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Virus-based medicines

Production of vectors makes gene therapy advance, as an alternative treatmentfor diseases like brain cancer

EUGENIA COSTANZI-STRAUSS / ICB-USPGlioma before and after applying the virus: the way to the first clinical protocol for the gene therapy of cancer in BrazilEUGENIA COSTANZI-STRAUSS / ICB-USP

Brought into existence about ten years ago, gene therapy is a delicate frontier area that deals with the correction of defective genes connected with pathologies. On this front line, researchers from the Institute of Biomedical Sciences (ICB) of the University of São Paulo (USP) are drawing up a protocol for gene therapy of brain cancer. Under the coordination of Eugenia Costanzi-Strauss, they use the strategy of gene transfer mediated by a virus to induce the death of tumor cells. Some viruses have the ability to introduce genetic material into human cells: modified in the laboratory, these viruses are vectors of gene transfer, and serve to carry therapeutic genes. The group has already produced four systems of these vectors.

Outside Brazil, almost 600 protocols for gene therapy have now been planned or carried out, and they have served to help with the treatment of about 3,500 patients – 96% from the United States and Europe, but none from Latin America, which is stimulating the researchers. As to effective results, there is insufficient data for an analysis.”Besides being a very young methodology (the first protocol was published by the Science magazine in 1995), gene therapy is only applied in terminal cases, when all the traditional treatments have been used and failed”, says Eugenia.

Accordingly, few cases have resulted in a reduction in the size of the tumor, and few patients have survived longer, but there have been gains. “Some patients have reported that, after gene treatment, they have felt less pain and their quality of life has improved.” And gene therapy has had great success in another area: the treatment of serious immunodeficiency with retrovirus.

The challenge is to put the right gene in the right cell, and to make it express itself properly. According to Bryan Eric Strauss, an American who is Eugenia’s husband and the main collaborator of the group, everything depends on virus vectors being developed: “When a virus penetrates a cell, its genetic information is added to the cell’s genome. This is the natural process. Our intervention consists of manipulating the genetic information carried by the virus, and also the way it expresses itself in the cell”.

Strauss is working to perfect these vectors and now has a collection of viruses, which he intends to process: four families modified by using one he brought from the University of California, United States, plus another two families under development. “I believe that our manipulated viruses will be dealt with like medicines.” Important, for him, is that all this technology is being developed in Brazil, from the discovery of new genes and the production of the therapeutic viruses to drawing up protocols for therapy. Abroad, biotechnology companies are now producing a variety of virus. “Instead of importing them, Brazil can become a center for innovation and supply”.

Brain tumor
The group is working on gene therapy for the glioblastoma, a brain tumor with the worst of prognostics, since it does not respond to conventional treatments. The aim is to inhibit the tumor with suppressor genes: they control the cell cycle, preventing the disorderly reproduction of cells that characterizes cancer. Present in normal cells, these genes have been lost by tumor cells. The issue is to replace them with viral vectors. “We are using modified retroviruses (made up by RNA, ribonucleic acid), which are the vehicles capable of carrying the genes that interest us into the target cells, and once they are there, to promote their expression”, says Eugenia.

The researchers constructed recombinant viruses, carriers of genes that suppress the tumor, and demonstrated that there is no single suppressor gene capable of inhibiting all the cases of glioblastoma. They also built and cloned bicistronic retroviruses – able to transfer and to express two suppressor genes simultaneously, so inhibiting the proliferation of cells in a wide range of action. And they also established animal models, which showed that viruses are efficient vectors, capable of transferring and expressing genes in vivo. “Now that the foundations have been laid”, says the researcher, “we are starting to draft the first clinical protocol for the gene therapy of cancer in Brazil, and a painstaking methodology for the production of therapeutic viruses intended for human beings”.

To put the vector together, two molecular sequences or plasmids are combined: one loaded with the therapeutic gene or genes; another, with genes that give the set the characteristics of a virus. Both are coupled together by packaging cells with a high capacity for synthesizing protein. “When the plasmids are combined, the result is a totally manipulated virus, able to communicate the genes of interest to the target cells”, is how Strauss sums it up. “This virus is incapable of replication: it infects the cells just once, which guarantees that the process does not get out of control”.

In cancer, the genes of interest are p16, p21, p53 and pRb. Present in healthy organisms, they are frequently lost by the tumor tissue and have to be replaced. P53 and p16 are particularly important: the former causes cells to die, and the second induces their aging. Without the inhibiting action of both, cancerous cells grow uncontrollably.Infected by a bicistronic virus that carries the two genes, the cells receive a strong stimulus to stop growing, to die even, and they become vulnerable to radiotherapy. Important: this manipulated virus is now available in the arsenal of the Gene Transfer Laboratory of the ICB.

A cure for the heart
Another important contribution was to manipulate the LTR plasmid component – which sets off the expression of the DNA sequence that comes next, to prevent the viral vector expression from decaying. “To do so, we use the p53 gene itself, used to cause the death of tumor cells”, says Strauss. Using p53 to kill cancerous cells is now a common procedure. “The originality of our contribution lies in turning to the same gene to increase the expressiveness of the viral vector, an extremely complicated technology, all of it developed in Brazil”.

One of the partnerships that the group entered into was with José Cipolla Neto, also from the ICB, a specialist in the induction and removal of tumors from the brains of animals. Once the tumor is removed by surgery, the neighboring tissues are given an injection of suppressor genes to avoid recurrence, which is frequent with glioblastomata. On concluding his post-doctorate studies at the ICB, Strauss moved: he went to the team of José Eduardo Krieger, at USP’s Heart Institute (Incor), which is testing on animals an innovative procedure, for the cardiac cells to produce therapeutic proteins.

“Our interest is to develop viral vectors capable of making the cardiac cells express certain proteins – in particular, the IGFI, associated with growth, and the VEGF, linked to the formation of new vessels”, Krieger discloses. Once the cells are transformed into producers of these substances, someone who has had a heart attack or lost heart tissue can have an improvement in the remaining cells. The procedure is like the one used by the people from the ICB in cancer therapy, but with the opposite objective: “They need to kill cells, but we want to help cells to survive”. The method is already being tested on animals. The group removes muscle from rat skeletons, separates the stellar cells [M7] (which have no specific function), and transforms them in vitro into viral vectors, for them to produce proteins of interest to them. The material they obtain is injected into the guinea pig’s myocardium. A marker protein gives the changed cells a greenish blue color: accordingly, after the animal has been sacrificed, it can be seen, through histological cuts, how many cells have been successfully installed.

“We want to make a critical numbers of transformed cells ‘stick’  in place and remain there long enough to promote cell growth and to produce new vessels”, explains Krieger. “But it is not so simple: even if the original cells have been taken from the individual himself, they undergo rejection, for being infected with the viruses”.

The group from Incor went into a partnership with the ICB’s Biotechnology Center to produce therapeutic viruses of a pharmacological quality. Other groups in the country have projects in the area, with different strategies and models. “This is a field of research that is maturing in Brazil”, says Eugenia. These groups will soon be faced by the task of transferring technology to the patient’s hospital bed. “To meet this challenge”, he concludes, “all the groups need to validate their genetic tools and to produce them to a pharmacological quality, to make it feasible for clinical protocols to be drawn up”.

The project
Efficient Transfer of Control Genes from the Cell Cycle Mediated by a Perfected Retrovirus Platform of the PCL System, – Applications In Vitro and In Vivo (nº 98/15120-0); Modality Regular research benefit line; Coordinator Eugênia Costanzi-Strauss – Institute of Biomedical Sciences of the University of São Paulo; Investment R$ 317,928.41