#apaperaday: The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy.

Today’s pick was read on the plane and is from Wasala et al in Human Gene Therapy on the finding that gRNA levels determine genome editing efficiency in mice. Work from Duan’s group doi 10.1089/hum.2021.130.

Genome editing requires an enzyme to cut DNA (cas9) and a guide RNA (gRNA) to tell cas9 where to cut. These components are usually delivered to muscle with AAV vectors. Cas9 is big so it fills one vector. The gRNA or gRNAs (for dual cut) go in the other AAV.

The authors showed in the past that in long term experiments gRNAs became depleted much more than cas9. Here they wanted to test if that also occurred after a short term (is it a general effect or a duration effect?) They treated 4 week old mdx mice and analyzed after 3 months.

Mice were treated with either equal amounts of cas9 and gRNA AAVs or in a 1:3 ratio. Authors showed also short term gRNA genomes are depleted preferentially (33% left compared to cas9). The 1:3 ratio resulted in 1:1 vector copies, while 1:1 ratio resulted in 1:0.4.

Not surprisingly the 1:3 ratio resulted in better editing and dystrophin restoration and better histological and functional effects. Authors discuss that not only they found a preferential depletion of gRNA vectors. Also others found it appears to occur in systemic treatment. Was shown for 3 different Duchenne mouse models, with AAV6 and AAV9, for saCas9 and spCas9 and for single and dual cut approaches in young and old mice.

The mechanism is not clear yet. Authors speculate that it could be the hairpin palindromic sequence of the guide, a reduced packaging efficiency for gRNA vectors (though that seems less likely as others found no difference for this)

The fact that gRNAs are the bottleneck is unexpected as Cas9 is the catalytic enzyme and the guides are reused. However if levels are lower they become the bottleneck of course. No editing without a guide.

While the mechanism is yet unclear it is good to know there is a preferential depletion of gRNA vectors for preclinical optimization & towards possible clinical trials. The question is whether using a higher dose for gRNA vectors is possible in humans due to tolerability issues.