Gene therapy for myotonic dystrophy type 1 also requires epigenetic intervention – Removing a genetic mutation alone appears to be insufficient

Correcting a genetic mutation does not automatically repair the epigenetic changes. Rick Wansink discovered this during research into myotonic dystrophy type 1. The finding, published in Nature Communications, also has consequences for the development of therapies for other similar conditions.

Myotonic dystrophy type 1 (MD1) is a hereditary muscle disease in which the muscles become increasingly weak and tense muscles relax more slowly than normal. Other organs may also function less well. The disease is caused by an error in the DMPK gene. At the end of this gene is a three-letter piece of DNA (CTG) that is repeated hundreds, sometimes thousands of times and often becomes longer during life. The longer this piece of ‘stuttering DNA’, the more serious the complaints. Within families with DM1, the disease starts at a younger age with each new generation and the symptoms are also more severe, because the ‘tail’ of CTG repeats becomes longer and longer.

Stuttering DNA

Rick Wansink, molecular cell biologist at Radboud university medical center, has been researching diseases caused by such pieces of stuttering DNA for decades. “Most hereditary disorders are caused by mutations in a gene that cause the associated protein to no longer work properly,” says Wansink. “The situation is very different with diseases such as myotonic dystrophy. There the problem is caused by the long RNA tail, which is created during the translation of DNA into protein information. In the case of myotonic dystrophy, all kinds of other well-functioning proteins can stick to the RNA tail, causing them to no longer work. In this way, the RNA tails disrupt more and more processes in the cell, causing it to function increasingly poorly.”

Molecular adhesive tape

Wansink’s group investigates how these processes lead to disease at the molecular level and how you can do something about it. Which strategies are likely to achieve treatment? Because to date there is no therapy available for this disease, even though there are several in the pipeline. A pipeline that is based, among other things, on previous work by Wansink: “My group was one of the first to introduce therapeutic antisense oligonucleotides in DM1 and the first to delete the CTG repeats in DM1 in human cells via CRISPR/Cas9 technology. Antisense oligonucleotides – AONs – are molecular adhesive tapes with which you can tape off the disruptive pieces of RNA, allowing the processes in the cell to proceed normally again. Using CRISPR/Cas9 technology – molecular scissors – we have shown that you can cut the repetitive tail from the DMPK gene.

Block or cut

Wansink’s fundamental research has consequences for the practical development of therapies. He recently investigated whether it makes a difference whether you use AONs, those molecular patches, to cut the RNA tail into pieces or block its effect. “If you block the tail, it loses its magnetic attraction to all kinds of proteins,” says Wansink. “If you cut the tail into pieces, you actually expect the same effect. However, it is better to opt for a blocking sticker, as research by PhD student Najoua El Boujnouni has shown in a collaboration with biochemist Roland Brock. The sticker that encourages cutting not only sticks to the RNA tail of DM1, but also to RNA of other proteins. It is therefore less picky and causes side effects in the cell. We had not estimated it that way in advance. So I would rather start a trial with a blocking sticker than with a breaking sticker.”

Epigenetic packaging

Wansink’s group recently published research in Nature Communications that builds on previous CRISPR/Cas research. The tail can be removed from the DNA with the molecular CRISPR/Cas scissors. This is a potentially curative treatment, but application in the clinic still requires the necessary research. It is not yet known exactly what role epigenetics plays, the ‘packaging’ of the genes. In myotonic dystrophy type 1, the repetitive CTG stretch in the genes becomes longer and longer. In the patient himself, but also in subsequent generations. In the most serious, congenital form of the disease, the increasing stuttering of the DNA is also accompanied by methylation, with epigenetic changes that package the DNA in a different way. Wansink wanted to know what role epigenetic packaging plays in – possibly therapeutic – changes in the DNA and contacted Rachel Eiges in Jerusalem, a specialist in the field of epigenetics surrounding stuttering DNA.

Differentiation makes a difference

Wansink: “The research with the group in Israel quickly showed that if we remove the DM1 mutation in human stem cells with CRISPR/Cas, that separate epigenetic packaging immediately disappears. But once those stem cells have developed into myoblasts or fibroblasts, into muscle or connective tissue cells, we can remove the genetic mutation that causes DM1, but the epigenetic packaging remains intact! If we turn those muscle or connective tissue cells into stem cells again, it will work again. Rachel Eiges and I came to the conclusion that specific enzymes apparently maintain epigenetic changes in those more mature, differentiated cells, but not in stem cells. That is an important insight. Not only for myotonic dystrophy, but also for similar hereditary conditions such as Huntington’s disease, fragile X syndrome and various forms of spinocerebellar ataxia.”

Locked packaging

Wansink’s research puts an end to the generally accepted idea that correcting a genetic mutation always involves reversing the epigenetic changes. The fact that a difference has now been found between the response in stem cells and more mature cells suggests that differentiation of cells functions as a kind of epigenetic lock. Wansink: “Correction of the DNA stuttering in cells of patients with myotonic dystrophy type 1 can solve some, but not all, associated problems. Our discovery may help develop new strategies for treating myotonic dystrophy type 1 by targeting the epigenetic machinery in addition to the genetic mutation.”

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Publication in Nature Communications: Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus – Tayma Handal, Sarah Juster, Manar Abu Diab, Shira Yanovsky-Dagan, Fouad Zahdeh, Uria Aviel, Roni Sarel-Gallily, Shir Michael, Ester Bnaya, Shulamit Sebban, Yosef Buganim, Yotam Drier, Vincent Mouly, Stefan Kubicek, Walther JAA van den Broek, Derick G. Wansink, Silvina Epsztejn-Litman & Rachel Eiges