Genomic medicine corrects common LOPD defect in cells, mice
Precision approach seen promising for targeting 'fundamental defect'
A genomic medicine designed to correct the genetic defect found in up to 90% of people with late-onset Pompe disease (LOPD) worked as intended in patient-derived muscle cells and a mouse model, according to a study.
The study, “Splicing correction by peptide-conjugated morpholinos as a novel treatment for Late-Onset Pompe Disease,” was published in Molecular Therapy Nucleic Acids. It was funded by Sarepta Therapeutics, and all study authors are affiliated with the company.
Pompe disease is caused by mutations in the GAA gene that lead to a deficiency in functional acid alpha-glucosidase (GAA), an enzyme that breaks down the large sugar molecule glycogen to provide cells with an energy source.
Consequently, GAA accumulates in cells, especially muscle cells, and causes damage. LOPD symptoms such as progressive muscle weakness and breathing issues emerge in childhood or adulthood.
Current therapeutic options, such as enzyme replacement therapy, can have limited efficacy in LOPD, according to the authors.
Addressing an unmet need
“Given the widely recognized unmet need for these patients, a precision genetic medicines approach for these patients would come closer to addressing the fundamental defect,” the researchers wrote.
As with most genes, individuals have two copies of GAA, one inherited from each biological parent. For Pompe to manifest, mutations must occur on both copies. Up to 90% of people with LOPD have a mutation called IVS1 on at least one copy of the GAA gene, which is usually accompanied by a different type of mutation on the other.
For a protein to be made from the information in DNA, production of an intermediate molecule called messenger RNA (mRNA) that can be used as a blueprint by a cell’s protein-making machinery is required.
Proper formation of mRNA depends on splicing, a process in which certain regulatory segments of genetic material (introns) are cut out, and the remaining protein-coding sections (exons) are strung back together. Improper splicing leads to a faulty mRNA template and disturbed protein production.
IVS1 causes a GAA splicing defect, in which a protein-coding segment called exon 2 is mistakenly removed, either completely or partially, in most mRNA transcripts, producing an mRNA not able to make GAA enzyme. About 10%-15% of mRNA will still have normal splicing and be able to yield GAA.
The researchers tested a potential treatment approach to correct the splicing defect caused by IVS1. Called a peptide-conjugated phosphodiamidate morpholino oligomer (PPMO), the therapy essentially contains a lab-made strand of genetic material that can influence gene splicing, linked to protein fragments that enhance its cellular uptake.
When given via an infusion into the bloodstream, the PPMO is taken up by cells and binds to GAA to correct the splicing defect and enable more normal GAA production.
After screening several PPMO candidates in lab studies, the scientists identified a favorable candidate called RC-3003 that led to increased GAA enzyme activity without significant toxicity.
In skeletal muscle cells derived from people with LOPD, PPMO treatment led to about a fourfold increase in correctly spliced GAA and a more than twofold increase in the GAA enzyme levels and activity.
To further assess the relevance of the approach, the scientists developed a mouse model of LOPD with the IVS1 mutation.
As in the cells, PPMO treatment increased GAA gene activity and helped correct the splicing defect in muscle tissue. The treatment did not result in signs of liver or kidney toxicity, and no adverse safety signals were identified.
“Together, the data outlined here demonstrate the mechanism of action and successful GAA restoration in LOPD patient iPSC myotubes [patient-derived muscle cells], coupled with the ability to penetrate muscle tissue and similarly correct pathogenic GAA splicing in vivo [in a mouse model,” the researchers wrote.
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