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#apaperaday: Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Humanized DMD Mouse Model

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Humanized DMD Mouse Model.

Today another @biorxivpreprint , from Chey and @Fatwa_Adikusuma et al on generating a single copy humanized Duchenne mouse model using our hDMD mouse model as a starting point. So I am biased and also can give spoilers from our group. DOI 10.1101/2024.03.25.586713

Dystrophinopathies are caused by mutations in the HUGE dystrophin (formally DMD) gene. Lets reflect on its size of ~2.2 megabases – this means the DMD gene is 0.75 mm long, if we use the 0.34 nm distance between basepairs. i.e the size of a small ant!

The information contained (2,200,000 basepairs) is also HUGE. Book 5-7 from Harry Potter contains less letters than the DMD transcript (i.e. RNA). Back to the paper: there are currently mutation specific approaches in development for Duchenne to restore dystrophin.

Exon skipping using antisense oligonucleotides (ASOs) target the DMD transcript (RNA), while gene editing using CRISPR/Cas9 targets the DMD gene (DNA). Both try to make the genetic code readable again so an internally deleted, partially functional dystrophin can be produced.

The challenge with mutation specific approaches is with testing in animal models, usually the human version will not target the model system DMD gene due to species differences in the DNA. Thus a humanized model was produced decades ago by Johan Den Dunnen. Generation and characterization of transgenic mice with the full-length human DMD gene.

This model contains a full human DMD copy from a yeast artificial chromosome integrated in mouse chromosome 5. It produces human dystrophin which can compensate for lack of mouse dystrophin when crossing the hDMD mouse with an mdx mouse.

The challenge of this model is that it produces dystrophin, so with mutation specific approaches you cannot test for dystrophin restoration or reduction in pathology and functional deficits. Furthermore, with ASOs biodistribution is different for dystrophic vs healthy muscle.

Thus, my group generated the hDMDdel52/mdx mouse which has a deletion of exon 52 in the human gene in a mouse dystrophin negative background. Skipping exon 51 or editing it out, restores dystrophin production. This mouse took FOREVER to make. A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

Homologous recombination (tool back then) succeeded in creating a deletion, but then upon clone expansion and more checks, exon 52 would magically reappear as well. After 1000s of clones, we resorted to talen technology and then managed at last to make the deletion exon 52 model.

Then later we discovered the cause of our failed recombination attempts: the hDMD transgene is present in 2 copies, with a tail to tail integration. So using recombination we had a deletion of one copy but not the other… Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model

The 2 copy model (or 4 copies when you breed the mice homozygous) can be used for optimization of human specific exon skipping ASOs targeting exon 51 or 53. However, authors argue that for gene editing having multiple copies makes things more challenging.

e.g. when editing is too efficient you will remove everything between the target sites. Because the genes are in a tail-to-tail orientation the resulting gene remnants will not produce dystrophin.

Thus authors set out to remove one of the copies. To this end, they first did short and long read sequencing. This confirmed the duplication as there were 2 times more reads from human chromosome X than from mouse genome. Using the reads authors could construct the insertion (see image).

As one can see, there are also still yeast sequences present at both sides of the hDMD genes and between the 2 genes there are also yeast sequences. As the neo cassette was present only once, authors made guide RNAs targeting neo and hygro to remove one copy.

Authors injected these guides and Cas9 into hDMD embryos and this resulted in 1 male founder with a single copy of the hDMD gene, from which authors generated a line. The single copiness was confirmed with sequencing.

Authors confirmed the mouse was able to produce dystrophin by western blot & immune fluorescence. Authors note dystrophin levels produced by the single copy hDMD mouse is less than that of mouse dystrophin, probably because the human promotor does not function optimally in mouse.

The mice had normal strength and no pathology in muscle and normal CKs (elevated creatine kinase (CK) levels in blood are a marker of muscle damage) as expected. Authors discuss that they managed to delete a huge amount of DNA with gene editing (indeed, 0.75 mm ???? and respect!)

Authors mention in the discussion that with the single copy hDMD model it will be easier to make deletion mouse models. However, CRISPR/Cas9 is incredibly efficient and we have since been able to generate deletion models for exon 44, 45, 51 & 53 (both exons deleted for each).

Authors discuss that their data shows that there were no mouse genes disrupted by the integration of the human gene and yeast DNA into mouse chromosome 5. Assembling the sequence was challenging due to the presence of the yeast but also the double copies.

Authors discuss they were unable to fully create the sequence map. However, they already managed more than we did so again respect. This works also outlines that what used to be very very difficult with homologous recombination became possible with talens and easy with CRISPR.

Looking forward to seeing the final product as a published paper but appreciate the authors already sharing this!