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From slave to master

RNA molecule takes over the place of DNA as a promise for fighting diseases

CÁRCAMOEach one of our cells keeps the recipe for its functioning in the DNA molecule, the double strand of deoxyribonucleic acid in the form of a spiral staircase. For them to work, though, the cells depend on the action of a family of molecules that are simpler and more versatile, generally made up of a single strand of ribonucleic acid, RNA. At every moment, a specific type of RNA called messenger copies the instructions contained in the genes and sends them to the place where they will be read to originate proteins, the fundamental components of living beings. It is a more complicated task than it seems, since the route covered by the messenger is full of obstacles and traps. Like Captain Gulliver, dominated by the tiny inhabitants of Lilliput, in the novel by Jonathan Swift, the messengers are frequently intercepted and gagged, sometimes even dismembered, by another variety of molecules, even smaller: the micro-RNAs, which act associated with a complex of proteins. The recipe for producing micro-RNAs lies in portions of the DNA that until recently were thought to have no function – thus known as junk DNA. “Junk that is de luxe”, explains molecular biologist Carlos Menck, from the University of São Paulo (USP), who estimates that between 30% and 40% of the human genome is dedicated exclusively to producing RNA with the essential function of regulating almost everything that happens in the cells. In experiments with the Caenorhabditis elegans worm, used as a biological model for more complex living beings, the American researchers Andrew Fire and Craig Mello demonstrated in 1998 that small injected molecules of RNA would block, with efficiency, the interpretation of certain cell commands – a process that they baptized as RNA interference. Put simply, the RNA would silence the genes, preventing the production of proteins.

The work of Fire and Mello earned the pair of them the 2006 Nobel Prize in Medicine and revealed to geneticists and biologists a new strategy of taking over the cell command and thus try to combat, in an efficient and definitive way, problems of a genetic origin, like cancer. Using micro-RNAs as a mold, researchers from Europe and from the United States recently started to produce in the laboratory RNA molecules specifically designed to interfere in the functioning of certain genes. These artificially created molecules are the so-called interference RNAs, or simply RNAi, which, like DNA, are made up of a double strand and not a single one. Just as the micro-RNAs, the RNAi intercept and destroy the cell information before they are processed and originate proteins. With the assistance of this tool, teams from USP and from the State University of Campinas (Unicamp) are beginning to understand better how certain forms of epilepsy, cancer and cardiovascular ailments arise. They are also taking the first steps to ascertain the potential of these molecules for controlling these health problems, besides others caused by viruses, bacteria, protozoa and worms.

“RNAi has a great therapeutic potential”, explains geneticist Iscia Lopes-Cendes, from the School of Medical Sciences at Unicamp. Tests recently concluded in her laboratory indicate that RNAi may help to fight infestation by Schistosoma mansoni, the worm that causes schistosomiasis, a problem that affects about 200 million people in the world. Iscia’s team prepared copies of RNA to deactivate a gene that is essential for the metabolism of S. mansoni and applied them on mice infested with the worm. In six days, the number of parasites in the mice was 27% lower, a reduction that the researchers consider promising as a starting point. Despite the hopeful result, the geneticist shows caution. “It still be proved that the worms really died as a direct consequence of the inhibition by RNAi”, she explains. For this reason, she is now investigating the effects of the RNAi applied directly on the parasites in isolation, kept on glass slides. Besides being effective, this technique promises fewer unwanted effects in the cases in which the intention is to fight invading microorganisms like viruses, bacteria, protozoa or worms, because it is possible to design an RNA molecule exclusive to the genes of the parasite, without anything corresponding to it in human beings.

Before the experiments, though, they had to learn how to make the copies of RNAi. While studying for the doctorate he concluded in 2005, in Iscia’s group, biochemist Tiago Pereira developed a computer program capable of designing RNAi molecules to measure. It was quite an advance for the group from Unicamp. Available free of charge from the laboratory’s website on the Internet, the program created by Pereira now makes it possible for Brazilian researchers interested in using RNAi no longer to depend exclusively on foreign companies. Anyone who intends to silence a specific gene can use Unicamp’s program to design the desired RNAi and send the specific sequence of this molecule to international companies specialized in producing large quantities of them.

Controlling diseases
Some of the RNAs designed by Iscia’s team have now arrived at the laboratory of cardiologist Kleber Franchini, in another building of the School of Medical Sciences at Unicamp. Intrigued with the increase in the heart caused by arterial hypertension, Franchini ordered RNA molecules to interfere in cell processes that lead to the growth and deterioration of the heart in patients with arterial hypertension, besides other cardiac diseases. Right away on the first day after injecting RNAi into the mice with hypertension, he observed a 70% fall in the levels of some proteins that regulate cell division, which prevented the functional problems that arise from the increase of the heart. Better still, this effect lasted 15 days, suggesting that RNAi may, in future, become a way of preventing the exaggerated growth of the cardiac muscle, which in extreme cases impairs the pumping of the blood to the organism and can lead to death.

In São Paulo, at the Heart Institute (InCor), biologist Luciana Vasques uses this technique with the objective of solving a problem arising from an operation that is part of the routine for cardiologists: bypass vein grafts, to replace obstructed arteries of the heart by lengths of the saphenous vein, removed from the thigh. Transplanted to the heart, the vein may react to the new environment by bringing about the multiplication of muscle cells in its walls, which become thicker and may impair the passage of the blood. To inhibit this thickening, Luciana tested RNAi molecules capable of deactivating a gene involved in the proliferation of cells in blood vessels. The treatment reduced by 70% the multiplication of cells in rats in vitro. Luciana is now trying to discover the effects of this therapy on live rats.

CÁRCAMO From the problem to the solution
By preventing the functioning of the genes, RNAi does more than deal with a problem. It can also reveal its origin. At Unicamp, Iscia has been using RNAi to understand how epilepsy arises. She silenced genes activated at different stages of the cerebral development of mice and found that different forms of epilepsy originate at specific stages of life. Wilson Araújo da Silva, a biologist from USP in Ribeirão Preto, is trying to identify how the mechanism of regulation by RNA activates or switches off genes at unsuitable moments and so lead to the emergence of different types of cancer. Currently with the Ludwig Institute at Memorial Sloan-Kettering Cancer Center, in New York, Silva designs RNAi molecules to silence genes associated with tumors like those of the breasts, lungs and skin. “The silencing of genes will probably not replace surgical procedures, but it will make it possible to retard the development of certain types of cancer”, comments Silva.

Although years of research are necessary before silencing is available for people, the results obtained already make it possible to classify RNAi as the great promise of genetics for curing diseases. It is a position that years ago had already been occupied by gene therapy, which was trying to replace defective genes by other, healthy, ones, but has still not worked as was expected. Biologists and geneticists are betting on RNAi for two reasons: it is a cheaper technique than gene therapy, and, in the experiments already carried out, silences as many as 90% of the genes chosen as a target. If it does in fact work out, the silencing of genes may mean an injection of prestige into the Human Genome Project. Regarded as one of the great investments of science at the end of the last century, the sequencing of human genes generated a certain disappointment for not producing an immediate impact in the medical area. Today, it is possible to delve into a person’s genome and identify genes that indicate a propensity to diseases.

The optimism that surrounds the therapeutic potential of RNAi can be measured by the investment of the industry, which finances between 30% and 40% of Franchini’s work at Unicamp. Over 30 pharmaceutical and biotechnology companies are now seeking to use these molecules in medicine. One example is Sirna Therapeutics, created to develop treatments based on RNAi and in December 2006 bought by Merck, one of the world giants of the pharmaceutical industry. In the word’s of Sirna’s CEO, the company intends to be “ready to change modern medicine, potentially to stop diseases before they can progress and, in some cases, to turn around the very process of the disease”. Sirna intends to deal like this with any human disease – perhaps an exaggeration of optimism, but for some diseases RNAi has in fact proved to be effective in clinical tests.

Knots to be untied
Before this promise becomes a reality, though, several knots remain to be untied. The most important is to determine whether RNAi really is safe for human beings. Just as it may interrupt the functioning of genes associated with diseases, silencing is also capable of affecting other genes responsible for important functions of the cells, such as controlling their proliferation – a disorder that may give rise to cancer. Another difficulty is to make the RNAi molecules hit the right target, since once injected into the bloodstream they are usually dispersed through the organism before concentrating in the kidneys, from where they are excreted without having hit their target. They are trying to get round this problem by indicating the use of therapeutic RNA for diseases that can be treated by means of localized applications, such as injections made directly into the eye to fight macular degeneration of the retina, the use of sprays against asthma, or applications of vaginal creams against infections.

All over the world, ways are still being sought for increasing the stability and the durability of the RNAi in the organism. Often, the effects that it produces are still ephemeral – these molecules do not multiply inside the cells and can be degraded by enzymes specific to RNA. One strategy adopted by some researchers, like Luciana, from InCor, is to insert them into the genetic material of a virus that is innocuous for human beings. These viruses invade the cells that they hit and insert their genetic material into the genome of the host. These cells now begin to make copies of the therapeutic RNA, along with the copies of their own genes, with a possible permanent effect. Others, like Iscia, from Unicamp, prefer to make localized applications of molecules bare of RNAi, with small alterations that increase their stability.

“One of our concerns is to find out what happens in the organism in the medium and long term”, Iscia says. Despite these knots, common to any area of science at the initial stage of development, geneticists are showing their optimism and believe that, shortly, sufficient will be known about the functioning of RNAi to overcome these difficulties. “The science of RNAi is just beginning; for this reason, research projects in the area have to be induced”, explains Carlos Menck, who coordinates one of the four laboratories that make up USP’s Gene Therapy and Vaccine Center, founded about three years ago. Until this occurs, he is acting on his own account and trying to round up his colleagues, who are still working on the same theme, though in an isolated manner. “The silencing of genes with RNAi may give the center real unity, since it lends itself to the inquiries of all its researchers”, he says. Attitudes like this may perhaps succeed in making Gulliver arise and take control over the Lilliputians.