Therapy corrects common GAA splicing defect in lab studies
Study finds approach may also correct defects caused by rarer mutations
A therapeutic approach designed to correct the cellular defect caused by a common mutation in people with late-onset Pompe disease (LOPD) worked as intended to increase activity of the acid alpha glucosidase (GAA) enzyme in lab studies, according to a study.
People with Pompe disease are lacking in GAA due to mutations in the GAA gene, and this mutation in particular, called c.-32-13T>G, affects an important process called splicing.
The therapy was designed to correct the splicing defect and restore GAA production. Beyond c.-32-13T>G, it could also correct splicing defects caused by other, less common mutations, according to the scientists.
The researchers said the study provides proof of concept for their approach to boosting GAA levels in Pompe patients.
The study, “Rescue of common and rare exon 2 skipping variants of the GAA gene using modified U1 snRNA,” was published in Molecular Medicine.
Splicing defect
The lack of functional GAA in Pompe disease means that the sugar molecule glycogen, which GAA is normally responsible for breaking down, consequently accumulates in cells and damages them.
While the existing standard of care, enzyme replacement therapy — which provides a version of the missing enzyme — can lead to clinical gains for Pompe patients, outcomes can be variable, especially in people with LOPD, according to the authors. That means a identifying a broader range of therapeutic options, particularly ones personalized to a patient’s specific disease-causing mutation, is helpful.
Hundreds of GAA mutations have been linked to Pompe disease, and each affects the gene — and subsequent production of GAA — in different ways. c.-32-13T>G is common in LOPD, with about 90% of patients having the mutation in at least one copy of their GAA gene, according to the researchers.
This mutation interferes with splicing, a process important for protein production. When the information in DNA is being used to make a protein, a template molecule called messenger RNA (mRNA) is produced, which is then read by a cell’s protein-making machinery.
For mRNA to be properly formed requires splicing, in which certain segments that don’t directly encode protein production (introns) are cut out, and the remaining protein-coding segments (exons) are strung back together to form the mRNA template. When splicing is abnormal, a faulty mRNA is produced, disturbing protein production.
When the c.-32-13T>G mutation is present, some mRNA transcripts are normal, but in most, abnormal splicing causes a protein-coding segment of GAA called exon 2 to be removed when it shouldn’t be. This results in production of a nonfunctional mRNA that can’t make GAA enzyme.
The researchers developed a tool to correct this splicing defect. Called a U1 snRNA, it is designed to bind to the site where splicing should occur in exon 2, and recruits cellular machinery that will enable the process to happen normally.
A few versions of the therapy were developed in lab experiments. The most effective one led to a 2.5-times increase in the number of mRNA transcripts produced that properly included exon 2, and stopped production of a nonfunctional mRNA.
When tested in cells derived from the skin of a Pompe disease patient carrying the c.-32-13T>G mutation, the treatment increased the amount of normal GAA mRNA by 1.8-fold, in turn increasing GAA enzyme activity by up to 70%.
Because many patients carrying the c.-32-13T>G mutation have some remaining GAA activity, the researchers wrote, the increases from the U1 snRNA treatment “would be enough to exceed the threshold activity needed to prevent pathological [disease-related] glycogen accumulation and achieve a beneficial effect in clinical settings.”
The U1 snRNA was also found to be able to correct splicing defects caused by some other types of GAA mutations that affect exon 2, although effects varied by specific mutation.
Additional experiments indicated the treatment approach was not likely to have strong unintended effects on genes other than GAA.
“The data presented here suggest that the use of modified U1 molecules … represents a promising strategy for rescuing normal splicing of transcripts carrying both common and rare splicing variants affecting GAA exon 2,” the team concluded.
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