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Antisense and Gene Therapy Options for Duchenne Muscular Dystrophy Arising from Mutations in the N-Terminal Hotspot

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Prof. Annemieke Aartsma-Rus is taking on a challenge by reading and commenting on a paper a day. She shares her insights, findings and thoughts via her @oligogirl Twitter account. See below the overview of March 2022.

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Prof. Aartsma-Rus reads and comments on the paper titled: Antisense and Gene Therapy Options for Duchenne Muscular Dystrophy Arising from Mutations in the N-Terminal Hotspot

A review paper from Wilton-Clark and Prof. Toshi Yokota published in genes focusing on antisense oligonucleotide and gene therapy approaches for Duchenne patients with deletions in the N-terminal part of dystrophin. Doi 10.3390/genes13020257

Duchenne is caused by lack of dystrophin while Becker patients produce partially functional dystrophins. The focus of this paper is the beginning of the protein: the N-terminal part contains the most important actin binding domain.

75% of Duchenne patients have a deletion or duplication of one or more exons. Most of these cluster between exon 43 and 55, but a minor hotspot exists between exons 2-22 (encoding the N-terminal domain). Small mutations occur throughout the gene so also in the N-terminal domain.

Authors focus on how exon skipping and micro-dystrophin gene therapy can potentially help patients with N-terminal mutations. They start with exon 2 duplications which combine AAV gene therapy and exon skipping by delivering a modified U7 snRNP “antisense gene”.

U7 snRNP normally contains a protein part and an RNA part and is involved in modifying histones (the system that prevents DNA from getting tangled). However researchers have discovered you can replace the original RNA of U7 with an antisense RNA sequence of choice—> antisense gene.

U7 genes are very small: 4 fit within a self complementary AAV vector! Authors describe a method for AAV U7 snRNP antisense gene exon skipping they claim is developed by Astellas. However this exon 2 skipping gene therapy is developed by Kevin Flanigan and Wein.

This U7 snRNP AAV approach focuses on exon 2 skipping for Duchenne patients with an exon 2 duplication. As was presented by Kevin Flanigan Duchenne Parent Project Italy’s 2022 conference. Currently 3 patients have been treated so far.

Brief summary: 3 patients treated, muscle biopsy shows low level dystrophin restoration for 2 older patients. Patient 3 was 7 months old at treatment. No biopsy data yet but CK dropped dramatically suggesting improved muscle fiber integrity.

Authors discuss the challenges of AAV delivery, with significant safety concerns (liver toxicity, myocarditis, nerve toxicity and severe nausea and dehydration). Furthermore patients with antibodies against AAV are excluded from trials (and for now also treatment in the future).

Authors outline research is ongoing into new AAV serotypes to have a lower chance of pre-existing immunity and a reduced inflammatory response. I think the main goal is also to allow treatment with lower doses which likely will be much more tolerable.

Moving on to exon skipping using ASOs. Authors claim this can result in Duchenne patients behaving like Becker patients. I know I used to make claims like this but no more: 1. ASO treatment occurs when a lot of damage has accumulated. This can not be undone.

Earlier treatment likely will result in more benefit. 2. Levels of dystrophin restoration with ASOs that are approved are very low and suboptimal. This may still slow down disease progression and thus benefit Duchenne patients. However, it is important to provide realistic information.

Approved ASOs target exons 45, 51 and 53 and are approved in the USA and Japan (exon 53 only). This does not apply to patients with N-terminal mutations. Preclinical studies in IPSCS differentiated to cardiac cells show reading frame restoration for such mutations improves deficits.

Contractility+calcium handling improved, especially when an exon 3-9 deleted dystrophin was produced. It would be interesting to compare functionality of these dystrophins with in-frame deletions in major mutation hotspot, which contains N-terminal actin binding domain.

I would expect the latter to be more functional. Authors highlight their dog studies showing that a dystrophin lacking exon 6-9 improved muscle quality (MRI) and muscle function after treatment with exon 6 and 8 ASOs.

Exon skipping is mutation specific and most exons apply to small groups. Authors propose multiexon skipping as a potential solution for increasing applicability. They outline the regulatory hurdles of showing safety for individual ASOs and the combination.

However they do not discuss that multiexon skipping for most patients will mean they also receive ASOs that target exons deleted for them, so not therapeutic. The question is whether regulatory agencies will accept this (potential side effects for compound know to be ineffective).

Authors also summarize the microdystrophin gene therapy developments. For a more complete and up to date overview I again refer to my Italian Duchenne Conference tweets. Briefly, Pfizer, Sarepta and Solid are conducting clinical trials (and Genethon – not mentioned by authors).

A point I want to highlight is that in placebo controlled trials with micro-dystrophin patients receive the gene therapy after a year. So participation in a trial means treatment (unless a patient becomes AAV seropositive during the placebo year – hopefully this won’t happen).

Authors again stress the side effects seen for AAV gene therapy, mentioning also the patient in the non-ambulant Pfizer microdystrophin trial who sadly passed away. At the Italian Conference it was mentioned he had severe dehydration.

Authors state that microdystrophin may be a therapy for patients with mutations in the N-terminal domain. However, I do not agree. Currently patients with deletions in this area are excluded from microdystrophin trial participation, likely due to risk of anti-dystrophin immunity.

The microdystrophin has the N-terminal actin binding domain which patients with deletions in the N-terminal part miss. So this domain is seen as a foreign (immunogenic) epitope. I anticipated authors would discuss this aspect.

All in all a nice complete review. Authors are not to blame that information presented at the Italian Conference is not included as this was not known when they wrote the paper. However disappointed about them not discussing N-terminal microdystrophin immunity problem.

About Professor Annemieke Aartsma-Rus

Prof. Dr. Annemieke Aartsma-Rus is a professor of Translational Genetics at the Department of Human Genetics of the Leiden University Medical Center. Since 2013 she has a visiting professorship at the Institute of Genetic Medicine of Newcastle University (UK).

Her work currently focuses on developing antisense-mediated exon skipping as a therapy for Duchenne muscular dystrophy. In addition, in collaborative efforts she aims to bridge the gap between different stakeholders (patients, academics, regulators and industry) involved in drug development for rare diseases.

In 2013 she was elected a member of the junior section of the Dutch Royal Academy of Sciences (KNAW), which consists of what are considered the top 50 scientists in the Netherlands under 45. From 2015 to 2022, she was selected as the most influential scientist in Duchenne muscular dystrophy by Expertscape.

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