25.11.2016 change 25.11.2016

Biophysicists from the University of Warsaw remodel gene therapy

Researchers at the University of Warsaw have developed a method that can change gene therapy. The compounds they designed allowed the researchers to watch and better understand the mechanisms of protein reproduction in the cell, which should allow for the creation of better therapeutics.

Disease often interferes with the production of proteins in cells of the affected organism. Sometimes not enough necessary proteins are produced or they are incorrect, but overproduction can also be harmful. An extreme case is cancer.

One way of dealing with such problems is gene therapy - providing a body with genetic material encoding proteins that support the proper functioning of cells.

Initially there were attempts to use DNA as the genetic material. Unfortunately, due to the very complex structure of DNA, there is a high risk that the supplied genes will solve some of the problems, but will result in new, sometimes more serious issues.

Doctors have high hopes for mRNA - particles that are smaller and simpler, easier to prepare in the laboratory. In addition, unlike DNA, mRNA does not modify the genetic code permanently.

mRNA molecules are natural polymers produced in the cells. They are genetic code carriers and serve as templates for the production of new proteins.

mRNA molecule lives in a cell for a few minutes to several hours and then is degraded by enzymes. The short lifetime of the mRNA - also the one prepared for therapeutic purposes - restricts its use. Doctors would like the mRNA used in medicinal preparations to "live" longer than its natural counterpart.

The team from the Department of Biophysics, Institute of Experimental Physics at the Faculty of Physics, University of Warsaw has significant achievements in the study of therapeutic mRNA. Researchers at the University of Warsaw for years have been studying the possibility of using mRNA with a modified cap (the fragment initiating protein biosynthesis) in gene therapy.

According to the Faculty of Physics, University of Warsaw release sent to PAP, initiator of research on the mRNA modification is Prof. Edward Darżynkiewicz (Faculty of Physics UW), and the leader of the team working on the therapeutically important modifications is Prof. Jacek Jemielity. Team members are a dozen researchers - doctors and students, and the main animator of work at the Faculty of Physics UW is Dr. Joanna Kowalska.

Recently published results show that the new compounds - designed and produced at the University of Warsaw - have longer life and are more effective than their natural counterparts. They can also be produced at low cost.

Warsaw research continues, with the objective to find even more useful cap analogues, develop technology for mass production of therapeutic mRNA and to understand the natural processes of protein synthesis.

"7-methylguanosine cap is located at one ends of the mRNA (so-called 5th end) - explained Prof. Darżynkiewicz. - The cap structure in cytoplasm is recognized by the factor eIF4E initiating protein biosynthesis, or translation. This stage determines the speed of the whole complex sequence of events that ends with producing the protein in the cell. The presence of the cap also protects mRNA against premature degradation by enzymes - nucleases. Unfortunately, the cells have a natural mechanism to remove the cap by so-called decapping enzymes (for example Dcp1/Dcp2). A few years ago we discovered that using modified cap analogues can make the 5th end of mRNA resistant to degradation and increases productivity."

"In the paper published recently in the journal Nucleic Acids Research we presented a new class of modified caps - an improved version of those that are currently in clinical trials - said Prof. Jemielity. - The modification involves replacing the oxygen atom with sulphur in several positions. The resulting cap binds unusually effectively and has a high resistance to degrading enzymes. The modification results in a much higher amount of the therapeutic protein."

For patients, the availability of the drug is very important. Conventional enzymatic synthesis methods are inefficient. "When in 2010 we had to prepare for the first 4 grams of cap for clinical trials, it took half a year. This amount was sufficient to treat only 12-13 patients" - reminded Dr. Kowalska.

Meanwhile, the potential annual demand can be estimated in kilograms. "We noticed the so-called click chemistry - said Sylwia Walczak. - It is a method of synthesis of complex compounds using chemical building blocks - semi-finished products. Each block has at least one fragment, which - when it encounters its counterpart in another molecule - connects to it like a clothing clip."

When studying the possibility of using this method, Polish scientists obtained 36 new variants. "Two of them, entered into mRNA, worked as well as a natural cap" - added Anna Nowicka.

"For the first time we were able to design and obtain compounds that imitate a cap and are capable of inhibiting the activity of the enzyme Dcp1/Dcp2 that cuts off the cap from the mRNA, which determines the destruction of mRNA - boasted Marcin Ziemniak. - (...) Simply put, the enzyme complex swallowed our indigestible bait imitating a caps in mRNA and was frozen shortly after binding. Together with colleagues from the University of California in San Francisco, using the method of X-ray diffraction (...), we determined the structure of the enzyme complex responsible for cutting off the cap".

The results were published in two prestigious scientific journals: RNA and Nature Structural and Molecular Biology. They will allow scientists to better understand the mechanism of cap degradation (decapping). "We are confident that it will allow us to develop even better inhibitors of the mRNA decapping process" - believes Prof. Jemielity. - They will be useful not only for further research on the processes of mRNA degradation, but we expect that they will find therapeutic use, for example as adjuvant therapy in cancer vaccines and in antiviral therapies".

The researchers emphasised that the problems, which they tackle, require a multidisciplinary approach. "We have access to well-equipped laboratories, but many other teams in the world also have similar equipment. Our advantage, however, is human capital, which allows to combine expertise in the fields of (bio) physics, chemistry, and molecular and cellular biology. Conducting research on the border of three different fields and the ability to look at the same research problem from different perspectives is very stimulating intellectually and results in completely new ideas and solutions, which would be much more difficult to reach by looking at the problem from only one side. I believe that in this area it is a unique approach, not only in our country but also worldwide" - concluded Dr. Kowalska.

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