The RNA Revolution / Christine Gorman and Dina Payne Maron

RNA, long considered only a housekeeping molecule in the cell, turned out to be a breakthrough in a new world of medical treatment

Since the discovery of the double-stranded structure of DNA in 1953, molecular biology has been characterized by more participants than heroes of a Russian novel. Biologists have identified tens of thousands of molecules that direct and shape the organized chaos that prevails in the body's cells and have derived thousands of drugs and treatments from these findings.

For decades, the stars of the drama came from two camps: DNA, or deoxy-ribonucleic acid, which serves as a repository of genetic information that hardly changes over time, and proteins, which perform the handiwork of the genes. Research on proteins has led to medical breakthroughs, such as synthetic insulin, interferon and a new generation of anti-cancer drugs, and gene therapy that uses modified DNA segments paves the way for the development of treatments for hemophilia, hereditary blindness and other diseases that previously had no solution [ See: The second act of genetic healing, Ricky Lewis, Scientific American Israel, June-July 2014].

However, in the march of medical progress, a third type of biological molecule was forgotten: RNA, or ribonucleic acid, which, like its more famous sister, contains genetic information but its chemical stability is lower. Thus, enzymes often break it down in the turbulent chemical environment of the cell.

Although scientists have known for a long time that RNA is involved at a certain point in almost every cellular process, they attributed to it a secondary role in the picture of DNA and proteins. In the 50's and 60's of the 20th century, biologists thought that RNA was a kind of stepdaughter that transmits messages, coordinates supplies and generally keeps cells in order. This image was preserved for several generations.

However, all this was before the RNA molecule received an image makeover that happened like a magic wand. A series of discoveries in the late 20th century revealed new forms of RNA that were not modest housekeepers at all. Rather, RNA molecules knew how to regulate the behavior of DNA molecules and proteins and cause certain molecules to increase or slow down their action. Using such RNA molecules, scientists can try and develop new treatments for cancer, infectious diseases and a wide range of chronic diseases.

In the last decade, scientists rushed to realize the new discoveries. The pace of discoveries has increased, dozens of start-up companies have been established that aim to benefit from the findings, and now some promising treatments are also on the horizon.

Meanwhile, economic interest has increased from a meager trickle to a multi-billion dollar influx of investment. Recent transactions include the launch of Editas Medicine at the end of 2013, which received $43 million in venture capital. The company focuses its efforts on the "hot" and cutting-edge RNA technology, known as CRISPR. An older company, Alnylan Pharmaceuticals, founded in 2002, received $700 million in January 2014 to develop, among other things, a line of RNA drugs to treat fatal conditions of the blood system, liver diseases and immune system disorders.

The funding comes in "waves", says Robert McCloud, VP of Oncology and Research at the pharmaceutical company Isis, which has raised approximately 3.8 billion dollars since it was founded in 1989. Its leading product, Kynamro, was approved by the US Food and Drug Administration (FDA) in 2013 as an RNA drug for people suffering from a rare genetic disorder that significantly disrupts their ability to process cholesterol and puts them at a particularly high risk of having a heart attack or stroke.

As in any rapidly developing field, along the way there were some obstacles and detours, and not every discovery will stand the test of time. And yet the researchers are giddy with excitement. They feel as if they have discovered a new continent and will explore it in search of possible breakthroughs.

supporting role

It is easy to understand why molecular biologists attributed the roles of the stars to DNA and proteins and not to RNA. The main building blocks of DNA - adenine, thymine, cytosine and guanine, or A, T, C and G - make up the basic instruction book of almost every living thing in our world. And one of the most important processes that DNA manages (using a code) is the creation of proteins.

Proteins, for their part, shape the three-dimensional structure of the cells and allow them to carry out most of their functions: they provide the freshness and youthful appearance of the skin and the strength of the heart over time. They also turn the DNA on and off in response to signals from the environment, determine the best way to utilize sugar in the cells and regulate the ability of nerve cells to transmit messages to each other in the brain. The vast majority of drugs today, from aspirin to Zoloft, affect proteins, either by blocking their activity or by changing the amount in which they are formed.

The fact that most drugs affect the activity of proteins does not necessarily mean that researchers can develop a drug for every target protein. The most common drugs are small molecules that can survive ingestion and passage through the acidic stomach. Upon absorption from the digestive system, they must fit into the active sites of the target proteins like a key fits into a lock. However, there are certain groups of proteins for which this traditional approach is not suitable: those that hide their active site deep inside narrow channels or those that do not have an active site because they are part of the internal skeleton of the cells and therefore they do not "respond to drug treatment," according to McLeod.

The new RNA drugs are designed to overcome exactly this barrier that until recently was not clear how to overcome it. Biologists have known for a long time that RNA serves as a competent intermediary that copies, or reproduces, the DNA instructions into a complementary sequence (it matches C to G, for example) and then translates the code into three-dimensional proteins. RNA of this type, known as messenger RNA (mRNA), is created in the nucleus and migrates to the cytoplasm, where structures called ribosomes, in cooperation with another type of RNA, transfer RNA (tRNA), read the message and connect amino acids (compounds containing nitrogen) into long chains that become proteins. But RNA can do much more than that.

A Star Is Born

The ground for the RNA breakthrough was prepared in 1993 with the identification of the first microRNA molecules [the scientists who did this were awarded the Wolf Prize for Medicine in June 2014 at the Mishkan Knesset in Jerusalem - the editors]. MicroRNAs are atypically short RNA segments that bind to messenger RNA strands and prevent the ribosomes from progressing in protein assembly [see box on next page]. The cells apparently use microRNA molecules to coordinate the schedules in the production of many proteins, especially in the early stages of the organism's development. Five years later, researchers reached another breakthrough when they showed that various short RNA molecules effectively silence the translation of genes into proteins by cutting the messenger RNA. This discovery, which was a milestone, won the Nobel Prize in 2006.

At that time it seemed that everyone, and not just RNA experts, was interested in using this molecule, which was once ignored, to influence the way proteins are created. The process of cutting messenger RNA by short RNA segments is called RNA interference (RNAi) and the RNA molecules active in the process are called short interfering RNA (siRNA). Also, a wide variety of scientists realized that they could solve the problem of proteins that do not respond to drug treatment by advancing the focus of activity from the final product, the protein, to the stage where RNA is involved in the chain of processes for the production of proteins.

More than 200 experimental studies that deal with microRNA or siRNA are currently registered in the US government's database for clinical trials and are intended for the diagnosis and treatment of almost everything, from autism to skin cancer. Among the promises they make: treatment of those infected with the Ebola virus, which causes an extremely deadly disease, which terrorism experts fear could be used as a biological weapon, and the hepatitis C virus, which causes chronic hepatitis in approximately 150 million people worldwide and is the main cause of liver cancer [see box on the left and box on the other side of the page ].

What in the future?

While drugs containing microRNA or siRNA are advancing in the race to clinics, a new generation of female stars is now waiting for a workout. These future drugs should act even earlier in the chain of processes, on the DNA molecule itself. One of the approaches is based on the CRISPR sequences found in bacteria, and it was enthusiastically called "CRISPR madness" in the scientific journal Science. Another approach, based on long molecules of non-coding RNA (lncRNA), still raises doubts about its usefulness.

CRISPR sequences are strange sequences of repetitive DNA segments found in bacteria and bacteria-like organisms. These sequences interact with a group of proteins associated with these sequences, known as Cas. CRISPR sequences and various Cas proteins create a system of bacterial defense against viruses.

These proteins have one function: to cut the DNA of the virus in half and destroy it. Complementary strands of RNA guide the proteins to their target: certain segments of viral DNA. But where does this RNA come from? Like a microscopic version of Jiu-Jitsu, the cells take the RNA from invading viruses and make a double agent out of it that guides the Cas proteins to the exact spot where they are supposed to cut.

Although CRISPR elements were first discovered in 1987 in bacteria, scientists began to adopt this system for a wide variety of animal tissues, including humans, only in 2012. Creating a guide RNA in the laboratory allows researchers to direct Cas proteins with perfect precision to the desired site in the nucleus to cut the DNA molecules. In fact, they turned the bacterial defense mechanism into a precise tool for gene editing.

Such a precise technology could bring about a revolution in gene therapy, and this may happen sooner than we think.

Today, medical researchers can only randomly inject corrective DNA into patients with defective genes in the hope that at least some of the genetic material will find its way and start acting in the right place. Full development of CRISPR/Cas technology could change the situation and allow researchers to choose precisely where to change the patient's DNA. "As early as 2015 we should see some experiments with the CRISPR method," says George M. Church, professor of genetics at Harvard Medical School, co-founder of Editas and scientific advisor to Scientific American. "The method comes, in fact, ready," he adds. "You can take it from bacteria almost without the need for changes. And almost any guide RNA we want will reach the site where it works. It is fast and allows for permanent changes in the DNA."

Church hopes that Editas will move into clinical trials immediately after the animal study is completed. Other CRISPR-centric companies have recently been established, including Caribou Biosciences and Egenesis.

The most controversial discovery among the recent discoveries in the field of RNA is related to lncRNA. These long RNA sequences, first described in 2002, come from the nucleus and at first glance they looked like messenger RNA, but they lack the sequence of letters needed to start the translation process.

Why does the cell need all this excess RNA? Part of this RNA was undoubtedly created from the reproduction of early versions of genes that have broken down over time and no longer function. (One of the most surprising discoveries of the genetic revolution is that almost all the DNA found in the nucleus is copied, not just the parts that code for proteins.) Other RNA segments are probably evidence of attacks from the distant past by certain types of viruses that could integrate their genetic material into DNA of the cell to pass it on to their future generations.

But what if part of the lncRNA represents a way of gene regulation that was not known before, one that does not require potentially dangerous DNA mutations and that does not depend on proteins as main players? DNA is folded like origami, says John Rinne, an RNA researcher at Harvard University. You can make an airplane or a crane from two identical pieces of paper, and it is possible that lncRNA has some role in keeping the DNA folded in the right order. And just as an error in origami can create a wingless paper crane, so, for example, an excess of lncRNA may cause cancer even if there is no mutation in the cell's genes.

Another possibility that is being studied is that lncRNA molecules can bind themselves to different parts of the DNA molecules, change their three-dimensional structure and expose them to activity or hide them.

Many other types of non-coding RNA molecules may serve as important genetic regulators, or alternatively they may be genetic ghosts. One of the difficulties in studying non-coding RNA molecules is that they do not produce proteins, so it is more difficult to prove that they do something important. "I think it's just too early, this is just the beginning," says John Mattick, senior researcher of non-coding RNA molecules and director of the Green Institute for Medical Research in Australia. "A whole new world is revealed here."

When one considers the wide variety of RNA compounds that have been designed and tested, it seems that the most attractive feature of these molecules is their simplicity. Unlike proteins, whose three-dimensional structure must be precisely characterized in order to develop effective drugs, RNA fundamentally consists of a two-dimensional sequence (if we ignore for the moment some of the three-dimensional shapes that RNA molecules can create). "It turns a three-dimensional problem, where a small molecule has to fit with energetic precision into a site on a protein like a key to a lock, into a two-dimensional, linear problem," says McLeod. Thanks to the Human Genome Project, researchers already know most of the important sequences in the genome. All they have to do is synthesize the complementary RNA strand, and hitting the target is guaranteed.

Of course, efforts are still required to put the plan into action, but for now at least the magic staff is working.

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in brief

Three of the most important complex molecules in living things are DNA, RNA and proteins. For decades, biologists attributed the most important activity in the cell to DNA and proteins; RNA was certainly important but was considered a provider of support services.

A series of discoveries at the end of the 20th century revealed several forms of RNA that have active regulatory roles in cells: they determine which proteins will be produced and in what quantity and even silence some genes.

These new insights allow scientists to create a new world of experimental drugs against bacteria, viruses, cancer and various chronic diseases that will be more precise and effective than the drugs available today.

A new shot against hepatitis C / Kristin Gorman

MicroRNA in liver cells is a target for a drug that may neutralize a deadly and silent enemy

25 years ago no one had heard of the hepatitis C virus. Today it is the main cause of liver cancer and a major reason for liver transplants. The virus kills about 350,000 people a year in the world. In the US, more people die from hepatitis C than from AIDS.

The infection can be cured, but the treatment is accompanied by debilitating side effects. The conventional treatment with interferon and ribavirin causes fever, headaches, exhaustion, depression and anemia. Such treatment can last up to 11 months and eliminate the infection in 50% to 70% of the patients. Recently, when they also started using protease inhibitors, a type of medicine that was first used against HIV, the cure rate improved and the treatment time decreased. Unfortunately, the new drugs only work against the hepatitis C strain common in North America, Europe and Japan, and are not as effective in the rest of the world.

RNA drugs may improve the situation. In 2013, researchers showed that an experimental drug called miravirsen, which acts on microRNA molecules in liver cells, significantly reduced the amount of hepatitis C virus in most of the patients who received the treatment, in some of them even below the diagnostic threshold. The experimental drug consists of a short sequence of DNA whose "letters" precisely complement the sequence of letters found in the microRNA, thus enabling the drug to precisely target the target.

This microRNA, known as miR-122, plays a role in the production of many proteins in the liver. But unlike most microRNA molecules, it apparently does not inhibit the production of proteins but accelerates it. When the hepatitis C virus manages to enter a cell, it binds itself to miR-122 to ensure the creation of many copies of the virus. Blocking miR-122 also blocks virus culture.

The main side effect is redness at the injection site, which eventually disappears. Since the treatment is directed against a target found in the host cells and not against one of the virus proteins (like protease inhibitors), the treatment should be effective against all hepatitis C strains.

Although the trial was designed to last only four weeks (the infection eventually returned in all treated patients), there is reason to believe that longer treatment with mirvirsen would be more effective. "The thinking is that if we block the virus culture for as long as necessary it will be possible to cure the disease," says Harry L. A. Jensen, senior scientist at the Institute for General Research in Toronto and co-author of this study published in the New England Journal of Medicine.. The experiments continue.

Christine Gorman She writes about health and medicine.

Defeating nature's terrorists / Ferris Jaber

A treatment based on RNA can stop the Ebola virus

People who contract the Ebola virus first think they have the flu: fever, chills, muscle aches. Then the bleeding starts. As the virus attacks the cells throughout the body to replicate itself, it takes over and causes damage to the liver, lungs, spleen and blood vessels. Within days the organs fail and the patients go into a coma. Some outbreaks, mainly in central and western Africa, killed up to 90% of the people infected.

This dire prospect may change. Thomas W. Geisberg, now working at the University of Texas Medical Branch in Galveston, and many collaborators treated six monkeys, previously infected with the virus, using molecules of short interfering RNA (siRNA)) and saved their lives. In January 2014, it was reported that the treatment was successful in the first safety test in a healthy human volunteer. One of Geisberg's colleagues, Jan McClellan, and his team from the pharmaceutical company Takmira in Burnaby, British Columbia, received a $140 million grant from the US Department of Defense to continue developing the treatment.

Together, the scientists engineered an siRNA that would prevent the Ebola virus from producing a certain protein without which it cannot replicate itself. "If you eliminate this protein, in theory you eliminate everything," Geisberg says. The researchers are designing another siRNA to thwart the production of a second protein that the virus uses to weaken the immune system of those infected. There is no danger that the siRNA will interfere with normal cellular activity because the viral target protein is not found in the cells of humans or other animals at all.

McClellan and his colleagues wrapped the siRNA in bubbles of fat that can easily pass through cell membranes. They injected the drug into some rhesus macaques that had been infected with the Ebola virus less than an hour earlier. In one experiment, two out of three monkeys that received four treatment doses survived the week after exposure to the virus. In a second experiment, designed to test the effectiveness of a larger dose, all four monkeys that received seven injections of siRNA survived. Tests showed that molecules of the virus in the blood of the treated monkeys were considerably less than those found in untreated infected animals. The macaque monkeys passed the siRNA injection safely, and those that survived remained healthy even 30 days later.

The study was "a milestone," says Gerry Covinger of the University of Manitoba, who is developing another antibody-based treatment for Ebola. He believes that Geisberg and his team are "leading the effort toward clinical development."

Paris Jaber He is an associate editor at Scientific American.

More on the subject

Small silencing RNAs: An Expanding Universe. Megha Ghildiyal and Phillip D. Zamore in Nature Reviews Genetics, Vol. 10, pp. 94-108, February 2009.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724769/

Developing MicroRNA Therapeutics. Eva van Rooij et al. in Circulation Research, Vol. 110, no. 3, pp. 496-507, February 3, 2012. http://circres.ahajournals.org/content/110/3/496.long

Control of Gene Expression by CRISPR-Cas system. David Birkard and Luciano A. Marraffini in F1000 Prime Reports, Vol. 5, Article No. 47; November 1, 2013.

http://f1000.com/prime/reports/b/5/47

The article was published with the permission of Scientific American Israel