Special purpose virus

Elena Trukhina, "Science in Siberia" Photos provided by Anastasia Yunusova

In the numerous kingdom of viruses, there is an absolutely outstanding representative – the smallpox vaccine virus, which is part of the orthopoxvirus family. He played a major role in ridding humanity of smallpox, which has claimed millions of lives since ancient times. Now this disease has been defeated all over the globe, including thanks to our hero. But it is clearly too early for him to "retire": scientists have high hopes for him in the field of cancer treatment.

Anastasia Yunusova, PhD student at the Institute of Chemical Biology and Fundamental Medicine SB RAS (Microscopic Research Group) says:

– Our laboratory conducts research on infectious agents that can fight cancer together with colleagues from the Vector World Health Center.

Due to their specificity – the ability to multiply in tumor cells – the idea that they can be used in the treatment of oncological diseases appeared quite a long time ago. When the oncolytic abilities of infectious agents were discovered (for the first time – in one of the adenoviruses), researchers began to study many other viruses and found that there was a whole panel of similar objects.

Oncolytic viruses are a collection of different viruses that are not pathogenic to humans, to normal tissues in their body, but they multiply and cause the death of rapidly dividing tumor cells.

– I am dealing with the smallpox vaccine virus, – Anastasia Yunusova continues. – What is it good for? At one time, when there was a smallpox eradication program, it was the smallpox vaccine virus that was used for vaccination. Accordingly, it is now well known how this agent behaves in the human body, and there is an array of knowledge that will eliminate side effects from therapy, if they occur. In addition, it has a natural ability: it is "more willing" to multiply in tumor cells. The basic experiment shows that if you take different cells – a culture of normal and a panel of cancer cells – and add a suspension of the virus, then it will multiply more actively in tumor cells. Why there is such selectivity is being investigated, the mechanism of this very important phenomenon is still unknown to scientists.

An important condition for research is that scientists need a live virus, but the original strain, as it is, is not suitable for therapy, it must be weakened, which is achieved by molecular biology methods due to deletion (that is, removal) of some genes from the virus genome. Firstly, it reduces the likelihood that the agent will multiply in normal cells as well. Secondly, its specificity to tumor cells increases.

― For example, one of the recombinants that we use in the study is the smallpox vaccine virus, which does not have a gene responsible for the production of thymidine kinase (an enzyme that plays a key role in DNA synthesis), – Anastasia notes. – This enzyme is necessary for virus replication at the moment when DNA doubling occurs. The smallpox vaccine virus is self–sufficient, it is very complicated, it has its own proteins, including thymidine kinase. Accordingly, if the virus has this gene, when it enters the cell, it does not depend on the proteins that it contains. When the virus does not have its own thymidine kinase, it uses a cellular protein for replication. Since tumor cells divide rapidly, this enzyme is always in excess in their cytoplasm, and in normal ones it appears cyclically. Accordingly, when the gene encoding this enzyme is removed from the virus, it is naturally preferable that it will multiply in tumor cells. The multiplication of the virus in the cell leads to its destruction, thus the tumor cell dies.

In addition to weakening the pathogenic properties of the virus by removing genes, it is possible, on the contrary, to insert certain genes into the genome of the virus so that, while it replicates, it expresses molecules that affect tumor cells. For example, there are a number of proteins that trigger apoptosis (cellular "suicide") in the cell. It is believed that this process is the most preferable way of cell death for use in therapy, since the inflammatory process does not develop at the same time. The essence of apoptosis is that a cascade of reactions is triggered in the cell, as a result of which enzymes are activated that specifically begin to destroy DNA and proteins in the cell.

From the point of view of morphology, the nucleus is compacted in cells, the cell is fragmented, that is, it is destroyed, but all its components remain enclosed in membranes. The contents of the cytoplasm do not come out – this is usually what causes an inflammatory reaction – and in the case of apoptosis, the cellular "remains" are tightly packed. These apoptotic corpuscles are then absorbed either by neighboring cells or macrophages. It turns out a "clean" cleaning of the fabric.

Since modern technologies allow this to be done, the idea arose to genetically modify the virus in such a way that its genome contains a gene for a protein that triggers apoptosis. The virus, after penetration into the cytoplasm, will replicate, while a protein that triggers apoptosis will be synthesized in the cytoplasm of the cell. And, in addition to the multiplying virus, a protein will also act in the cell, which induces apoptosis and causes the death of the tumor cell.

– The protein expressed by the smallpox vaccine virus in our studies is called apoptin, – says Anastasia Yunusova. – Many studies confirm its ability to trigger apoptosis in tumor cells, the mechanism of its action is known. We studied the effect of the virus producing apoptin on mice that were vaccinated with a human tumor, and we were convinced that a virus expressing apoptin, compared to the fact that it does not have such a property, but also has an oncolytic effect, is preferable, since it leads to the destruction of tumor cells and to the "shrinkage" of the tumor.

Interestingly, this effect was not associated with apoptosis of tumor cells. The explanation of this phenomenon was found using electron microscopy and immunohistochemistry (detection of proteins in cells at the microscopic level). The protein apoptin, in order to start apoptosis, must be in the nucleus at some point.

― Our study also showed that apoptin did not penetrate into the nucleus, remaining in the cytoplasm, – Anastasia explains. – A phenomenon was observed: the tumor shrinks, the cells die, and apoptosis does not occur. We have been thinking for a long time, what explains this: judging by the observations, the reproduction of the virus in the cell is ahead of the action of the apoptin protein, and the cells die from the reproduction of the infectious agent, apoptin "does not have time" to start the process of apoptosis. However, apoptin, being in the cytoplasm, modifies cell death in such a way that, roughly speaking, it is mummified. Apparently, the protein binds to the components of the cytoskeleton, often with filaments (intracellular cytoplasmic fibrillar protein structures), and in an electron microscope we observed that yes, the cell died, but the cytoplasm inside it is filled with filaments and is a kind of "mummy". That is, apoptosis did not occur, but a variant of cell death developed in which there is no inflammation and edema.

– For comparison, we had two groups, – explains the researcher. – In the first, mice that were vaccinated with a human tumor were treated with a non–recombinant virus, and in the second with a virus expressing apoptin. In the first experimental animals, tumors were destroyed, and at a very rapid pace, but cellular detritus remained at the site of the malignant neoplasm, traces of edema and cavities filled with fluid were observed. The second rodents had no such effects, the tumor "shrank". With the help of a control group in which saline solution was injected into mice with a vaccinated tumor instead of a viral suspension, we observed the size dynamics: during the experiment, the tumor increased significantly, whereas in experimental groups of rodents, the tumor size was significantly smaller. On the 55th day, in mice injected with the recombinant virus, the tumor practically disappeared, a small scar remained in its place. In comparison with tumor nodes in mice of the control group, this result is significant and promising.

Despite the impressive results of the experiment, the researchers plan to proceed to preclinical trials only after they fully understand all the mechanisms of action of the virus expressing apoptin.

– In our experiments, special mice are used – "nudes", whose immunity is suppressed, which allows them to instill a human tumor under their skin, – explains Anastasia Yunusova. – Accordingly, this is a very approximate model. In experimental animals, a malignant neoplasm is convenient to observe, palpate and inject a drug into it. The treatment consists in delivering the medicine directly to the tumor and analyzing its effect. In order to move on to clinical and preclinical trials in the future, a lot more experiments need to be done: to administer the drug parenterally (bypassing the gastrointestinal tract, through the skin, muscles, blood vessels, etc.), you need to use experimental animals with "normal" immunity and different variants of tumors to see how the immune system reacts the system for the introduction of the drug and the destruction of the neoplasm. That is, we still have very long studies ahead of us.

Studies of oncolytic viruses are conducted in various countries, and clinical trials of a number of drugs (rigvir and reolysin) based on them are being conducted in the USA. Currently, it is obvious that different viruses "prefer" different types of tumors, which means that it is impossible to create a universal virus that would treat all cancers. Most likely, in the future there will be a certain set of drugs based on various viruses that "specialize" in certain types of malignant neoplasms, which can become an effective tool for oncologists.

Portal "Eternal youth" http://vechnayamolodost.ru  28.10.2016