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#apaperaday: A cell-penetrating peptide enhances delivery and efficacy of phosphorodiamidate morpholino oligomers in mdx mice

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: A cell-penetrating peptide enhances delivery and efficacy of phosphorodiamidate morpholino oligomers in mdx mice

Today’s pick is from Molecular Therapy on Nucleic Acids by Gan et al on efficacy studies of a cell penetrating peptide conjugated to a phosphorodiamidate morpholino oligomer targeting mouse exon 23. Study from Sarepta. Doi 10.1016/j.omtn.2022.08.019.

4 phosphorodiamidate morpholino oligomers (PMOs) are FDA-approved to treat Duchenne patients with eligible mutations (needing exon 51, 45 or 53 skipping). However approval is based on low level of dystrophin restoration. FDA label says functional effects have yet to be confirmed.

It is clear there is room for improvement however with the largest increase in efficacy expected from improving delivery of PMOs to skeletal muscle and heart. One way to improve PMO delivery is to add arginine rich peptides. This is improving ALL delivery, not specific delivery.

The challenge of arginine rich peptides is that while they are very efficient to improve delivery, they are also toxic with a small therapeutic window in humans. So the question is: what comes first: efficacy or kidney toxicity? As such the peptides need to be optimized.

Here authors present a proprietary peptide (they cite a patent but that has hundreds of peptides in it, so difficult to know which one this is, but authors do mention it is the same one as used in SRP-5051, the pPMO currently in trials for exon 51 skipping).

The pPMO tested in this paper however targets mouse exon 23, to restore dystrophin in the mdx mouse model. First a dose response revealed very good exon skipping and dystrophin restoration in muscle and heart after a single dose of 80 mg/kg.

The 40 mg/kg also is efficient in muscle but less so in heart. The 40 and 80 mg/kg doses also improved function. Authors then studied the durability of effect: exon skipping levels went to (close to) 0 between 30 and 90 days, while dystrophin was still detectable after 90 days.

Finally authors performed a repeated dose study with 1, 2 or 3 monthly doses of 40 mg/kg intravenous. As expected this resulted in cumulation of dystrophin levels with repeated injections up to ~50% in quadriceps and diaphragm and ~10% in heart.

The same dosing regimen of regular PMOs resulted in very low dystrophin levels in quadriceps and barely detectable dystrophin levels in diaphragm and no dystrophin restoration in heart. So it is clear the peptide outperforms the unconjugated PMO.

In the discussion authors discuss that their pPMO can achieve dystrophin restoration in diaphragm and heart, while PMO cannot. This is important as most Duchenne patients currently die due to respiratory and heart failure. However, note heart dystrophin levels were not very high.

Authors also discuss the ‘how much dystrophin is needed’ question. The short answer is ‘we don’t know’. This is a multifaceted question, where parameters like time of intervention, which dystrophin is produced, how the dystrophin is distributed over fibers etc also play a role.

Also we know you need more dystrophin to improve strength than function (at least in mice). There are 2 aspects authors do not discuss here: 

  1. Safety. In the trial with SRP-5051 hypomagnesemia was observed at doses of 30 mg/kg in some patients. Patients are supplemented with magnesium – however, hypomagnesemia could be the result of excretion of magnesium via the kidney due to renal toxicity. This is something that bears further study. Note that hypomagnesemia was also found for single dose of PepGen’s pPMO at 20 mg/kg. We have to wait how this develops. I hope the therapeutic window allows dosing at a level that is tolerable for the patients and results in higher amounts of dystrophin (SRP-5051 achieved up to 6.5% dystrophin – Pepgen pPMO was only used in healthy volunteers so far).
  1. Authors here test a mouse dystrophin exon 23 pPMO, which means they have to extrapolate results from one ASO to another (i.e. SRP-5051) and from one species to another. Using a humanized mouse model in my opinion would be a better choice as it would allow testing of SRP-5051

For those interested in the humanized deletion exon 52 model. 

  • https://pubmed.ncbi.nlm.nih.gov/33362201/ 
  • https://pubmed.ncbi.nlm.nih.gov/29466448/

For full disclosure: I am on the SAB of Sarepta (with remuneration to Leiden University Medical Center, not me). I do not think that prevents me from providing a fair review of the paper.

All in all I appreciate the diligent studies the authors have done AND I appreciate them sharing the results with the community. Would have preferred them testing the exon 51 skipping version in the humanized mouse – maybe that will come in the future.