Sleep, my beauty
The "sleeping beauty" genetic editing system was taught to work almost without misfires
Polina Loseva, N+1
German biologists have improved the "sleeping beauty" genome editing system – a method of delivering genes to a cell based on mobile elements. The new version of the method avoids uncontrolled embedding of the "cargo" into the genome. It works on both adult and stem cells, and the CAR-T gene therapy technology with its use becomes faster and safer. The work was published in the journal Nature Biotechnology (Querques et al., A highly soluble Sleeping Beauty transposase improves control of gene insertion).
The easiest way to edit the genome is to use viruses, which owe their existence to the ability to "write" their genes into someone else's DNA. All approved methods of gene therapy work on the basis of neutralized viruses, or viral vectors. However, this method has its disadvantages: for example, viruses are immunogenic, and even a neutralized version of them causes immune aggression, so rejection of edited cells is possible.
Recently, methods of "point editing" are gaining more and more popularity – for example, the CRISPR/Cas9 system or "zinc fingers". These systems allow you to embed the "cargo" not in a random, but in a strictly defined place on the chromosome. But they also have drawbacks: they miss every now and then (this is called inappropriate editing), and they also do not allow you to embed a sufficiently large load into the genome.
There is also a third direction of genome editing – using mobile genetic elements, or transposons. These are sections of DNA that are able to cut themselves out of one place of the genome and embed into another – for this they encode the enzyme transposase. Many such sequences in our genome have lost activity over time. But in 1997, scientists restored one of the ancestral variants of transposase, which "fell silent" in the fish genome millions of years ago – and named it "sleeping beauty", in honor of a long "sleep" and a sudden "awakening".
"Sleeping Beauty" has already been tried on human cells, and some technologies with its use have reached the 1-2 phase of clinical trials. This editing system is arranged as follows: a plasmid (ring DNA) with a transposase gene is injected into the cell and another plasmid with a "load". The cell produces a transposase, and that recognizes the "load", cuts it off from the plasmid and makes breaks in the DNA, in place of which the load is embedded.
This system works efficiently, but there is one difficulty with it: it does not allow you to control the work of the transposase – how many times it will be able to embed the "load" into the genome and whether something superfluous will get with it. Therefore, cells that are edited in this way are then cultured for a long time – up to a month – to make sure that there are no unnecessary mutations in them. But not every patient – especially when it comes to CAR-T therapy, when a receptor for tumor proteins is "implanted" into immune cells – can wait so long.
Irma Querques and her colleagues from the European Molecular Biology Laboratory (EMBL) in Heidelberg have developed a new version of the system based on the "sleeping" beauty. They proposed to introduce into the cell not a plasmid with the enzyme transposase, but a protein in its pure form. Previous attempts to achieve this have failed, because the transposase sticks together in lumps and passes poorly through the membrane.
The researchers replaced two amino acids in the composition of the transposase, which made the protein more soluble in water. They placed the resulting "hyper-soluble" transposase into cells using electroporation and compared the result with a conventional plasmid: the pure protein gradually disappeared from the cells within a day, and the plasmid remained in place for at least 5 days. This means that the action of the "improved" transposase will be short, and therefore, it will be able to introduce fewer unnecessary mutations into the genome.
The scientists tested their method on HeLa tumor cells, then on embryonic stem cells and on hematopoietic cells. And only after that we decided to apply it in real technology – CAR-T. The researchers took human T cells and inserted into them a chimeric receptor gene that recognizes B-cell lymphoma. The embedding efficiency turned out to be about 20-30 percent – about the same as in the real CAR-T technology, which is used in the clinic and which is based on viral gene delivery.
In culture, the edited cells destroyed more than 60 percent of the tumor cells in their path, and also cured mice with lymphoma within a week. At the same time, in each T-cell, scientists found about 5 gene embeddings in DNA – this is two times less than if a ring DNA with a transposase gene got into the cell. In addition, in about a quarter of cases, the gene was embedded in the "safe" places of the genome – this is about as often as if it was inserted randomly, and 8.5 times more often than HIV-based vectors.
The authors note that their new system can advance editing using transposase in clinical trials. It works safer than previous similar methods, but allows you to carry more weight than point editors like CRISPR/Cas9. The ideal option, according to the researchers, would be a synthetic design that combines CRISPR/Cas9 accuracy and a low number of transposase misfires.
The debate about the safety of genetic editors continues. Recently, for example, scientists have shown that the offspring of edited animals does not carry unnecessary mutations. And they also came up with a new editing method, which turned out to be even more accurate than the classic CRISPR/Cas9 system.
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