New genome editing techniques have opened up the number of potential mutations that could be addressed in the inherited muscle wasting disorder Duchenne muscular dystrophy (DMD), while also reducing the probability of inducing off target effects.

A proof-of-concept study published in the April 30, 2021, issue of Science Advances describes the use of base editing and prime editing – both of which can perform more targeted edits than CRISPR-Cas9 – to override the exon 51 deletion in the dystrophin gene.

That was shown to restore production of dystrophin in 96.5% of muscle fibers in transgenic mice that carried the DMD mutation, and in induced pluripotent stem cell-derived cardiomyocytes from DMD patients, in vitro.

The research is the first to demonstrate prime and base editing can be used to increase expression of a functional protein. As such, it also offers promise for other rare inherited disorders caused by faulty or inadequate production of an essential protein.

Current approaches to exon skipping in the DMD gene require a specific construct for each exon. The use of base and prime editing will override this requirement, according to Eric Olson, professor of molecular biology at University of Texas Southwestern Medical Center, who led the research.

"Thousands of different mutations causing DMD have been identified, but they tend to cluster into certain parts of the dystrophin gene," Olson said. "The power of our method is that you don't need a new gene editing strategy for every patient with a new mutation; you can correct multiple different mutations with a consolidated approach."

Olson previously reported using CRISPR-Cas9 to correct the exon 51 deletion, the most commonly occurring mutation in the DMD gene, causing around 13% of cases of the disorder. Based on this research, Olson went on to found Exonics Therapeutics of Boston, Massachusetts, to apply CRISPR-Cas9 gene editing in the treatment of DMD. The company was acquired by Vertex Pharmaceuticals in 2019 for USD 245 million upfront and milestones of USD 1 billion. Vertex is working on multiple different preclinical CRISPR-Cas9 constructs to address the many DMD-causing mutations.

Although CRISPR-Cas9 is a more straightforward way of inducing exon skipping or restoring the open reading frame, it involves a double strand DNA cut. That creates the possibility of generating larger deletions or chromosome rearrangements, which could be damaging.

In this latest research, Olson and colleagues describe using an adenine base editor fused with a Cas9 nickase to precisely modify a splice site of exon 50 of the DMD gene, converting an adenine to guanine. That restored dystrophin expression by skipping exon 50, allowing exon 49 to splice to exon 52.

The researchers also exemplify the use of a prime editor in which nCas9 was fused with a reverse transcriptase to precisely insert two nucleotides into exon 52, restoring the open reading frame and allowing the dystrophin gene to be translated, generating functional protein.

Deletion of exon 51 prevents production of 78 amino acids from the central rod domain that forms the majority of the mass of a dystrophin molecule. However, exon 50 encodes only 36 amino acids in the central rod, and as a result the dystrophin protein generated by splicing exon 49 to exon 52 contains 97% of the 3,685 amino acids found in the full length molecule, and is expected to be highly functional.

"Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome," the researchers wrote.

"This method makes it possible to correct large deletions in the DMD gene by specifically swapping [a single nucleotide]," said Olson. "That level of specificity and efficiency is remarkable."

"A finer scalpel"

Base or prime editing is essentially, "a finer scalpel" said Francesco Muntoni, co-director of the center for neuromuscular diseases at University College London, one of the leading experts on DMD, who led the clinical development of antisense oligonucleotides as molecular patches to override missing exons in the DMD gene. Olson has produced "enticing results" that "demonstrate the efficiency of the two different techniques," Muntoni said, commenting on the research.

To assess the specificity of nucleotide editing Olson et al screened for off target editing at 8 sites predicted to have the potential to be affected. Genome sequence analysis did not reveal any notable editing at any of these sites.

The researchers reasoned that while nucleotide editors can edit all base pairs within a defined window, these bystander edits occur in non-coding introns or in the to-be-skipped exon, meaning they do not affect the final transcript.

The advantages of greater precision are tempered by the size of the nucleotide editing constructs, which are too big to fit into a single adeno-associated viral vector. To overcome this, the researchers used a system that allows protein fragments made by separate vectors to link up within the cell.

Although that strategy was successful, it involved using an amount of AAV9 that is too high to extrapolate from intramuscular administration in mice to systemic administration in humans, as the researchers acknowledge. "Improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD," they said.