Cheer for us
Why do we need transgenic mice for the COVID-19 vaccine
Sergey Kuznetsov, Daria Spasskaya, N+1
The Institute of Gene Biology of the Russian Academy of Sciences is creating the first "batch" of genetically modified mice that would be sick with COVID-19 like humans. In June, the animals will go for experiments near Novosibirsk – to the Vector virological center, to which such manipulations are allowed by Rospotrebnadzor. Alexey Deikin, head of the Center for Collective Use "Genomic Editing" of the IBG RAS, told N+1 about why "coronavirus" mice are needed, and what difficulties researchers may face when creating them.
Why mice
Mice and rats are weakly susceptible to infection with the new coronavirus. Theoretically, you can give them such a large viral load that they will have symptoms, but this will absolutely not correspond to how a person is sick: most likely, they will just start losing weight, nothing more. This problem was encountered back in 2003, when there was an outbreak of the first SARS. Then, too, genetically modified mice were created that would be sensitive to this virus.
There are practically no laboratory animals that could be used as full-fledged models for studying coronavirus infection. SARS-CoV-2 coronavirus can infect ferrets, monkeys, there is evidence that hamsters are infected – and are able to infect each other.
But for full-fledged work on the development of funds from COVID-19, two conditions must be met.
The model organism should reproduce the "human" course of the disease – its entire pathogenesis, which is peculiar to humans.
To get statistically significant results about the safety and efficacy of a drug or vaccine, to detect all, even very rare side effects, thousands or even tens of thousands of experimental animals are needed.
And in the case of SARS-CoV-2, both of these conditions are not yet met for any model organism.
The same ferrets do not have the usual respiratory symptoms for humans, they do not cough. They have a gastrointestinal phenotype of the disease. And in humans, intestinal symptoms are far from the most frequent and not the most important. Therefore, testing human medicine on ferrets will not be quite indicative.
The pathogenesis of the disease in monkeys is more similar to human, but here a second problem arises. It is very important that animal experiments are reliable, so that we can see the real mortality, the real probability of side effects.
Imagine that the vaccine has a probability of some kind of severe side effect of one case in 10 thousand. You conducted an experiment on a thousand animals, on a thousand people, and did not see this effect.
And then 100 thousand people receive this vaccine – and this is a potential ten deaths. And if we are talking about millions of people? The only reliable tool here may be mass tests on laboratory animals.
It is impossible to imagine an experiment even on a thousand monkeys. Now, for example, Vector has bought 20 monkeys, and there are no more monkeys in Russian nurseries. Even on a thousand ferrets, the study is unrealistic, because these are very aggressive animals that are difficult to contain. But a thousand mice can be placed in a room with an area of 20 square meters.
Difficulties of transgenesis
The "old" SARS-CoV uses the ACE2 receptor on the surface of the epithelium of the upper respiratory tract and lungs to penetrate into the cell. In 2005-2007, several model mice were created, which, unfortunately, turned out to be unsuccessful. In all cases, scientists tried to express human ACE2 by transgenesis in the mouse body, but under different regulatory elements that would cause the receptor to be synthesized in larger or smaller amounts. The researchers did not think about the tissue–specific expression of ACE2 – that is, so that it appears only, in fact, in the lungs. Therefore, the receptor was expressed in places where, in theory, it should not, for example, in the brain.
Due to the fact that the genetic cassette with ACE2 was inserted accidentally, these mice got sick very differently after infection. Depending on the number of embedded copies of the gene, they either showed no symptoms at all, or vice versa, they died in agony on the second or third day after infection - primarily from brain damage.
The Chinese made a transgenic mouse in which the expression of human ACE2 was controlled by a human promoter. This was to ensure its more correct operation, corresponding to the work of the gene in humans. These mice were infected with the first SARS, but they were very weak: among the symptoms were only weight loss and lethargy. And they become infected with the new coronavirus even more weakly – the disease proceeds with almost no symptoms.
In 2013, there were reports that for the virus to enter the cell, not only the ACE2 receptor is needed, but also a second protein, TMPRSS2. This is a serine protease that activates a protein on the surface of the virus, thereby triggering an alternative pathway for the virus to enter the cell.
In cell culture, it was shown that an inhibitor that prevents the binding of TMPRSS2 to the virus blocks the infection of cells. This means that the path associated with the interaction of TMPRSS2 and ACE2 may be even more important than penetration using ACE2.
This is confirmed by an article in Nature published on April 26, where the expression profiles of ACE2 and TMPRSS2 are compared. It was shown there that it is those cells where these two genes, two proteins are expressed simultaneously, that suffer. These are the epithelium of the upper respiratory tract, alveolocytes of the second type, corneal cells of the eye, somewhat smaller in the intestine and in the testicles. These two receptors are increasingly mentioned together when it comes to COVID-19. But to date, there is no animal model with these two "humanized" genes.
We believe that a desirable – I don't know how ideal – animal model of COVID-19 should be characterized by three characteristics.
First: that they have both genes, ACE2 and TMPRSS2.
The second is that these genes should be expressed in the same way as in humans, in certain tissues.
Third, so that they are not expressed when we do not need it, that is, outside the virological laboratory.
Coronavirus by signal
The ability to "turn off" the ability to get infected with coronavirus in a pandemic is necessary – a laboratory assistant can cough on these mice, and if they are so sensitive, they will get sick and die without any sense. And according to some reports, very many people have an asymptomatic disease.
Our concerns are confirmed by the situation in the Netherlands, where mink farms were closed on the 20th of April. Minks are sensitive to infection with this virus: they became infected from the staff, began to die en masse. Farms are closed – to the point that there is no one to take out manure.
So that this does not happen in our nurseries, where mice are bred, or does not happen during transportation, we create mice where the expression of human genes is triggered by a signal.
Here we use a technology using the viral enzyme Cre-recombinase. This protein recognizes certain sequences in the genome, which are called loxP sites, and uses them to cut out a piece of DNA. That is, if we surround some genetic element with these loxP and then turn on the expression of Cre-recombinase, this element will be cut out.
This technology is already 20-30 years old. There are a large number of mice that express Cre recombinase in different organs and tissues. There are mice in which, for example, Cre-recombinase is expressed in lung tissues, but it is not expressed in the liver. Moreover, there are mice where the expression of this Cre-recombinase is triggered only after, for example, the mouse was fed the antibiotic doxycycline. You don't need to create them, you can just buy them.
We are collecting such a genetic construct. It will consist of coding sequences of two human proteins, TMPRSS2 and ACE2. There will be a regulatory element between them, which will ensure their simultaneous work in the cell. And in front of them there will be a stop cassette surrounded by loxP sites. This construct is inserted into the mouse genome in the area where its own TMPRSS2 gene is located.
When a cell differentiates, becomes, for example, an alveolocyte of the second type, it triggers the expression of a gene characteristic of it, this TMPRSS2. But when the TMPRSS2 promoter starts working, it starts scanning our design. And two human genes start working under the mouse promoter. But they will start working only when the stop cassette, which is located between them and the promoter, is removed. And it will be removed only after crossing with the corresponding Cre-mice.
The final mouse has two human genes in one chromosome under its own mouse TMPRSS2 promoter, blocked by a stop cassette. And a Cre recombinase is inserted into the other chromosome, which is expressed under the action of the antibiotic doxycycline.
Such a mouse will be brought to Vector, given doxycycline, and then Cre-recombinase will turn on in her body. It will remove the stop tape in front of human genes in the lungs. The lung promoter will start working, these genes will appear on the surface of the lung cells, and the mouse will become sensitive to the coronavirus.
Scheme for creating a Cre loxP mouse. Illustration: Vladislav Maslov.
Procedure of actions
The first stage of the work is molecular biological cloning, the assembly of genetic cassettes. We have already received the design with ACE2, many thanks to colleagues from Moscow State University and Peter Vladimirovich Sergiev, who was able to synthesize this gene himself. We are working with a gene that we obtained de novo in Russia. Now we are working on the development of the TMPRSS2 gene. It's a little more complicated.
The second stage is the microinjection of cassettes into fertilized eggs of mice.
The technological sequence looks like this: first we get female classical laboratory mice, small, immature – and cause them to superovulate.
From each such 12-gram female, we can get up to 50 eggs. When the sperm penetrates into the egg, a male pronucleus is formed, there is already a female pronucleus in the egg. And the male pronucleus in the egg approaches the female pronucleus, they merge, and division begins. At the moment when there are two pronuclei in a cell, we introduce into it the genetic construct that we have created. This is a matrix for repair, that is, we will embed copies of this design into the genome of mice. In addition to it, we introduce another plasmid – it contains a gene editor, the CRISPR/Cas9 system. The guide RNA is encoded in it, which indicates to Cas9 where to cut – in the TMPRSS2-promoter region. Thanks to this scheme, our cassette will be inserted not just anywhere, but in a specific place of the genome.
The egg is evaluated for viability, cultivated for some time. If the cell has survived, it is transplanted to the recipient female. It is surgically transferred to the funnel of the oviduct, that is, to the very beginning of the reproductive path of the mouse. The mouse is sewn up, left on the contents. Three weeks later, she gives birth to mice.
On May 18, we are due to have the first transgenic mice that will have only the ACE2 gene. TMPRSS2-mice will be born before the end of June.
Depending on how effectively this whole system has worked, we find the modification we need in the genome of these mice. The first offspring (the primary transgen) does not go to work. They are crossed among themselves, descendants are obtained, and they are already characterized – how many copies of the desired gene, where that got up. And already they are crossing with Cre-mice. And now the descendants of transgenes and Cre mice containing both transgen and Cre recombinase will go on to work on infection with the virus.
We expect the birth of mice with two genes already – both ACE2 and TMPRSS2 – in early June. We can send them to the "Vector", or their descendants. In any case, they will be there at the end of June. The final answer to the question of whether these mice will get coronavirus and what symptoms they will have will be available only there.
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