20 February 2020

Curing genetic disease in human stem cells

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Whereas the CRISPR-Cas technology developed in 2012 cuts out a defect in a gene and replaces it with a new piece, the latest CRISPR technology works differently. The aim is to repair the error in the DNA without cutting it. This theoretically makes it a safer form of genetic editing. Scientists from Utrecht have shown for the first time that this technique can effectively and safely repair the DNA of stem cells derived from cystic fibrosis patients in the lab. The results of this study were published in the scientific journal Cell Stem Cell on the 20th of February.

The new CRISPR enzyme variant developed in the United States in 2018, is a technique that works more precisely and makes fewer errors, explain researchers Maarten Geurts from the Hubrecht Institute and Eyleen de Poel from UMC Utrecht. Maarten: “With the traditional form of CRISPR-Cas, a certain piece of DNA is cut away, after which the cell has to repair itself, hopefully by replacing it with a ‘good’ piece of DNA that is made in the lab and presented to the cell. With the new form of CRISPR-Cas, called base-editing, the mistake in the DNA is identified, but not cut and replaced; it is repaired on site.”

Previous research has shown that this new method of genetic correction is safe in stem cells of rice and mice. The Utrecht study has shown that CRISPR base-editing also works effectively and safely on human stem cells. The results of this research led by professors Hans Clevers (Hubrecht Institute) and Jeffrey Beekman (UMC Utrecht) were published in Cell Stem Cell.

Mini intestines
The foundation Hubrecht Organoid Technology and UMC Utrecht jointly manage a biobank with so-called intestinal organoids. These are a kind of mini-intestines, grown from stem cells from patients with Cystic Fibrosis (CF). These organoids are used to investigate what exactly goes wrong in cystic fibrosis. In addition, they are used to determine which medicine works for which CF patient, but also to test new treatments, such as CRISPR-Cas. For the current study, they organoids were used to investigate whether CRISPR base-editing works on the DNA in human stem cells, and whether it can be used to cure CF in the mini intestines.

What goes wrong in CF?
CF is caused by an error, also called a mutation, in the CF gene (CFTR), resulting in a non-functional gene. Like all genes, the CFTR gene is a small part in a large DNA molecule, or chromosome. Each chromosome consists of a long series of four types of building blocks – nucleotides – called A, T, G, and C. The order in which these nucleotides are linked together determines the genetic information. The nucleotides form a long series of three-letter words that give the cell the information it needs to make a functional gene product. The gene involved in CF consists of around a quarter of a million letters. Most CF patients miss three letters, which is exactly one DNA word.

For a smaller group, there is a premature stop codon. Normally, a stop codon is a three-letter DNA word at the end of the sequence of a gene, that signals to the cell that the gene ends there. Due to a mistake in the three-letter word, for example due to an A at the place where normally a G should be, a stop codon can occur too early. This makes the cell think the gene ends while it doesn’t. This could be remedied by changing the A in the stop codon back toG.

These two groups of CF patients therefore have a different flaw in their CFTR gene but with the same result: no working CFTR protein is formed by the cell. In the body, CFTR is needed for the transport of chloride and water through the mucosal cells of many organs. If that does not happen, the mucus in the organs is not hydrated enough and it becomes tough. As a result, various organs such as the lungs, pancreas and reproductive organs do not work well and the function of these organs gradually declines.

For the large group of CF patients, with the missing DNA word, effective medication is now available. For the smaller group however, with the error in the stop codon, there is no medication. That is why the new CRISPR base-editing technique was tested on stem cells from CF patients with the premature stop codon.

How can CRISPR base-editing help?
Maarten explains what the new CRISPR-Cas technique does in the CFTR gene. “The Cas part of the traditional CRISPR-Cas technique, identifies the wrong piece of DNA and cuts it away. By adapting this Cas part, it still identifies the DNA mistake in question, but does not cut it away anymore. The goal of CRISPR base-editing is to restore the CF gene by restoring the wrong A back to a G. The CRISPR-Cas base-editing enzyme therefore has a protein attached to it that destroys the A in such a way that it resembles a G. The cell – which repairs broken nucleotides itself – now recognizes it as G and repairs it as G. Thus, the stop codon is no longer seen as a stop codon and the CFTR gene can be completely restored.

In this way, we succeeded in repairing the CFTR genes with a stop codon in mini intestines from the biobank: the cells in the repaired mini intestines produced the correct CFTR protein.” Maarten says that the great thing about CRISPR base-editing is that it appears to only do what it should do. “Because that is the biggest disadvantage of traditional CRISPR-Cas technology: it appears that it not only cuts out the “error”, but also cuts away small pieces of DNA that are very similar to the error. This results in unintended DNA damage. The new technology is more accurate, recognizes the wrong DNA sequence better and only changes the DNA there. This has also been confirmed in our investigation, we have not found any additional DNA damage in the repaired mini intestines. CRISPR base-editing therefore not only appears to be effective, but also safe.”

Organoids with cystic fibrosis (left) that do not swell due to a mutation in the CFTR-gene and organoids in which this mutation is repaired (right) that do swell because the CFTR-gene is functional again. Credit: Eyleen de Poel en Maarten Geurts, copyright UMC Utrecht and Hubrecht Institute.

How to proceed?
The demonstration that this technique works in the laboratory does not mean that patients can already benefit from it. Eyleen: “This research represents a big step towards genetic repair of diseases in patients, however, a big challenge that remains is to direct the CRISPR enzyme to the right places in the body. CF is not the easiest condition in that regard, because it affects so many different body functions. The reason we used CF cells for our research is because we have this unique biobank with mini intestines. In conditions affecting a single organ or cell type, such as sickle cell anemia, the use of CRISPR-Cas has been promising. Further research is needed to see whether this new CRISPR base-editing technique can also be applied in the clinic. But partly due to our research, we may already expect the first clinical application within the next five years.”

In addition, there are of course ethical aspects to the genetic editing of cells. Eyleen: “We focus on repairing broken cells. Theoretically, this technique can also be used for changing cells, for example, in embryos. If this is possible, that does not mean it is also desirable. A societal discussion is needed to determine how we should deal with this as a society.”

Eyleen de Poel and Maarten Geurts. Copyright UMC Utrecht.

CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. M.H. Geurts*, E. de Poel*, G.D. Amatngalim, R. Oka, F.M. Meijers, E. Kruisselbrink, P. van Mourik, G. Berkers, K.M. de Winter-de Groot, S. Michel, D. Muilwijk, B.L. Aalbers, J. Mullenders, S.F. Boj, S.W.F. Suen3, J.E. Brunsveld, H.M. Janssens, M.A. Mall, S.Y. Graeber, R. van Boxtel, C.K. van der Ent, J.M. Beekman†, H. Clevers†. Cell Stem Cell 2020.
*These authors contributed equally – †These authors are co-corresponding authors

Hans Clevers is group leader at the Hubrecht Institute and the Princess Máxima Center for Pediatric Oncology, professor of Molecular Genetics at the UMC Utrecht and Utrecht University, and Oncode Investigator.

Jeffrey Beekman is group leader at the UMC Utrecht and part of the Regenerative Medicine Center Utrecht, and professor of Cellular Disease Models at the UMC Utrecht.