To date, there is no drug or vaccine against the SARS-Cov-2 coronavirus. In the United States, Stanley Qi from Stanford University and his team of researchers are working to develop a prophylactic cellular defense system against SARS-Cov-2 that protects human cells from infection by the virus by destroying the genes of the virus. The scientists have their invention in the journal Cell presented [1].
Li calls this artificial cellular defense system PAC-MAN – or “prophylactic antiviral CRISPR in human cells ”. CRISPR stands for C.lustered Regularly I.nterspaced Shoard Palindromic Repeats. These are short, repeating DNA sequences in the DNA of certain bacteria. If the bacterium is infected by a virus, a so-called bacteriophage, the Cas proteins of the bacterium split the DNA of the invading virus into small fragments. These are then inserted between the CRISPR sequences. If the virus is infected again, the DNA between the CRISPR sections is rewritten into RNA: this so-called lead RNA then “checks” the DNA of the newly penetrated virus. If this matches the DNA between the CRISPR sequences, it is cut by the Cas proteins. The bacterium has become immune to the virus attack.
“But man is created to look at and imitate nature ”(“ Homo autem ortus est ad mundum contemplandum et imitandum ”)
Cicero, De natura deorum, II, 37.
Like all CRISPR systems, PAC-MAN consists of an enzyme – in this case the enzyme Cas13d and four strands of lead RNA (22 nucleotides long) that direct Cas13d to the specific nucleotide sequences in the coronavirus that it is designed to destroy. These nucleotide sequences are highly conserved sequences that are found in many coronaviruses, not only in SARS-CoV-2. These highly conserved sequences are found in the coronavirus genes responsible for the protein RdRP and encode nucleocapsid proteins.
RdRP is necessary for the multiplication of the virus; the nucleocapsid proteins protect the virus RNA from being broken down by human RNAses. By destroying these nucleotide sequences, PAC-MAN can stop the multiplication of SARS-CoV-2, since both the multiplication of the viral RNA and the production of viral proteins are disrupted.
PAC-MAN is particularly useful for a patient whose immune response to the virus is weak and who e.g. B. does not form antibodies against the virus. Antibody formation takes a few weeks and in some cases this can be too long. In addition, the antibodies are often directed against surface proteins of the virus, which can change their shape very quickly and are then no longer recognized by the antibodies. The virus escapes from the immune system through this so-called antigen drift. At PAC-MAN, the use of several lead RNAs in parallel could significantly reduce this problem and give the cells an additional weapon against the virus.
In the lungs, gas exchange takes place in the alveoli: carbon dioxide is absorbed from the blood into the alveoli and oxygen is released into the blood from the air we breathe. If SARS-CoV-2 enters the lungs, the lungs of an infected person can become inflamed and filled with fluid, blocking gas exchange and making it difficult for the patient to breathe and connecting to a ventilator in the intensive care unit.
The researchers tested PAC-MAN in cell culture, on the human lung epithelial cell line A549. First they brought PAC MAN into the cell line, then 24 or 48 hours later they infected the cells with an artificially produced weakened SARS-CoV-2 virus. The scientists then measured the amount of virus protein and virus RNA in the infected cells.
According to Qi, PAC-MAN did very well. It reduced the amount of synthetic SARS-CoV-2 by 90%. Next, the scientists plan PAC-MAN in organoids of human lungs and animals, e.g. B. ferrets and rhesus monkeys to test against a natural SARS-CoV-2 virus. Unlike in the experiment, the geneticists primarily want to know how well PAC-MAN works in cells that are already infected with a natural SARS-CoV-2 virus.
However, it will take years before clinical studies with PAC-MAN can be carried out, so it will not play a role in combating the current Covid 19 pandemic. For LI, the biggest obstacle to therapeutic use is getting PAC-Man into the lungs through the airways. Li therefore teamed up with Michael Connolly of the Lawrence Berkeley National Laboratory at the Department of Energy. Connolly wants to transport PAC-MAN into the lungs with so-called lipitoids using nanoparticles. Lipitoids are non-toxic to the body and can transport PAC-MAN into cells by encapsulating them in a tiny nanoparticle that is only a billionth of a meter wide.
“Effective transport of lipitoids coupled with CRISPR targeting could provide a very effective therapy for combating viral diseases, not only against SARS-CoV-2, but possibly also against new strains of virus with pandemic potential,” said Connolly.
further reading
[1]. Timothy R. Abbott, Girija Dhamdhere, Yanxia Liu, Xueqiu Lin, Laine Goudy, Leiping Zeng, Augustine Chemparathy, Stephen Chmura, Nicholas S. Heaton, Robert Debs, Tara Pande, Drew Endy, Marie F. La Russa, David B. Lewis , Lei S. Qi. (2020) Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell, 181 (4): 865-876. DOI: 10.1016 / j.cell.2020.04.020
–
