#apaperaday: Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene

Today’s pick is from Molecular Therapy Nucleic Acid by Happi Mbakam et al on prime editing to correct a stop mutation in exon 59 in cultured cells from a Duchenne patient DOI: 10.1016/j.omtn.2022.09.022

Prime editing uses a modified Cas protein (DNA scissor). Unlike genome editing it allows the substitution of one base into another, or a small insertion or deletion. Thus it allows correction of small mutations. Here authors focus on c.8713C>T in the DMD gene (nonsense mutation). 

Like Cas9, prime editing relies on specific sequences, where the enzyme will nick the DNA. The selected mutation is challenging as the edited base is 13 bp away from the nick site. Authors test first in healthy HEK293 cells whether they can edit at this location.

The efficiency is ~5.5-6.5% in cultured cells. Authors used different cas9 proteins and modified the PAM sequence, or vary the distance between nick and edit, the sequence surrounding the edit and the type of edited nucleotide (with >3 pages of different sequences)

In an optimal setting editing occurred at 60-75%. They discovered that several parameters determine the efficiency of the edit: the type of nucleotide, the distance between edit and nick and the PAM sequence. While this is important to know, in real life you cannot influence this

So this insight will help to identify and prioritize target sequences. Authors finally use their optimized system to correct the mutation in immortalized myoblast cultures from a Duchenne patient and manage to achieve this in 17% of cells and restore dystrophin in vitro

Authors discuss that it is unclear why the type of nucleotide and its relative position and environment influences the editing efficiency. However, it is clear that it does. While this work shows prime editing is feasible in cultured cells, clinical applicability is far off still

Authors argue that even though in vivo efficiency likely will be quite low, this may not be a problem because muscle fibers are multinucleated and it may be enough to target a subset of nuclei. However, authors have to take into account that dystrophin does not travel far.

So if you edit one nuclei, dystrophin will only be restored in the area immediately surrounding that nucleus. As such editing only a small subset may only have a limited effect. For now, in vivo prime editing was not tested in this study.

I do commend the authors for the systematic approach by which they tested many different combinations of cas9s, target sequences and guides. As prime editing is a new approach, this information is crucial to allow optimization and prioritization and feasibility assessments.