#apaperaday: Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy

Today’s pick from PNASNews is again from the Prof. Toshi Yokota group. This time a research paper on peptide conjugated ASOs for Duchenne exon skipping in cultured cells and our own hDMDdel52/mdx. Doi 10.1073/pnas.2112546119.

Exon skipping ASOs have been approved for Duchenne but they restore only low amounts of dystrophin because of low delivery of to muscle. Furthermore exon skipping is mutation specific with each ASO applying only to a small group of patients.

Here authors propose D9 conjunction as a method to improve delivery of ASOs. D9 is an arginine rich peptide but some arginines are D rather than the normal L orientation (chirality again). Furthermore they propose multiexon skipping as a method to increase applicability.

See #apaperaday from yesterday about regulatory concerns for multi exon skipping. Here authors focus on the fact that 11 ASOs are needed for skipping exon 45-55. First they try to reduce the number of ASOs aiming to skip blocks of exons with less ASOs.

This idea is not new. I am a bit disappointed our previous papers on this were not cited: multi exon skipping here, here,and here. And, even more relevant one on intron removal dynamics here and here.

Suggest the authors to have a look at the last paper for optimizing their ASO mix. Below are the different mixes the authors used. Using the different mixes they observe different levels of exon 45-55 skipping in control and patient cell cultures.

Authors quantify the levels using image J of RT-PCR agarose gel images. This is not quantitative for two reasons: 1. Small products bind less ethidium bromide —> underestimation. 2. Small products will have amplification advantage —> large overestimation.

Also there is spontaneous exon 45-55 skipping seen (we saw this as well in our paper van Vliet et al). This makes optimizing the skipping very difficult (combination of background exon 45-55 skipping and very unreliable quantification).

My summary from the results: some mixes may increase exon 45-55 (dystrophin restoration was confirmed). but the extent is difficult to quantify. What is interesting is that some of the mixes work for control cells and some mutations but not for all.

Then authors first try exon 51 skipping with a DG-ASO in our hDMDdel52/mdx mouse. This results in low levels of dystrophin restoration (nice for single injection). More dystrophin was seen after multiple injections with this ASO, leading also to increases in strength and function.

Finally authors use combinations of ASOs with intramuscular injections. Here they see very minor increases in dystrophin (0.3% increase). However it is not sure if this is only due to exon 45-55 skipping or also exon 53 skipping (53 ASO in mix, 51 ASO is not).

Authors discuss that their mix of 5 ASOs can induce exon 45-55 skipping. This is better than the original mix of 11 ASOs but still a lot, and also there is a reduction of efficiency. Authors discuss the differences with exon 23 skipping (higher dystrophin levels).

I agree this is likely due to a mix of factors: exon 23 skipped transcripts and proteins are more stable. Also I want to add that exon 23 skipping PMO ASO is more effective than all other PMOs I have tested so far in vivo (~10fold – reason is ???).

Authors discuss their DG9 conjugate may be more safe than previously tested arginine rich peptides. I hope this is the case but the arginine rich peptides were also safe in mice, so the fact that these are for now does not mean much for human safety.

So all in all I have quite some comments and considerations about this work. I like to think this is not because they forgot to cite some of our relevant previous papers but are general concerns but will provide this disclaimer (I am human and may be petty) for transparency.

 

Pictures by Annemieke, used with permission.