‘Reserve gene’ switching on for gene therapy

A portion of the genes in our DNA serves as a recipe for proteins. Errors in these recipes form the basis of hereditary diseases, in which mutated proteins lose their function. To address this, current gene therapies focus on repairing defective genes by introducing new genes or elements from outside.

Now, researchers from the Hubrecht Institute, Erasmus MC, and Sanquin have discovered a new method for gene therapy. In doing so, they made use of the fact that not all genes are active at all times — some can be switched ‘off’. They managed to reactivate helpful but dormant genes by bringing them closer to the genetic switches on the DNA, known as enhancers. They achieved this by cutting out the intervening stretch of DNA using CRISPR-Cas9, a technique they call ‘delete-to-recruit’. Geneticist Wouter de Laat, who recently joined UMC Utrecht, led the research at the Hubrecht Institute: ‘We have shown that a specifically reactivated gene can take over the function of the defective gene in hereditary blood disorders such as sickle cell disease and beta-thalassaemia.’

Reactivation

In patients with sickle cell disease and beta-thalassaemia, the adult globin gene is broken. That stretch of DNA normally codes for the production of haemoglobin, a protein in red blood cells that is essential for oxygen transport. But alongside it lie two foetal genes as well. These are responsible for haemoglobin production during the foetal stage of life, after which they are switched to inactive.

A few years ago, researchers demonstrated that an inactive foetal gene could be switched back on by creating a loop in the DNA, reducing the distance between the enhancer and the foetal gene. This causes a cell with a defective adult globin gene to nonetheless begin producing globin. ‘That this was possible came as a complete surprise to me,’ says De Laat. ‘It was a source of inspiration for us. We are now showing for the first time that you can achieve the same result with our “delete-to-recruit” technique, in which we remove the intervening DNA.’

Precisely why the inactive gene switches on due to the nearby enhancer remains not entirely clear. Enhancers are known for attracting transcription factors — proteins that promote the transcription of DNA into RNA for the production of new proteins. ‘The classical idea is that a nearby enhancer increases the concentration of relevant transcription factors, leading to greater transcription at active genes,’ says De Laat. ‘Inactive genes, on the other hand, are switched “off” by transcription-repressing factors. Our results therefore seem to suggest that the transcription-stimulating factors from the nearby enhancer override the transcription-repressing ones.’

Stem cells

De Laat’s research group collaborated with Sanquin and Erasmus MC to test their technique in red blood cells. ‘Sanquin also had the ambition to bring gene therapy for sickle cell disease and beta-thalassaemia into the clinic,’ says De Laat. As an organisation that conducts blood research and is responsible for the blood supply in the Netherlands, Sanquin provided primary stem cells from healthy donors. ‘We wanted to investigate whether our deletion technique could be applied as gene therapy in stem cells.’

The researchers succeeded in applying their technique to the stem cells, which subsequently matured into red blood cells that produced foetal globin. The results were even more successful than anticipated. ‘We thought the deletions would be too inefficient to use as a therapy,’ says De Laat. ‘But it turned out to work in more than half of the stem cells. The resulting foetal globin production was just as effective as with other established gene therapies.’

Scaling up

Before the deletion technique can be deployed as a therapy, CRISPR-Cas9 must more efficiently edit as many stem cells as possible whilst keeping them viable, says De Laat. ‘That remains a major challenge.’

Beyond scaling up, safety considerations also present a significant hurdle. ‘The last thing you want is for the Cas9 protein to start cutting in places where it shouldn’t. We still need to conduct further research into the safety of our technique. It is therefore estimated that it will be another two to three years before we can actually offer this as a therapy.’