Pre-clinical validation of FDA approved molecules able to rescue GAA pre-mRNA splicing of c.-32-13T>G mutants as therapeutic agents for late-onset Pompe Disease

Enzyme replacement therapy (ERT) is the only approved treatment for Pompe disease (PD). Alt-hough ERT improves cardiac function, and lifespan in patients affected by the infantile PD pheno-type, limitations of this approach are becoming evident in particular in patients affected by the late onset forms of PD (LO-PD).
In recent years, our research has been focused on finding solutions for mis-splicing alterations in-duced by the c.-32-13T>G mutation of GAA.
Indeed, the possibility to restore/increase normal splicing of the GAA exon 2 of transcripts carrying the c.-32-13T>G mutation is particularly appealing considering that: (i) almost all LO patients carry this mutation in at least one allele and (ii) some patients express up to 30% of normal GAA activity and just a little increase in exon inclusion might be enough to achieve a beneficial effect in clinical settings.
We already provided in vitro evidence showing that it is possible to increase the expression of normal spliced GAA mRNA of c.-32-13T>G mutated alleles, by targeting a specific silencer within exon 2 with a combination of Antisense Morpholino Oligonucleotides (AMOs). Most importantly us-ing this strategy we have shown that in the context of the c.-32-13T>G mutation, a 70% increase of the residual GAA activity is enough to partially correct the intracellular glycogen storage. Although these results are encouraging, further studies would be needed to test the efficacy and the delivery of the AMOs to skeletal muscle in vivo. However, no animal models of PD carrying this particular mutation are available, making pre-clinical studies extremely difficult.
An alternative promising approach to restore normal splicing of mutated transcripts is the use of small molecules that modify splicing patterns. These therapeutic agents offer several advantages compared to nucleic acid based therapies, including lower manufacturing costs and oral delivera-bility. In addition, many small molecules are already approved by FDA; therefore they could be easily translated from the bench to the bedside.
Thus, with the aim of identifying small molecules able to rescue/increase exon 2 GAA inclusion we have set up a splicing reporter system and screened a library of 1270 FDA approved compounds. We have already identified several compounds capable of significantly improving exon 2 inclusion in the presence of the c.32-13T>G mutation. Furthermore, treatment of patient fibroblasts carrying the c.-32-13T>G mutation with the most promising hit in the screening analysis, resulted in a 4-fold increase in exon 2 inclusion.
Therefore, the aims of this project will be to validate the effect of this compound and other com-mercially available analogs on exon 2 inclusion, GAA activity and glycogen storage in myotubes from LO-PD patients carrying the c.-32-13T>G mutation and to elucidate the mechanism of action and the metabolic pathways altered by the drug. This information will then be used to search for other types of FDA-approved drugs that acting on the same pathways that might be more effective in promoting exon 2 inclusion.
The results of this research will provide pre-clinical data to support the use of FDA approved ther-apeutic agents in LO-PD.