The Near and Long Term: A Couple of Promising Advances Due to Recent Focus on Coronavirus Research
The novel coronavirus SARS-CoV-2 which causes Covid-19 has dominated headlines, attention, and pretty much every aspect of the world so far this year. As the world struggles with the first truly global pandemic in over one hundred years, it’s very easy to focus on one of two things: The doom and gloom of the outcomes of the disease itself, or the progress on the over 200 vaccines in development around the world.
First US case of Covid-19, virus in blue. Image Credit: CDC/ Hannah A Bullock; Azaibi Tamin
The unprecedented focus of the scientific community around the world on this disease, however, has driven some incredibly interesting advances that have flown under the radar for most people. In this article I will be discussing two scientific advances that have come about because of the focus on corona-viruses; one is a future novel therapeutic approach to corona-viruses in general, and the other is a potential near-future prophylactic measure against coronavirus infection. I will begin with a brief background on one of the tools used to create these advances.
A brief background on CRISPR:
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is so named because the initial evidence of the entire system was found in bacterial DNA where there was a section of code that followed the aforementioned name. In case you were wondering what exactly a “palindromic repeat” might be, a palindrome is something that reads the same both backward and forwards. In DNA the code is made entirely out of 4 bases: A, T, C, and G. In this code T always pairs with A, and C always pairs with G.
Example of palindromic repeats, No copyright, Researchgate.net
A short palindromic repeat of these might be a segment of code, that, when folded in the middle, matched up so that the AT and CG pairs are together. An example might be the following, with the “x” designating the middle point that would “fold”, ATGCTxAGCAT or TAxTA, or any number of other similar patterns. Eventually, it was found that these short palindromic repeats were derived from DNA fragments of various viruses that normally preyed upon the bacteria. In the interest of keeping this brief, we shall skip most of the story of the discovery of this system (For which two women were awarded the Nobel Prize this year!). These short DNA fragments work as a sort of “defense library” that when paired with an associated protein called Cas (CRISPR Associated Protein with many subtypes denoted by number) work like a GPS (the guide RNA) attached to a Bowie Knife (the Cas protein) that can hunt down viruses invading the bacterium, use a guide-RNA to match the viruses DNA to that of the CRISPR library, and if a match is found, cut up the viral DNA before it has a chance to damage the bacterium.
CRISPR-Cas9 complex, created in BioRender.com
The most common form of this is the CRISPR-Cas9 unit, which has gained a lot of fame during the last decade because some very brilliant scientists realized that they could use this tool to target any DNA, by feeding the protein a short guide-RNA sequence that they engineered, and modify the Cas protein to not only cut but also replace the DNA code at the target site. Because of this most people who have heard of CRISPR know it as a tool that scientists can use to edit DNA inside a living organism. This is an incredibly powerful tool, but it is important to remember that originally the entire system originated as an immune system for bacteria.
A Long-term goal of pan-coronavirus protection:
A group of researchers, mostly from Stanford, published a paper titled Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza in May 2020 in the journal Cell. The researchers decided to engineer a “novel” use of CRISPR technology, by attempting to integrate its original bacterial use in bacteria, as an immune system, into human cells. They used a subtype of protein called CRISPR-Cas13d and called this system PAC-MAN (Prophylactic Antiviral CRISPR in Human Cells).
The first thing that the researchers did was analyze SARS-CoV-2 genomes from 47 different patients along with known SARS-CoV and MERS-CoV genomes (the viruses that cause SARS and MERS, respectively) to find highly conserved regions of DNA. These are parts of the genome that tend to stay the same even across subspecies or through mutations, usually because they code for a very important function for the virus that cannot survive if it is changed. Once they had narrowed down conserved regions, they engineered a group of crRNAs (CRISPR RNAs) that function as the guide-target for the Cas13d protein.
They then tested the CRISPR-Cas13d system in human lung epithelial cells (the cells that make up the inner lining of the lung, where exposure to these viruses commonly leads to infection) and were able to show that the group of crRNAs that they engineered were able to inhibit SARS-CoV-2 fragment expression. At the time they were required to use fragments of SARS-CoV-2 due to a lack of availability (and the danger of using) of the live virus for testing and experiments. Using these fragments, however, they found that the crRNAs were able to reduce expression by up to 83% within these tissues.
In order to confirm the effectiveness of these tests on fragments, they then tested this on live H1N1 influenza virus in the same type of Human Lung Epithelial cells. Against this virus, they were able to show inhibition of H1N1 expression by up to 78%. This amount of inhibition is quite significant. If a human were able to prevent 80% of a virus from infecting their cells, it gives the rest of the body a much better chance to fight off any serious infection.
Finally, the researchers decided to see if there is a small group of crRNAs that would cover all fully sequenced corona-viruses. They analyzed all 3,051 known coronavirus sequences, 91,600 IAV (Influenza A Virus), and found a group of 6 crRNAs that covered 91% of coronaviruses, a group of six crRNAs that covered 92% of IAV strains, and a group of 22 crRNAs covered 100% of all sequenced coronaviruses.
While this paper is an early proof-of-concept antiviral strategy and has some important hurdles to clear before it could be used in any therapeutic way, it shows the incredible long-term promise of a potential cure for the common cold (and all other coronaviruses). The authors readily admit that this will not be able to help with the current pandemic, but they list a series of tests and steps to advance this type of treatment for the future and are hopeful at its potential for long-term viral protection.
A potential near-future molecular PPE for SARS-CoV-2 infection:
Camels, llamas, and related animals produce small proteins called nanobodies, which work similarly to the antibodies found in the human immune system. These molecules were discovered in the 1980s and since then have been the subject of many types of research. While several labs have been working on finding ways to fight SARS-CoV-2 with these antibodies, I will be focusing on the pre-print paper An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation, a study by UCSF researchers that was posted to the pre-print server www.bioarxiv.org. While the study has not yet been peer-reviewed, it still shows promise for a potential protective therapy for Covid-19 in the future.
Nanobodies are quite interesting and offer a novel avenue for researchers. They are much smaller than human antibodies and have a relatively simple structure, which makes them much easier to engineer in the lab. The small structure also makes it possible to mass-produce using yeast or E. coli. Finally, their small size makes them much more stable, which enhances the possibility of transport and storage of any future medicines.
Created in BioRender.com
The researchers first sifted through a staggeringly large number of synthetic nanobodies, looking for high interaction with the Spike protein on the SARS-CoV-2 virus. This Spike protein is what you have almost certainly seen in any image of a coronavirus. The SARS-CoV-2 Spike protein has a triple head that is normally “closed” but that opens up to bind to the ACE2 receptor in human cells, working like a key that unlocks our cells and lets the invading virus inside. Without this Spike key, the virus is unable to penetrate our cells and no infection can occur. Having found some 21 of the nanobody configurations that attached best to the Spike protein, they further narrowed this down to the two best candidates and engineered a new triple nanobody (A three-part conglomerate of the best candidates). This had an incredibly high affinity for attaching to all three heads of the Spike protein, while they are still closed, preventing the Spike from opening as well as preventing any of the open spikes from attaching to the ACE2 receptor.
These newly engineered triple nanobodies were tested against the SARS-CoV-2 virus and found to be extraordinarily effective as deactivating the virus, even in incredibly small doses. In order to further test the possibility of these nanobodies as an actual medicine, the researchers subjected the nanobodies to high temperatures (up to 50 degrees Celsius for an hour) and also converted them into a shelf-stable powder and an aerosol, all without any loss of effectiveness against the virus. This demonstrated the feasibility of making a cheap and easy-to-produce, shelf-stable inhaler or nasal spray.
The study still needs to undergo peer review, and these specific nanobodies, called “Aeronabs” by the authors, would need to undergo clinical trials for safety before they could possibly come to market for us to use. Despite this, the initial research has gone much further than most potential drugs, and outside of the slim possibility of the off-target interactions, these nanobodies show incredible promise as a daily “molecular PPE” that could be used to prevent infection by SARS-CoV-2 and change the outlook of this entire pandemic.
Silver linings and reasons for hope:
During this pandemic, it is all too easy to fall into an attitude of despair. Botched and unequal national responses and a proliferation of anti-science rhetoric have contributed to fatigue that affects us all. It is important, however, to remember that in our lifetimes we have never seen specifically-targeted research and funding for that research on this kind of international scale. The advances being made on a world-wide scale will carry on long past the end of this pandemic. There are breakthroughs being made even now, that we have not yet heard of, that will change the course of science as we know it. In this article, I have tried to (briefly) introduce two such advances that may stimulate your optimism and hint toward the brighter future that so many scientists are working toward.