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#apaperaday: Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled:  Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy

Today’s pick is from Biophysics Reviews on delivery challenges for CRISPR/cas9 tools for Duchenne by Harumi Padmaswari et al. Doi 10.1063/5.0131452.

Duchenne is caused by lack of dystrophin due to mutations in the dystrophin gene that make the code unreadable. Readable mutations allow production of partially functional dystrophins and are associated with the later onset and less progressive Becker muscular dystrophy. Exon skipping aims to make the dystrophin gene code readable so Duchenne patients can make a Becker like dystrophin. However, currently this requires weekly intravenous infusions as there is turnover of the exon skipping tools (ASOs), target transcripts and proteins.

Gene editing targets genes rather than transcripts, so each time a transcript is made, it is readable & a Becker dystrophin can be made. This requires delivery of tools for gene editing (cas9: DNA scissor & guideRNAs: GPS guiding the scissor to the correct location in the DNA).

It is not enough to deliver these tools to one muscle fiber in one muscle. Rather, because Duchenne affects almost all of our >700 muscles, delivery has to be to the majority of muscle fibers in all skeletal muscles. This is challenging.

Authors outline types of editing tools. Meganucleases & zinc fingers are laborious to produce. Talens are less laborious & can target all DNA motifs. Crispr/cas9 is easy to produce, but cannot target each region, only those close to a PAM sequence (protospacer adjacent motif).

Still generally the ease of using the CRISPR/Cas9 system outweighs the fact that Talens can target each region – especially since there are many PAM motifs in the genome. Paper contains a HUGE table summarizing gene editing efforts in Duchenne with different gene editing tools.

This also includes newer tools that do not rely on cutting the DNA, but can edit them (base editors) or add small pieces of DNA (prime editors). These can work for correcting small mutations, but can also make the code readable again.

The original gene editing tools cut DNA (double strand break) and there is a risk of off target effects (cutting DNA in unintended regions). For base and prime editors this risk is less, but there is a bystander effect: editing of other sequences in area that is targeted. So how to deliver these tools? To the majority of muscle fibers and muscles? In cultured cells people use transfection or electroporation but these methods cannot be used to deliver to muscle.

Most researchers have used the adenoassociated virus (AAV), that is also used for micro-dystrophin gene therapy. This is the only virus that can efficiently deliver to skeletal muscle. However, as cas9 is a big protein, you need 2 AAVs, one for the scissor and one for the guides.

Smaller scissor proteins (cas9) have been produced to overcome this challenge. However, these modified Casses have more target sites requirements so are less flexible. AAV does not efficiently target satellite cells, which means with time the edited muscle fibers will be lost.

Another challenge is that while AAV normally does not integrate, when a DNA break is present (induced by the scissor) it does and this can have negative consequences (such as cancer). Alternative viruses are available such as lentivirus. These can contain both the scissor and the guides but they do not infect muscle efficiently. Adenoviral vectors can be & can contain a lot of genetic information, but do not infect muscle efficiently either.

However, for adenovirus, modifications can be made so they target muscle better. As an alternative to viral vectors, synthetic delivery tools can be used, such as lipids and polymers. Lipid nanoparticles have been used to deliver locally to muscle (the covid-19 vaccines).

So far delivery body wide to skeletal muscle is not possible. Advantages of synthetic delivery tools is that they can deliver mRNA encoding for the scissor or the scissor protein. That means the scissor will only be around for a limited time & reduces the chance of unintended cuts.

Finally virus like particles are being developed that contain proteins/peptides that are used by viruses to deliver genes to skeletal muscles. Authors wonder whether these particles can lead to an immune response – like the one induced when using viral vectors.

Authors outline additional challenges, such as the fact that gene editing is a mutation specific approach, and that not only skeletal muscles are affected but also heart, smooth muscle and other tissues. Also, not only immunity to viral vectors is an issue, but also immunity to the scissors. Cas9 & related scissors are derived from bacteria: many people have immunity to these proteins.

Even if people do not have preexisting immunity, the proteins are immunogenic and a treated individual can generate an immune response. The risk here is an auto-immune reaction where the tissues expressing the scissor proteins are broken down by the immune system.

Work is ongoing to make the scissors less immunogenic. Also for non-viral delivery there may be immune responses, e.g. to mRNA or components in the particles or polymers (e.g. PEG can also elicit an immune response). It is clear that a lot more work is needed in this field. A very nice and comprehensive review paper that I enjoyed reading.