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Andrea Elena Dardis, PhD - "The common c.-32-13T>G splicing variant of GAA gene: from functional characterization to the development of new therapies for Pompe disease"

Head of the Laboratory of the Regional Coordinator Centre for Rare Diseases, Udine, Italy
When Jan 22, 2019
from 11:30 AM to 12:30 PM
Where Tigem, Vesuvius Auditorium
Contact Name
Contact Phone 081-19230659
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Abstract
Pompe disease (PD), is an autosomal recessive lysosomal storage disorder due to mutations in the GAA gene, which cause a functional deficiency of the alpha-glucosidase enzyme (GAA) and result in impaired glycogen degradation and accumulation within the lysosomes. Clinically, PD encompasses a continuous spectrum of phenotypes, ranging from infantile to late onset (LO) forms. Currently, the only approved treatment for PD is enzyme replacement therapy (ERT) with human recombinant GAA, which does not completely prevent disease progression.
The mutation profile of the GAA gene is heterogeneous; however, most patients affected by the LO phenotype carry the splicing mutation c.-32-13T>G in at least one allele, making this mutation an interesting target for therapeutic development. 
Therefore, we first performed an in deep functional characterization of this variant showing that it affects the binding of the U2AF65 splicing factor to the polypyrimidine tract of exon 2, leading to a complete or partial exclusion of this exon from the mature GAA mRNA. However, in the presence of this mutation, variable levels of the normal spliced GAA transcript and protein are produced.
Then, using a series of overlapping deletion constructs we mapped several splicing silencers within the exon 2 sequence and designed different antisense morpholino oligonucleotides (AMOs) to target those regions.  Using a minigene approach and patients fibroblasts we were able to successfully increase the inclusion of exon 2 into the mRNA and improve GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, treatment of patient myotubes with the AMOs combination resulted in a partial clearance of glycogen accumulation. These data suggest that in patients carrying the c.-32-13T>G mutation and retaining a substantial residual GAA activity, the AMOs effect on GAA might be enough to achieve a beneficial effect in clinical settings. These are very encouraging results and further studies will be directed to test the efficacy and the delivery of these AMOs to skeletal muscle in vivo.
An alternative promising approach to restore normal splicing of mutated transcripts is the use of small molecules that modify splicing patterns. We have developed a fluorescent reporter system suitable for highthroughput screening of molecules able to restore normal splicing of c.-32-13T>G mutant alleles. Using this approach we have screened a library of 1270 FDA approved compounds identifying several molecules able to partially rescue normal splicing.
In conclusion, our work has provided the bases for the clinical development of RNA based therapies and drug repurposing for the treatment PD patients carrying the c.-32-13T>G mutation. These therapeutic approaches alone or in combination with ERT, could improve the clinical outcome of this group of patients. 

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