Researchers use CRISPR and mini-organs to cure cystic fibrosis

Dutch scientist Hans Clevers and colleagues in Utrecht have cured cystic fibrosis in mini-organs grown from patient stem cells. Using the newly developed CRISPR-tool called base editors, they remove stop-codon mutations in the cystic fibrosis gene and turn it back into the original amino acid.

»I have never seen anything like CRISPR. Everything we try works. It's amazing,« says Hans Clevers, at the Hubrecht Institute, Utrecht University in the Netherlands.

On top of the editing success, the researchers found no evidence of off-target effects, which is one of the biggest safety concerns surrounding the use of CRISPR for gene therapy.

The research could pave the way for gene therapy based on two of the most promising new technologies CRISPR and mini-organs.

A mini-gut biobank

Mini-organs have attracted a lot of attention in the last decade, but what are they? Briefly, mini-organs or organoids are small pencil tip-sized lab-grown versions of organs, that are functionally similar and replete with the major cell types. The organoids can be grown from stem cells found in small tissue samples with the right nutrition and growth factors - 'the secret sauce' as Clevers calls it. Hans Clevers identified a cell in the gut that can give birth to all other intestinal cells and grew the first 'mini-guts' over a decade ago.

Collaborator Jeffrey Beekman at Wilhelmina Children's Hospital in Utrecht had the idea to develop the mini-guts into a cystic fibrosis functional assay.

The assay is currently used in the dutch health care system to predict if a costly drug called Orkambi by Vertex Pharmaceuticals will have an effect in individual patients.

»If you have a positive organoid test, you get the drug and so far it has always worked,« says Hans Clevers.

The problem is that it is challenging to predict if the drug will or will not work since many patients have a different version - cystic fibrosis can arise from over 2.000 mutations in just one gene, and many patients have rare or even unique mutations.

Over the years, the researchers have built a biobank that now holds mini-guts from 664 dutch cystic fibrosis patients - almost half of the country's cystic fibrosis population.

Erasing stop codon mutations with base editors

Having this unique resource Clevers and colleagues thought it would be ideal for studying if the latest CRISPR technology could be used to correct the mutations safely in human cells.

The so-called base editors were developed in 2018 and are Cas9 fusion proteins that can be guided to a specific sequence in the DNA and change one base-pair to a different base-pair. There are not base editors for all combinations but A-T to a G-C and vice versa exists and going through the biobank it turns out that about 20% of the patients could, in theory, be cured using the base editors. Importantly this includes patients with stop codon mutations for whom there currently is no medication.

Everything is done ex vivo

One great thing about organoids is that they are clonal, and everything is done in the lab outside the patient.

»So you can test for any off-target mutations before you transplant back to the patient,« says Hans Clevers.

»I think it's a significant advantage. You can do it hundreds of times and just pick the ones that worked exactly as you hoped. In vivo, you have to live with what comes out of the experiment.«

And another advantage working ex vivo is that circumvent problems with the immune system. The editing components are transferred without using viral vectors, and the Cas9 fusion-protein is long gone by the time the organoids would be transplanted back to the patient and come in contact with the immune cells.

So all in all, the combination of base editors and organoids seem to be an efficient and safe path to a cure for hereditary diseases using gene therapy.

Gene therapy cure for cystic fibrosis?

This begs the question of when the first cystic fibrosis patients may benefit from this organoid CRISPR gene therapy? It turns out the answer is more complicated than it might seem.

»If this is going to go into clinical trials, the field first has to show that organoids can be safely transplanted‚« says Hans Clevers.

Currently, there is no clinical practice for transfecting expanded cells whether it be induced pluripotent stem cell (iPS), embryonic or adult stem cells, or organoids. For each organ, the procedure would have to be worked out and optimized. But the first clinical trial using organoids is underway in Tokyo, Japan and maybe a first step for establishing a safe practice for transferring organoids back to patients.