The first formal study involving the gene-editing technique CRISPR/Cas9 has been welcomed by more than the usual CRISPR enthusiasts.
The trial based in Germany and backed by US company Vertex Pharmaceuticals will assess gene therapy ‘candidate’ CTX001 in patients with the condition ?-thalassaemia.
It was excellent news for market analysts keen to flag-up potential for fiscal returns and growth of shareholder value.
CRISPR has been lauded as the go-to method for gene editing, yet to be translated into results in terms of new therapies to combat obscure genetic diseases. And now the growing number of CRISPR sceptics have fresh ammunition.
A report published in July by the Wellcome Trust Sanger Institute (WSI) claims evidence that in some CRISPR/Cas9 experiments, sections of DNA appear to be scrambled. Some sections appear to be deleted and others appear to be replaced with new ones. It is a worrying conclusion for all involved.
This first formal trial in Germany, will see up to 12 adults (aged 18-36) with the inherited blood disorder enrolled and has a target date for completion on May 2022. The report itself is unlikely to be ready until some months after that.
? Thalassaemia is an ideal target. It is a well- understood genetic condition in which the production of haemoglobin, the iron-chelating protein in red blood cells, responsible for transporting oxygen to cells throughout the body, is severely reduced.
The underlying cause was identified some years ago as mutations in the HBB gene on chromosome 11 inherited in an autosomal recessive fashion. The severity of the disease depends on the nature of the mutations.
The aim of the trial, at Regensburg Hospital, will be to treat haematopoietic stem cells, cells taken from the bone marrow of participants. They will be genetically modified in the laboratory so they should be able to produce high levels of foetal haemoglobin.
These manipulated cells will then be reintroduced into the patient to hopefully produce large amounts of red blood cells containing foetal haemoglobin, to overcome the haemoglobin deficiencies.
Heather Nichols, for Vertex, said: “This is one important step of many toward bringing the promise of this new technology to patients with serious diseases like sickle cell disease and ? thalassemia.”
The CRISPR/Cas9 gene ‘editing’ technique evolved out of studies of antibiotics and the intricate mechanisms which bacteria employ to build resistance and to attack viruses which threaten them.
Central to CRISPR are two molecules, which effectively work as a pair of scissors and a microscopic sat nav. The mutant ? globin gene that drives the ? Thalassaemia is not altered, but rather switches on the foetal haemoglobin gene. The theory is that CRISPR-Cas9 introduces ‘small’ deletions into an “enhancer” gene called BCL11A in hematopoietic stem and progenitor cells extracted from patients.
But what should be made of the WSI findings that CRISPR may be messing up sections of DNA not being targeted for change?
Exploration of Cas9-induced genetic alterations has so far been limited to the immediate vicinity of the target site, leading to the conclusion that CRISPR–Cas9 was reasonably specific. WSI think that conclusion could be wrong.
In the article (Nature Biotechnology volume 36) the group say significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line were detected.
Professor Allan Bradley co-author said: “Changes in the DNA have been seriously underestimated. It is important that anyone thinking of using this technology proceeds with caution, and looks very carefully to check for possible harmful effects.”
Michael Kosiki, lead author on WSI, report, said: “The initial experiment used CRISPR/Cas9 as a tool to study gene activity. However, it became clear that something unexpected was happening. Once we realised the extent of the genetic rearrangements, started looking at different genes and different therapeutically relevant cell lines.”
Using long-read sequencing and long-range PCR genotyping, the indications are that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions. These extended over many kilobases. They suggest that CRISPR–Cas9 editing could actually have pathogenic consequences. Others have expressed concern.
Professor Maria Jasin, of Memorial Slone Kettering Cancer Centre, New York, who was not involved in the study says:
“This WSI study is the first to assess the repertoire of genomic damage arising at a CRISPR/Cas9 cleavage site. While it is not known if genomic sites in other cell lines will be affected in the same way, further research and specific testing is needed before CRISPR/Cas9 is used clinically.”
The progress of German trial will not be affected, but WSI’s report suggests CRISPR/Cas9 gene editing will not be given a free rein as the key technique for unlocking pathways to therapy for genetically based diseases.
Information on the trial can found at: https://clinicaltrials.gov/ct2/show/NCT03655678
By Dermot Martin