1 because it has several
properties that render it useful as a vector for cancer gene therapy,
Dr. Pawlik explained.
First, HSV replication itself is cytotoxic to cells, including tumor
cells. Progeny virion released from the cells can then infect
adjacent tumor cells. The cytotoxic effect of the HSV vector is
present even at an extremely low multiplicity of infection (MOI).
Further, he said, the HSV vectors can carry a great number of
transgenes in sequence, making it possible to deliver multiple
therapeutic transgenes to enhance therapeutic efficacy.
The HSV mutant, known as rRp450, expresses the cytochrome rRp450
trans-gene. The p450 component bioactivates the cancer prodrug
cyclophosphamide. As an HSV-1 vector, rRp450 also ex-presses viral
thymidine kinase, which bioactivates the cancer prodrug ganci-clovir (Cytovene).
In Vitro Studies
The virus cytotoxic efficacy was assessed in vitro by measuring
survival of a panel of colon carcinoma cell lines after exposure to
an increasing viral MOI. Even at a low MOI, rRp450 replication
effectively destroyed all colon carcinoma cell lines.
Cell lines were almost completely destroyed when as little as 1
viral unit per 100 tumor cells was added, Dr. Pawlik reported.
To examine the effects of the prodrugs on rRp450-mediated
cytotoxicity, the investigators infected each carcinoma cell line
with rRp450 using an MOI that would cause 50% cell destruction in
that cell line. Infected cells were then incubated in the presence of
the prodrugs, cyclophosphamide or ganciclovir, or a control.
Cyclophosphamide requires p450 to be activated to its cytotoxic
metabolite; therefore, it had no effect on cancer cell lines by
itself, but when it was added to the rRp450-infected cells, it
produced a 20% to 30% increase in cell destruction over the rRp450
When rRp450-infected colon carcinoma cells were in the presence
of cyclophosphamide, cell destruction was nearly complete, Dr.
Effects of Ganciclovir
In contrast, when ganciclovir was added to rRp450-infected carcinoma
cells, it actually diminished cell destruction.
These results indicate that these two prodrugs differ substantially
in their ability to potentiate rRp450-mediated oncolysis.
Cyclophosphamide enhances cell destruction, while ganciclovir reduces
the cytotoxic effects of this viral vector.
Further experiments showed that this difference in oncolysis of colon
carcinoma was probably due to a difference in the drugs effects
on viral replication. Ganciclovir significantly decreases viral
yield, and since viral replication produces tumor cell destruction,
ganciclovir reduces rRp450s oncolytic effects.
Experiments also showed that rRp450 has preferential replication in
colon carcinoma vs normal hepatocytes.
In a murine model of experimental colon cancer metastases, a single
injection of rRp450 into the portal vein effectively treated diffuse
liver tumors. Mock-infected animals had abdominal distension, bloody
ascites, innumerable tumor nodules, and livers that weighed twice
that of the treated animals. All rRp450-treated animals, in contrast,
appeared healthy, showing less than 10 tumor nodules per specimen.
The addition of cyclophosphamide to the rRp450 treatment further
reduced the number of tumors per liver specimen, Dr. Pawlik reported.
He concluded that the HSV-1 rRp450 mutant is highly oncolytic, with
viral replication able to destroy colon carcinoma cells. It
selectively replicates in colon
cancer cells vs normal hepatocytes; it bioactivates cyclophosphamide,
which augments its antineoplastic effects; and it significantly
reduces liver metastases via a portal venous dose.