Viruses are some of the most insidious creations in nature. They travel light: equipped with just their genetic material packed tightly inside a crystalline case of protein, they latch onto cells, insert their genes, and co-opt the cells’ gene-copying and protein-making machinery,using them to make billions of copies of themselves. Once formed, the new viruses percolate to the cell surface, pinch off inside minuscule bubbles of cell membrane and drift away, or else they continue reproducing until the cell finally bursts. In any case, they go on to infect and destroy other cells, resulting in diseases from AIDS to the common cold. Different viruses cause different diseases in part because each virus enters a cell by first attaching to a specific suction-cup like receptor on its surface. Liver cells display one kind of receptor used by one family of viruses, whereas nerve cells display an- other receptor used by a different viral family, so each type of virus infects a particular variety of cell. Cancer researchers have envied this selectivity for years: if they could only target cancer therapies to tumor cells and avoid damaging normal ones, they might be able to eliminate many of the noxious side effects of cancer treatment.
ADENOVIRUSES explode from a cancer cell that has been selectively infected in order to kill it. The viruses can spread to and wipe out other tumor cells.
Some scientists, including ourselves, are now genetically engineering a range of viruses that act as search-and-destroy missiles: selectively infecting and killing cancer cells while leaving healthy ones alone. This new strategy, called virotherapy, has shown promise in animal tests, and clinical trials involving human patients are now under way. Researchers are evaluating virotherapy alone and as a novel means for administering traditional chemotherapies solely to tumor cells. They are also developing methods to label viruses with radioactive or fluorescent tags in order to track the movement of the viral agents in patients.
Viruses to the Rescue?
ONE OF THE FIRST INKLINGS that viruses could be useful in combating cancer came in 1912, when an Italian gynecologist observed the regression of cervical cancer in a woman who was inoculated with a rabies vaccine made from a live, crippled form of the rabies virus. Physicians first injected viruses into cancer patients intentionally in the late 1940s, but only a handful appeared to benefit. Twenty years later scientists found that a virus that causes the veterinary disorder Newcastle disease shows a preference for infecting tumor cells and began to try to enhance that tendency by growing the viruses for generations in human cancer cells in laboratory culture dishes. Although critics countered that such viruses could be exerting only an indirect effect against cancer by generally activating an individual’s immune system and making it more likely to detect and kill cancer cells, reports continued to pop up in the medical literature linking viral infection and cancer remission. In the early 1970s and 1980s two groups of physicians described patients whose lymphomas shrank after they came down with measles.
The modern concept of virotherapy began in the late 1990s, when researchers led by Frank McCormick of ONYX Pharmaceuticals in Richmond, Calif., and Daniel R. Henderson of Calydon in Sunnyvale, Calif., independently published reports showing they could target virotherapy to human cancer cells grafted into mice, thereby eliminating the human tumors. (ONYX is no longer developing therapeutic viruses, and Calydon has been acquired by Cell Genesys in South San Francisco, Calif.)  Both groups used adenovirus, a cause of the common cold that has been intensively explored for virotherapy. (Other viruses under study include herpes simplex, parvovirus, vaccinia and reovirus.) Adenovirus is appealing in part because researchers understand its biology very well after years of trying to cure colds and of using the virus in molecular biology and gene therapy research. It consists of a 20-sided protein case, or capsid, filled with DNA and equipped with 12 protein “arms.” These protrusions have evolved over millennia to latch onto a cellular receptor whose normal function is to help cells adhere to one another. Adenoviruses are distinct from the types of viruses usually used in gene therapy to treat inherited disorders. Gene therapy traditionally employs retroviruses to splice a functioning copy of a gene permanently into the body of a patient in whom that gene has ceased to work properly. Unlike retroviruses, however, adenoviruses do not integrate their DNA into the genes of cells they infect; the genes they ferry into a cell usually work only for a while and then break down. Scientists have investigated adenoviruses extensively in gene therapy approaches to treat cancer, in which the viruses are armed with genes that, for example, make cancer cells more susceptible than normal ones to chemotherapy. In general, tests involving adenoviruses have been safe, but regrettably a volunteer died in 1999 after receiving an infusion of adenoviruses as part of a clinical trial to test a potential gene therapy for a genetic liver disorder.
Gene therapists have been working to tailor adenoviruses and other viral vectors, or gene-delivery systems, to improve their safety and reduce the chances that such a tragedy might occur again. It is perhaps even more essential for researchers, such as ourselves, who are investigating virotherapy to develop safer, more targeted vectors, because virotherapy by definition aims to kill the cells the viruses infect, not just insert a therapeutic gene into them. Killing the wrong cells could be dangerous. Adenoviruses bring with them characteristics that can make
VIROTHERAPISTS ARE DEVISING two main strategies to make sure their missiles hit their objectives accurately with no collateral damage. In the first approach, termed transductional targeting, researchers are attempting to adapt the viruses so that they preferentially infect, or transduce, cancer cells. The second method, called transcriptional targeting, involves altering the viruses so that their genes can be active, or transcribed, only in tumors
] Transductional targeting is particularly necessary because, unfortunately, adenoviruses bind more efficiently to the variety of normal tissues in the human body than they do to most tumor cells. We can reverse this pattern using specially generated adapter molecules made of antibodies that snap onto the arms of the virus like sockets on a socket wrench. By attaching carefully chosen antibodies or other molecules that selectively bind only to a specific protein found on tumor cells, we can render adenoviruses unable to infect any cells but cancerous ones. Once the antibody-bearing virus latches onto a targeted cell, the hapless cell engulfs it in a membrane sac and pulls it inside. As the sac disintegrates, the viral capsid travels to a pore in the cell’s nucleus and injects its own DNA. Soon the viral DNA directs the cell to make copies of the viral DNA, synthesize viral proteins and combine the two into billions of new adenoviruses. When the cell is full to capacity, the virus activates a “death gene” and prompts the cell to burst, releasing the new viruses to spread to other cells.
CELLS WITH VIRUSES TWO MAIN STRATEGIES are being explored for virotherapy, which is the technique of using reproducing viruses to kill tumors. In the first method, dubbed transductional targeting ( below ), scientists are attempting to engineer viruses such as adenovirus — which normally causes respiratory infections — to selectively infect and destroy only cells that have turned cancerous. They are attaching adapter molecules onto the viral outer coat proteins or directly modifying these proteins to try to prevent the viruses from entering normal cells and instead prompt them to home in 72 Adapter molecule on engineered adenovirus No infection or cell killing Receptor made only by tumor cells Targeted virus takes over cancer cell, making so many copies of itself that it kills the infected cell Cell bursts, and virus infects and kills other cancer cells Normal adenovirus receptor VIRAL DNA CELL DNA CELL DNA NORMAL CELL CANCER CELL Viral outer coat proteins on tumor cells.  The second approach ( below ) involves placing a snippet of DNA called a tumor-specific promoter next to one of adenovirus’s essential genes. The promoter acts as an “on” switch that permits the gene to function only in cancer cells. The engineered viruses can enter normal cells, but they cannot reproduce and kill them. Once they enter cancer cells, however, the tumor-specific promoter lets them make millions of copies of themselves and ultimately burst the cancer cells. They can then spread to — and destroy — other tumors. Tumor- specific promoter Promoter Infection occurs, but normal cell does not have switch to turn on viral gene. Virus cannot replicate or kill cell Cancer cell has switch to turn on viral replication genes Engineered adenovirus with tumor-specific promoter links to essential virus gene Cell bursts, and virus infects and kills other cancer cells
. Transcriptional targeting generally takes advantage of ge- netic switches (promoters) that dictate how often a given gene is functional (gives rise to the protein it encodes) in a particu- lar type of cell. Although each body cell contains the same en- cyclopedia of genetic information, some cells use different chap- ters of the encyclopedia more often than others in order to ful- fill their specialized tasks. Skin cells called melanocytes, for instance, must make much more of the pigment melanin than liver cells, which have little use for the protein. Accordingly, the promoter for the key enzyme for making melanin gets turned on in melanocytes but generally is off in most other body tis- sues. In the deadly skin cancer melanoma, the gene encoding this enzyme is fully functional, making the tumors appear black. We, and others, have engineered adenoviruses that have a promoter for the enzyme adjacent to genes that are essential for the viruses’ ability to replicate. Although these viruses might infect normal cells, such as liver cells, they can reproduce only inside melanocytes, which contain the special combination of proteins needed to turn on the promoter. Researchers are currently tailoring adenoviruses with a va- riety of promoters that limit their activity to particular organs or tissues. In liver cancers, for example, the promoter for the gene α -fetoprotein — which is normally shut down after fetal de- velopment — becomes reactivated. Adenoviruses containing that same promoter hold promise for eradicating liver tumors. Sci- entists led by Jonathan W. Simons at Johns Hopkins Universi- ty have tested the approach in men whose prostate cancer re- curred following treatment with radiation. The researchers used adenoviruses that had been engineered by Cell Genesys to contain the promoter for prostate-specific antigen, a protein made in abundance by prostate tumors. They administered the virotherapy to 20 men who received varying doses of the adenoviruses. In 2001 Simons and his colleagues reported that none of the men experienced serious side effects and that the tumors of the five men who received the highest doses of the vi- rotherapy shrank by at least 50 percent.
Other Strategies VIROTHERAPISTS MIGHT END UP combining the trans- ductional and transcriptional targeting strategies to ensure that the viruses kill only tumor cells and not normal ones. Adeno- viruses engineered to contain the promoter for the enzyme that makes melanin, for instance, can also replicate in normal melanocytes, so on their own they might cause spots of depig- mentation. And adenoviruses that are designed to bind to receptors on the surfaces of tumor cells can still invade a small proportion of healthy cells. But Is It Safe? Many approaches to virotherapy use adenoviruses, which caused a death in a clinical trial of genetherapy four years ago.
 IN SEPTEMBER 1999 18-year-old Jesse Gelsinger died after receiving an infusion of adenoviruses into his liver.
He had a mild form of an inherited liver disease called ornithine transcarbamylase deficiency (OTCD) and was participating in a clinical trial of a new gene therapy to use adenoviruses to ferry a corrected copy of the gene encoding OTCD into his liver cells.  Unfortunately, four days after an infusion of the viruses, he died of acute respiratory distress syndrome and multiple organ failure, apparently caused by an overwhelming immune reaction to the large dose of adenoviruses he had been administered as part of the trial.
 NOTE: Phase I tests are designed to evaluate safety in small numbers of patients. Phases II and III are intended to determine the appropriate dose and efficacy, respectively.
Although Gelsinger’s death was part of a gene therapy trial, the tragedy also has ramifications for the new field of virotherapy. Gene therapy uses crippled versions of viruses such as adenovirus to introduce a new gene into cells; virotherapy employs actively replicating viruses (which may or may not contain added genes) to kill specific types of cells. Both, however, rely heavily on adenoviruses. Gelsinger’s autopsy showed that the engineered adenoviruses had spread to his spleen, lymph nodes and bone marrow, and an examination of his records revealed that his liver function was probably too impaired for him to be a volunteer in the trial. A number of scientists have also suggested that he might have mounted such an extreme immune reaction because he had previously been infected with a naturally occurring adenovirus. Since Gelsinger’s death, gene therapists and virotherapists alike have focused on refining adenoviruses to make them safer. But researchers are still unsure why Gelsinger reacted so violently to the adenoviral infusions: a second patient participating in the same clinical trial tolerated a similar dose of the viruses. And dozens of other people worldwide have been treated so far with adenoviruses with no serious side effects.  A National Institutes of Health report generated in the aftermath of Gelsinger’s demise recommends that all participants in such clinical trials be monitored closely for toxic reactions before and after the infusion of therapeutic viruses.
It also stipulates that volunteers be screened for any predisposing conditions that would increase their sensitivity for the viruses. Fail-safe mechanisms would be expected to be less likely to harm normal cells.
There are no results at present, however, to demonstrate that a combination of approaches makes virus- es more targeted. A further strategy for targeting virotherapy makes the most of one of cancer’s hallmarks: the ability of tumor cells to divide again and again in an uncontrolled manner. Healthy cells make proteins that serve as natural brakes on cell division—notably, the retinoblastoma (Rb) and p53 proteins. As cells turn cancerous, however, the genes that code for one or the other of these proteins become mutated or otherwise inactivated. Certain viruses, including adenovirus, interfere with the braking mechanisms of a normal cell by making proteins that stick to and inactivate Rb or p53. They do this because they can replicate only in cells that are preparing to divide. Several research groups and biotechnology companies have engineered adenoviruses that fail to make the Rb or p53 blockers. Normal cells, which make these blockers, will stall the replication of these viruses by putting the brake on cell division. But these viruses will replicate in cells in which the Rb or p53 proteins are already disabled— cancer cells and kill them.  OncoVex attacks tumors in two ways. It features a modified cold sore
virus that replicates inside solid tumors, causing cancer cells to die.
 Secondly, the drug prompts the immune system to take out cancer cells.
In May 2009, BioVex impressed with Phase II data on the drug in melanoma
patients, showing that of 13 patients who had significant responses to
the treatment, nine had signs of the cancer completely wiped out.
Curiel is planning clinical trials of the approach for ovarian cancer.
Researchers are also arming therapeutic viruses with genes that make the cells they infect uniquely susceptible to chemotherapy. The technique involves splicing into the viruses genes that encode enzymes that turn nontoxic precursors, or “prodrugs,” into noxious chemotherapies. In one example, which was reported in 2002, André Lieber of the University of Washington and his co-workers designed adenoviruses to carry genes encoding the enzymes capable of converting innocuous prodrugs into the anticancer compounds camptothecin and 5-fluorouracil. The scientists engineered the viruses so that they could make the enzymes only in actively dividing cells, such as cancer cells. When they injected the viruses and the prodrugs into mice bearing implanted human colon or cervical cancer cells, they found that the viruses reproduced and spread in the tumors. Such “smart” virotherapies are the vanguard of the future. But physicians will also need to track the activity of virotherapies in a patient’s body to best assess how well the strategies are working and refine them further. Virotherapists are now teaming with radiologists to establish novel imaging technologies to easily measure how effectively a given virotherapy is replicating. The imaging strategies involve inserting a gene that governs the production of a tracer molecule into a virus or virus-infected cell. The tracer can be either a fluorescent protein that can be observed directly or one that binds readily to the radionuclides used in standard radiological imaging techniques. The fluorescent protein might work best for cancers that are accessible by an endoscope, such as cancers of the larynx. Physicians could peer into the endoscope and see exactly where the viruses and therefore, cancer cells are by looking for fluorescence. So far the approach has worked best with viruses that do not kill cells, however. Nevertheless, we are convinced that such sophisticated imaging technologies will enable scientists to draw more meaningful conclusions from future clinical trials of virotherapy. In 1995 gene therapy pioneer W. French Anderson of the University Of Southern California School Of Medicine predicted in this magazine that “by 2000 . . . early versions of injectable vectors that target specific cells will be in clinical trials.” These trials indeed began on schedule, as well as some he could not have envisioned then. We envision a substantial role for viruses. That is, therapeutic viruses in 21st-century medicine.