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CRISPR editing as a therapeutic strategy for Duchenne muscular dystrophy—anti-Cas9 immune response casts its shadow over safety and efficacy.

#apaperaday: CRISPR editing as a therapeutic strategy for Duchenne muscular dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: CRISPR editing as a therapeutic strategy for Duchenne muscular dystrophy—anti-Cas9 immune response casts its shadow over safety and efficacy.

Today’s pick is a commentary by JJ Dowling from the journal Gene Therapy on genome editing and cas9 immunity for Duchenne doi 10.1038/s41434-022-00323-8. This is a commentary on a previous paper-a-day

Duchenne is caused by lack of dystrophin due to mutations that prevent production of functional proteins. Mutations that allow production of partially functional dystrophins are associated with Becker (milder).

Different approaches aim to restore dystrophin, e.g. AAV micro-dystrophin (only the genetic code for the  bare essential domains of the dystrophin protein fit in AAV) or stop codon readthrough. Authors say stop codon readthrough so far is not very successful. 

But based on clinical trials suggesting a slower decline the European Medicines Agency (EMA) conditionally approved it already in 2014. Post marketing studies so far suggest a slower decline. Additional data is being collected as part of the ‘conditions of conditional approval’. 

Back to the commentary: Exon skipping and genome editing allow Duchenne patients to produce Becker dystrophins – where we know they are partially functional (which is not yet the case with micro-dystrophin). Exon skipping requires repeated dosing, while genome editing would be a 1 time treatment.

Genome editing is only in preclinical development so far however, due to several concerns. AAV is needed to deliver the gene editing components (cas9 or editors (that do not cut but edit) and CRISPR to guide the cas9/editor)). High doses of AAV can lead to severe adverse events.

These include thrombocytopenia, liver failure and sadly one micro-dystrophin AAV treated Duchenne patient died so far. Another concern is the irreversible aspect of genome editing. Not a problem if the editing is done correctly, but there is a possibility of off target edits.

The topic of the commentary and the paper by Dongsheng Duan is about yet another concern: immunity against cas9 or the other editors. These are bacterial proteins, so many people will have preexisting immunity as they encounter cas9 / editors.

It is estimated that ~80% of adults have preexisting immunity against cas9. It is not yet clear what percentage of children has this preexisting immunity. The work for Duan shows that in dogs puppies often do not have anti-cas9 immunity while some adult dogs do.

Doing local genome editing with intramuscular injections in dogs with preexisting immunity causes an anti cas9 immune response that results in reduced levels of dystrophin. After systemic treatment there was also an immune response but no reduction in dystrophin was seen so far.

JJ Downing outlines that while this is concerning, there are also limitations to the Duan study: the number of dogs is small (though results are robust), so far only 2 time points were assessed. As such it is not known what  longer term effects would be of systemic treatment.

There may be potential solutions, e.g. ex vivo modulation and stem cell transplantation would prevent anti-cas9 immunity. However, stem cell transplantation for Duchenne to restore dystrophin is not an option as delivery of stem cells to muscle is extremely inefficient as yet.

Other options may be better immune suppression or to develop newer cas9 proteins that are not recognized or to generate tolerance before the actual genome editing treatment. While the Duan paper outlines yet another concern for genome editing for Duchenne. The good thing is that now we know and we can try and find solutions – as is outlined by the author of the commentary.


Pictures by Annemieke, used with permission.