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#apaperaday: Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled:  Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells

Preclinical paper to align with my preclinical talk today @DuchenneUK New Horizon meeting. The picks is from @NAR_Open by Wang et al on gene editing for Duchenne. There is evidence of confetti making by Yuzu that I discovered while reading. DOI: 10.1093/nar/gkae057

Gene editing targets the DNA, creating permanent changes in the nucleus of the edited cell with effects lasting as long as the cell survives. However, traditional gene editing with Cas9 involves double-stranded breaks in the DNA at the target location, and potentially elsewhere.

Prime editing is another form of gene editing where only single-stranded breaks are induced. While Cas9 gene editing typically introduces deletions, prime editing can also add small pieces of DNA. Various prime editing systems have been developed.

These systems involve a nickase (to cut one strand of DNA), a reverse transcriptase enzyme (RT), a template, and guide RNAs to target the location of the nick. This means prime editing complexes are much larger than Cas9 and guideRNA complexes.

For muscle diseases like Duchenne, we need a tool to deliver the genes coding for the editing complexes. This is usually done using AAV, which can deliver genes efficiently to muscle. However, AAV is small, and the editors are too large to fit.

Authors solve this problem using high-capacity adenoviral vectors (AdV). These adenoviruses normally do not target muscles, but theauthors have modified them with proteins that improve muscle uptake. Additionally, AdV vectors can easily encompass all the genetic material needed for the editors.

For the scientific followers: authors did a very thorough job describing all the methods involved in making the editors and how they tested them. Authors aimed to reframe deletions of exon 48-50 and 45-51 in cultured cells by either adding 2 base pairs (bp) or deleting 1 bp.

They first tested whether the editors worked in control embryonic kidney cells (easy to manipulate). This worked well. Then they moved to patient-derived muscle cell cultures, using their AdV to deliver the editors. Indeed, they saw the anticipated edits.

Authors showed dystrophin restoration after the edits in the cultured muscle cells and also cardiomyocytes (heart cell cultures). They looked at whether there were unintended edits in sites similar to the target sites.

When using Cas9, there were unwanted edits, but with the prime editors, there were not. Authors also compared editing between myoblasts (proliferating cells) and myotubes (more muscle fiber-like cells that do not proliferate). Efficiency was higher in proliferating cells.

This is a problem because skeletal muscle is not proliferating in humans. However, authors outline that the AdV system can be repeatedly administered, potentially allowing editing levels to accumulate. Whether repeated treatment with AdV is feasible needs to be confirmed.

Authors outline that their system provides the option to deliver all the components needed for prime editing in one system. They also showed that providing 2 target sites in close proximity to the editors on both strands of the DNA improves editing efficiency.

Authors stress this is early-stage work, currently only in cultured cells. However, they are preparing to test this in humanized Duchenne animal models. That will be collaborative work with my group. Looking forward to this collaboration.