Russian scientists have improved the method of treating hepatitis B using CRISPR/Cas9 “molecular scissors”. Previously, it was believed that chronic hepatitis B could be cured by removing the main form of the virus’s DNA from liver cells. The authors showed for the first time that this is not enough, and even after the removal of viral DNA, a recurrence of infection occurs. The method they proposed makes it possible to completely rid infected cells of the virus, which can form the basis of new drugs against hepatitis B. The results of the study, supported by a grant from the Russian Science Foundation (RSF), are published in the journal Molecular Therapy: Nucleic Acid.
The hepatitis B virus is a dangerous infectious disease that kills about a million people every year. It affects mainly the liver and can lead to the development of cirrhosis and cancer. The virus is transmitted from person to person through blood and other bodily fluids. At the same time, it is very stable: the pathogen persists outside the human body on various surfaces for up to several weeks, and is extremely contagious – just one viral particle can be enough to infect an unvaccinated person. Russia has created one of the most effective hepatitis B vaccination programs in the world. Nevertheless, more than 316 million people are infected with hepatitis B in Russia and the world.
The currently existing methods of treating hepatitis B do not relieve sick people of viruses, but only suppress the reproduction of the pathogen in the body. The therapy is ineffective because in the nuclei of hepatocytes – liver cells – the circular DNA of the virus is preserved, which conventional drugs are not able to “detect”. The only known way to destroy it is to cut it with “molecular scissors”, or the CRISPR/Cas9 system. This tool accurately recognizes certain genetic sequences that scientists have specifically “targeted” it, and cuts them, thereby preventing the DNA of viruses from working. Therefore, if CRISPR/Cas9 is directed exclusively to sequences that are in the DNA of the virus, the system will be absolutely safe for humans.
Studies have shown that after the introduction of CRISPR/Cas9 into infected hepatocytes, viral DNA disappears from them, but reappears after a while. It turned out that the virus leaves behind two types of DNA molecules in cells. The first is closed circular DNA, which is effectively cut by “molecular scissors”. The second is relaxed, or broken in one strand, circular DNA, which serves as a precursor for the first type and has a somewhat different styling. CRISPR/Cas9 does not act on it, therefore, after “waiting out” the effect of the drug, the molecule turns into a full-fledged closed DNA, on the basis of which new viral particles are assembled.
Scientists from the First Moscow State Medical University named after I.M. Sechenov (Moscow) with Russian colleagues improved the CRISPR/Cas9 method for the treatment of hepatitis B, which made it possible to destroy both types of virus DNA in cells and prevent the recurrence of the disease.
Using molecular methods, the authors created the CRISPR/Cas9 system, which targets the closed circular DNA of the virus, and then tested its operation in artificial conditions – in human cell culture. The experiment showed that “molecular scissors” destroy more than 98% of the corresponding DNA molecules, while the CRISPR/Cas9 complex itself also decomposes in a day. This suggests that if applied to the human body, it will leave no traces after its work and will not be able to damage non-target areas in human DNA.
However, the study showed for the first time that if relaxed viral DNA was also present in the solution, after about two weeks, full-fledged molecules of closed viral DNA were created on its basis. To prevent this, the scientists proposed using a special inhibitor of DNA synthesis together with CRISPR/Cas9, a drug that has been used for many years in the treatment of patients with chronic hepatitis B.
The researchers confirmed the effectiveness of this approach in an experiment with a culture of liver cells infected with the hepatitis B virus. The day before the introduction of CRISPR/Cas9, they were treated with an inhibitor, and then the treatment was repeated for another six days in a row. This made it possible to completely get rid of the presence of both types of viral DNA and avoid the recurrence of the disease.
“The approach we proposed can form the basis of new effective drugs for the treatment of hepatitis B. Already, our team has developed a new, effective method for delivering CRISPR/Cas9 to liver cells, in which the system will only enter virus-affected cells. In addition, we have a large number of clinical trials to unequivocally verify the safety of such treatment for humans, ”says Dmitry Kostyushev, a participant in the project supported by a grant from the Russian Science Foundation, PhD in Biology, head of the laboratory of genetic technologies in the creation of medicines at Sechenov University.
The study also involved scientists from the V. A. Engelhardt Institute of Molecular Biology RAS (Moscow), the M. P. Chumakov Federal Scientific Center for Research and Development of Immunobiological Preparations RAS (Moscow) Medical and Biological Agency (Moscow), Research Institute of Occupational Medicine named after Academician N.F. Izmerov (Moscow), Central Research Institute of Epidemiology (Moscow), Moscow State University named after M.V. Lomonosov (Moscow) and the National Medical Research Center for Phthisiopulmonology and Infectious Diseases of the Ministry of Health of the Russian Federation (Moscow).