The replication of potentially harmful adenoviruses can be significantly reduced in human cells in cell culture by using the so-called CRISPR-Cas9 system ("gene scissors"). This method, which is used worldwide in science and research, thus also offers potential for future innovative therapies for the treatment of viral diseases. This finding is based on a study by the IMC Krems – University of Applied Sciences (IMC Krems) – in Austria, which has now been published in the renowned journal "Molecular Therapy Nucleic Acids". The study was funded by the Austrian Science Fund FWF.
Getting to the root of viral diseases remains a major challenge. Although there are isolated drugs that prevent virus replication in human cells, these are still a major exception. Against this background, members of the research group led by Prof. Reinhard Klein from the Department of Life Sciences at IMC Krems have now investigated the potential of molecular technologies to inhibit viral infections. The research group used a scientifically established method (CRISPR-Cas9) for the targeted modification of DNA to significantly reduce the replication of adenoviruses in human cell lines in cell culture.
Target: Viral DNA
This Nobel prize honored method allows DNA segments in mammalian cells to be altered in a highly controlled way. "Our consideration now," explains Prof. Klein, "was to explore the potential of this technique for combating viral infections, such as infections with adenoviruses, which frequently cause diseases of the respiratory tract, the digestive tract and the eyes." The goal here: to destroy a region of adenovirus DNA in infected human cell lines in such a way that the viruses can no longer reproduce.
An approach that was surprisingly successful, as Prof. Klein explains: "In fact, we succeeded in reducing the amount of infectious virus particles in the human cell lines by up to three orders of magnitude under certain conditions. A result that clearly confirms the efficiency of the method and further demonstrates that this technology is also potent enough to take on the large number of viruses produced in infected cells in the course of some viral infections."
The combination of several measures was decisive for this success. The first was the selection of the viral DNA sequence to be targeted by the CRISPR-Cas9 system. Here, Prof. Klein's team chose the E1A region of the virus, which is of particular importance at the very beginning of the viral replication cycle.
By virtually nipping viral replication in the bud, we managed to keep the amount of viral DNA in the cells low enough for the CRISPR-Cas9 system to work efficiently at all."
Prof. Reinhard Klein, Department of Life Sciences, IMC Krems
In fact, at the beginning of the study, the potential amount of viral DNA in the cells did cause the team some concern. After all, if the viruses multiplied rapidly, this could become so large that the CRISPR-Cas9 system would quite simply reach the edge of its capabilities. But the inactivation of E1A, which is responsible for the enormous increase in the amount of viral DNA in infected cells, prevented precisely this.
Success through combination
A second measure that contributed to the strong inhibition of viral replication was the use of combinations of so-called guide RNAs - strands of nucleic acid that allow targeting of the DNA target sequence. This resulted in more efficient silencing of the adenovirus E1A gene and thus increased the likelihood that the virus would be unable to replicate
Inhibition of viral replication was further enhanced by a combination with the DNA synthesis-inhibiting drug cidofovir even when applied in small doses. The amplification effect occurs because viral DNA replication is inhibited at two different levels: First, the amount of functional E1A product is decreased by CRISPR-Cas9 in the cell while, in a second step, the remaining viral DNA replication that is still taking place is inhibited by cidofovir.
The results of the internationally acclaimed study at IMC Krems underscore the very general potency of CRISPR-Cas9 and the possibility of using the technology to inactivate viruses as well. A principal, future, application against viral diseases has thus become theoretically conceivable. However, if this technology is actually to be used for the treatment of viral diseases in the future, numerous further questions must first be clarified and technical hurdles removed, as Prof. Klein emphasizes. An application is therefore still a long way off at the moment, but the theoretical possibility has been demonstrated.
Didara, Z., et al. (2023). Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene. Molecular Therapy. Nucleic Acids. doi.org/10.1016/j.omtn.2023.02.033.