The virus mutates? We have the tools to fight back
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Over the past two years, the COVID-19 pandemic transformed and accelerated scientific and medical research. Yet the ever mutating-nature of the virus dictates a need for an even shorter timeline from variant detection to clinical application. Several challenges hamper such progress, including sluggish and cumbersome procedures for acquiring samples of live SARS-CoV-2 viruses for basic and pre-clinical research. This bottleneck is limiting the ability to develop and test new medical interventions against rapidly emerging new SARS-CoV-2 variants.
In a new manuscript, we partnered with Twist Bioscience, the leading manufacturer of synthetic DNA molecules, to develop a safe, rapid, and accurate alternative to live-virus experiments. The method allows scientists anywhere around the world to safely study models of variants less than four weeks after a new variant is identified and sequenced — literally outpacing the spread of the variants themselves.
The call to action
As of today, three major barriers limit the ability to study live samples of new virus variants. The first obstacle is the need to find a collaborator with the live sample that is willing to share it. This is not so easy. Typically, the very first step of Covid-19 tests, after collecting the samples, is to destroy the virus using heat or a strong chemical. For obvious safety reasons, only special labs have access to samples bearing live viruses.
After that, there is a lot of understandable bureaucratic and legal red tape around the transfer of dangerous viruses. Lastly, after the samples arrive, the research must be conducted in a Biosafety Level 3 (BSL-3) lab — the 2nd highest biosafety level. The number of BSL-3 labs in each country is limited and their services are in high demand.
Many labs, especially outside the main research institutions, are desperate for alternatives to live-virus experiments. One option is to study pseudoviruses — a harmless synthetic virus with only select pieces of the infectious virus attached to it. This method is broadly used to study the infamous SARS-CoV-2 spike protein, via which the virus invades human cells.
Pseudoviruses are helpful if you search for ways to stop SARS-CoV-2 from infecting cells. But they are much less informative if you want to study other functions of the virus, like how it is replicating inside cells and affecting the cells’ machinery, and to find treatments preventing this replication.
We therefore decided to develop a replicon system. Similar to the viral genome, a replicon is an RNA molecule that encodes all the instructions needed for self-replication. But unlike the virus, replicons do not encode the instructions of how to invade other cells. Thus, similar to the famous “Hotel California” song, they can enter into cells but cannot leave to infect other cells. This bioengineering approach renders the replicon completely safe for research, without any risk of unintended infections or a need for BSL-3 labs and has already been developed for other viruses such as SARS-CoV-1 (the virus responsible for the SARS outbreak in 2003), Nipah, and Lassa viruses.
Safety first
To remove the infectivity instructions from the replicon genome, we engineered five layers of protection. The first two layers involved complete deletion of two genes, the Membrane (M) gene and the Envelope (E) gene. Without these genes, the replicon cannot produce the membrane and envelope proteins that are essential for encapsulation of the viral RNA, without which the virus is incapable of exiting the cell that it is replicating within.
As a third protection layer, we inserted a stop codon at the beginning of the spike (S) gene (in the 10th amino-acid of the Spike protein). A stop codon is a three-letter sequence that tells the cell’s machinery to stop translating RNA into protein. The other 8,801 letters of the S gene are still there, but the cell ignores them and doesn’t translate them into the Spike protein.
The fourth layer of protection included is the addition of a single nucleotide (equivalent to a letter) after the stop codon. When cells translate RNA into proteins, each three letters of the genetic code correspond to a single amino acid, the building blocks of proteins. By adding a letter, this entire three-letter step process is shifted by one letter. In this fashion, even if the three letter stop codon is ignored, the resulting amino acid sequence would be completely scrambled, losing any resemblance to the original spike protein and its function, often producing stop codons and other non-viable alterations.
The fifth and final layer of protection is another deletion of a piece of genetic code: the segment of the S gene encoding a region called the furin cleavage site (FCS). This region gives the Spike protein the flexibility needed to effectively attach itself to human cells. Multiple published studies showed that without the FCS, the virus loses its infectivity.
Each one of these layers alone would have been sufficient to mitigate the virulence of SARS-CoV-2. We incorporate all five of them for the abundance of caution. Our downstream experiments were then conducted in a BSL-2 facility, a lower biohazard level, which many labs globally have access to and were approved by the Safety Committee of the CRUK Cambridge Centre of Cambridge University that oversees the safety aspects of our experiments as a Cambridge University-based. The study was also reviewed and monitored by the Biosafety Committee created by Twist Bioscience
We also added a glowing gene into the replicon sequence. This gene is partly based on a jellyfish genome and partly on a deep-sea shrimp genome. It is a common ”reporter gene”, used routinely by us and other researchers as an easy way of counting the number of RNA replicons in a sample by measuring the amount of light it emits.
Building a scalable process
One of the main advantages of our collaboration with Twist Bioscience is that Twist’s high-throughput DNA synthesis platform enables the scalability of our pipeline. Thorough this partnership, we can create replicons from scratch, based solely on publicly available sequencing data.
We fully automated the entire replicon design process. After choosing the strain of interest, our pipeline program scans the National Center for Biotechnology Information (NCBI) public SARS-CoV-2 database to establish the most accurate genome sequence of the strain in question. Within a few minutes after choosing a viral strain, the program spits out a digital file containing the code for a replicon that is 97% identical to the SARS-CoV-2 sequence, yet completely lacking the live virus’s ability to infect cells, and is therefore safe for research.
Based on this file, our collaborators at Twist Bioscience can harness their massively parallel DNA synthesis array to produce DNA molecules of the replicon sequence. This DNA synthesis conceptually works like ink-jet printers, but instead of droplets of ink, Twist Bioscience’s machines print chains of nucleotides, the building blocks of DNA. We then assemble these molecules into one large piece and convert it into RNA molecules using special enzymes.
Once these RNA molecules enter into the cells (using special synthetic reagents since they cannot do it without help), they begin to replicate themselves, readying themselves for research. How do we know that? Because the cells start to glow. The green light from the reporter gene serves as a quantitative marker and allows us to confirm that the replicons are replicating.
We confirmed that replicons cannot exit the cells by collecting the media in which the transfected cells were growing and introduced it to other cells. These other cells are highly susceptible to viral infection when introduced to media containing viruses. Yet a highly sensitive PCR test did not show a single trace of the replicon genome in the susceptible cells. This is a clear indication that the replicon does not have any ability to infect new cells.
Using the replicon for research and development of therapeutics
To show the applicability of the replicon for medical research, we tested two different anti-SARS-CoV-2 compounds on cells infected with replicons. First, we tested the active ingredient of Merck’s anti-SARS-CoV-2 drug, molnupiravir. This antiviral causes the viral RNA to mutate until it loses its ability to replicate.
The second compound was our proprietary siRNA investigational therapeutic (more on that soon). The treatment is a cocktail of short interfering RNA molecules that specifically target the SARS-CoV-2 genetic material inside cells and disable it.
Both tests showed that once treated with the compounds, replicon-infected cells emit a fraction of the light that control cells do. The results are a clear indication that the replicon is useful for measuring the effectiveness of new treatments targeting the replication properties of SARS-CoV-2 variants.
A solution for the world
While our study focused on the Beta variant of SARS-CoV-2, the flexibility of the replicon system should allow for it to be used to produce other variants, such as Delta or Omicron. The vision is to expand this replicon program to the point that whenever a new variant of concern arises, a prototype will be generated and readily synthesized. We are currently in discussions on ways to expand the availability of the replicon system to interested groups globally. This safe and effective alternative to live-virus experimentation is now available to the research community as a whole.
