04 June 2009

RNA interference: Silencing genes

Viola Brick, Telegraph "Around the World": Shut up, gene, shut up!Control over the work of genes in the body is quite possible.
RNA molecules may simply leave them no room for expression.For many decades, DNA was considered the main "molecule of life".

And this generally correct opinion somewhat obscured the important role that another similar molecule, RNA, plays in a living organism. It was believed that in order to treat genetic diseases, it was necessary to make changes to the genes themselves, that is, to the structure of DNA.

The situation began to change with some important discoveries of the late XX – early XXI centuries, awarded the 2007 Nobel Prize. The idea turned out to be quite simple: if DNA contains "harmful" information that is difficult or impossible to correct, then this information can simply not be noticed. To do this, you only need to "adjust" the molecule that reads it accordingly – and this is RNA.

Attempts to implement such a "lightweight" program are now being made by various pharmaceutical companies. And although the difficulties they have already encountered are still much more numerous than the successes they can boast of, the results of their research inspire optimism.

Silence is goldA properly constructed interfering fragment of RNA can literally "turn off" an unnecessary gene and thereby stop the development of the disease at the earliest stage.

Interfering RNAs were called siRNA (Small interfering RNA), and in 2006 the Nobel Prize in Physiology or Medicine was awarded for the discovery of this phenomenon.

The first RNA-based drug was approved for clinical use back in 1998. This is fomivirzen (trade name "Vitraven"). Today, the drug is actively used to combat cytomegalovirus infection in patients with reduced immunity, including AIDS patients. Vitraven binds to one of the key genes of cytomegalovirus, stopping its expression, and suppresses the reproduction of the virus.

The first victory was followed by clinical trials of other drugs. So, in 2006, 8 years after the discovery of the phenomenon of RNA interference, the American pharmaceutical company Sirna Therapeutics began testing a new drug aimed at curing age-related blindness, the so–called age-related macular degeneration of the retina. The disease is caused by uncontrolled growth of blood vessels in the central part of the retina and leads to vision loss. Every year in the world, this diagnosis is made by more than half a million people, and these are only registered cases.

(Approximately as in the picture below, a person suffering from age-related macular degeneration of the retina sees a scene with boys. Photo: National Eye Institute, National Institutes of Health.)

However, the drug that the company tested was not approved. The attempt made by the American company OpkoHealth was also unsuccessful. The drug they created passed phase II of clinical trials, but a nonspecific effect was shown for it: the drug acted not only on protein synthesis, but also caused an immune response. To date, only one drug based on interfering RNA has been approved for the treatment of age–related blindness - "Makugen". It is injected into the eyeball, so that the drug acts locally, where its action is necessary.

The eyeball is not the only "convenient" organ for targeted pharmaceutical exposure. Alnylam Pharmaceuticals has started phase III clinical trials of a drug with interfering RNA, which is supposed to be used in the form of an aerosol or an inhaler. The target is epithelial cells in the lungs. The effect of the new drug is intended for the treatment of syncytial viral infection, which is especially common in infants and young children.

Another example of an ailment that they are trying to fight with the help of RNA interference is Huntington's disease. She is engaged in Phillip Zamore (Phillip Zamore), professor of the Faculty of Medicine at the University of Massachusetts Medical School. Huntington's disease is a genetically determined degeneration of the nervous system. The first symptoms appear in 35-50 years, when the structure of the huntingtin protein is disrupted in nerve cells. The physiological function of this protein is unknown, but in sick people, the shape of the structure of its molecule is disrupted, and it becomes toxic to nerve cells. In experiments, Zamore showed that injecting mice with siRNA, which stops the expression of the wrong protein, relieves the symptoms of the disease.

One of the difficulties with the development of drugs with RNA interference is that although the interfering RNA blocks the reading of a particular gene, it occurs throughout the body, in the cells of all organs. Each cell of the human body carries genetic information, which is enough to develop a new organism from scratch. But the formation of specific organs and tissues depends on which genes work in a particular place and with what force the genetic information is read. To get a good therapeutic effect, it is necessary that siRNA acts on a specific gene in a specific organ. In the meantime, it affects the reading of the gene throughout the body, therefore it affects many organs and tissues, having a side effect.

In addition, many diseases require systemic intervention. If we are talking about developing a medicine for the eyes or lungs, then everything is simple here: the medicine can be dropped into the eye or inhaled into the pulmonary pathways. To defeat, for example, cancer metastases or hypertension (which are not localized in themselves), a systemic injection of the drug is required – an injection into the blood so that the drug reaches all organs and tissues. But if we inject something into the bloodstream, then the blood carries it all over the body, does not keep it inside the vessels, the medicine penetrates into the capillaries and through their permeable walls can end up in the organs and tissues of the body. Here, the effect on the reading of the gene is undesirable, so a side effect is obtained.

Not so long ago, Alnylam Pharmaceuticals announced that it had received permission to test interfering RNA in humans for the treatment of hypertension and tumor processes in the liver. Preliminary tests on mice and monkeys allow us to hope that the systemic administration of siRNA can be effective without undesirable side effects.

When using RNA interference, it is very important to establish whether the disease has polymorphic gene regions. The fact is that the same gene may "sound" differently in different people, the sequence of the building blocks of DNA - nucleotides – may differ (although in the end the gene performs the same function for everyone). Such polymorphism, individual small variations in the DNA composition of a gene, is a real "headache" for doctors, because siRNA was created to recognize and affect a strictly defined sequence of nucleotides, and when polymorphism appears, siRNA may not recognize its target, and the drug will be ineffective. In this case, it remains only to do an individual genetic analysis for each patient and synthesize a unique drug based on it. But it's very expensive.
Not siRNA Unified…

In addition to siRNA, scientists are attracted to another class – microRNAs, small inhibitory RNAs (miRNAs). To date, more than eight thousand microRNAs are known, which by themselves do not carry information about the gene, but regulate its expression (reading genetic information) and the production of biologically active substances. The main difficulty is to establish the exact function of a particular microRNA.

microRNA can bind to the site where gene expression begins, and then expression will become impossible. As a result, the protein that is encoded by this gene will cease to be synthesized. For example, if microRNA blocks the reading of the gene responsible for the body's production of the hormone insulin, then theoretically it is possible to synthesize and introduce into the body a short RNA molecule that contains complementary nucleotides. Such complementary nucleotides bind to microRNA nucleotides. microRNA cannot sit on the gene, and accordingly, it does not block the gene's expression, insulin production is revived and theoretically a person is cured of diabetes.

The first results are promising. So, it became known that miR214 is responsible for the production of insulin by the pancreas. According to the work of Professor Guy Rutter from the Faculty of Medicine of Imperial College London, he managed to inactivate microRNAs with the help of small synthetic molecules to increase the production of insulin in the body.

In December 2008, the pharmaceutical company Regulus Therapeutics announced successful animal trials of synthetic molecules blocking miR-21, which is present in large quantities in the heart. Blocking miR-21 prevents heart attacks in laboratory mice. miR-122, which is necessary for the reproduction of the hepatitis C virus, has also become the subject of close study of this company.

Finally, the newest and most controversial approach is the use of short double–stranded antigen RNAs. Unlike traditional siRNAs, antigen RNAs do not act on informational RNA, but directly on DNA, on gene promoters (these are DNA regions that regulate the activity of adjacent genes). The detailed mechanism of this action is still being studied, however, it is assumed that the antigenRNA binds to a part of the gene that regulates transcription, that is, the transfer of genetic information from DNA to RNA. After that, the antigen RNA attracts special proteins. As a result, a large complex of RNA and proteins is formed on the gene site, which prevents the normal functioning of the gene. Given that many promoters are inhibited under normal conditions and keep the expression of the gene in a suppressed state, the use of antigenRNA can drown out the effect of the promoter and liberate the expression of the desired gene. To date, such a hypothetical pathway remains almost the only one to enhance gene expression, while other types of RNA allow only to reduce the activity of genes.

Classical RNA interference is the action of siRNA, the other short RNAs (microRNAs and antigenrnas) resemble interference to some extent by the mechanism of action, but do not fit into the usual scheme. Therefore, some scientists consider this to be interference, while others distinguish it into separate groups.

Pitfalls and reefs of RNA therapyDespite promising preliminary results, RNA therapy is still in the experimental stage.

The main problem is to make RNA stable, to protect it from degradation in living cells. Modern RNA-based drugs remain in the body for 14 to 30 days, which is enough to get a therapeutic effect, but it takes longer to treat chronic diseases. Theoretically, some chemical modifications of RNA can make it less vulnerable to internal RNA-destroying enzymes.

However, the problems do not end there. Even very short RNAs are not able to cross the blood–brain barrier - a barrier that protects neurons from the penetration of various substances from the blood that may be toxic to the brain. Unfortunately, RNA fragments also fall into the category of such molecules. To deliver RNA to the brain, you have to use sophisticated techniques: for example, you can use a non–pathogenic vector - a delivery molecule that, being absolutely harmless, is able to get into the hidden corners of the body and drag the RNA attached to it there.

It is known that adenovirus-containing vectors are usually preferred by nerve cells, so Beverly L. Davidson from the University of Iowa uses such vectors for targeted delivery of RNA, which disrupts the gene that produces the huntingtin protein. In experiments on mice, this approach gives good results, so the next step will be testing on monkeys.

Another interesting way of delivering RNA to the brain is used by Swami's group (Manhjunath N. Swamy) from the Medical Faculty of Harvard University (Harvard Medical School). During the experiment, RNA is bound to a short peptide that is isolated from the rabies virus. This glycoprotein selectively binds to acetylcholine receptors on nerve cells. Thus, siRNA appears on the surface of neurons and gets the opportunity to penetrate inside. In animal experiments, this method was used to defeat deadly encephalitis. RNA can be anchored not only on protein molecules, but also on lipids or artificial polymers. It remains only to make sure that such "carriers" are non-toxic to cells.

In addition to delivery to hard-to-reach organs, side effects of RNA therapy create a lot of problems. The difficulty lies in the fact that the side effect can be adequately traced only during the use of a new drug on humans: animal experiments in this case give only approximate knowledge or reveal only the most severe side effects.

Despite many difficulties, the development of RNA therapy is worth it, because it allows you to influence the work of organs at the most subtle level – at the level of genes. Changing the situation "at the root", at the earliest stages – so far the most reliable way to cure serious ailments, the only hope of many millions of people.

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