Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease

Abstract

pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-αglucosidase activity in patients, and a progressive decline in mobility and respiratory function. Enzyme replacement therapy is one therapeutic option, but since not all patients respond to this treatment, alternative interventions should be considered. One GAA mutation, c.-32-13T > G, impacts upon normal exon 2 splicing and is found in two-thirds of late-onset cases. We and others have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients with one c.-32-13T > G allele. We designed 20 oligomers and treated fibroblasts derived from five patients to identify an oligomer sequence that maximally increased enzyme activity in all fibroblasts. The most effective splice correcting oligomer was chosen to treat forced-myogenic cells, derived from fibroblasts from nine patients carrying the c.-32-13T > G mutation. After transfection, we show increased levels of the full-length GAA transcript, acid-α-glucosidase protein, and enzyme activity in all patients' myogenic cells, regardless of the nature of the mutation in the other allele. This data encourages the initiation of clinical trials to assess the therapeutic efficacy of this oligomer for those patients carrying the c.-32-13T > G mutation. Intronic variations that lead to aberrant splicing events, such as exon loss or the retention of intronic sequence in the mature mRNA in the form of pseudo-exons, have been reported in many genes 1,2. Most recently, Milasen, a splice switching compound targeted to the CLN7 pseudo-exon mutation in one Batten disease patient, was granted approval by the US Food and Drug Administration 3. There is growing interest in the use of splice switching antisense oligonucleotides (AOs) as therapeutic agents to treat serious inherited diseases. At present, three splice switching AOs, Vyondys 53 4 , Exondys 51 5 , and Spinraza 6 , have been approved by the US Food and Drug Administration as treatments for a subset of patients with Duchenne muscular dystrophy and spinal muscular atrophy, respectively. The late-onset form of Pompe disease, also known as glycogen storage disease type II (GSD II), presents as a suitable candidate for AO therapy, since approximately two-thirds of the adult Pompe patients harbour a common disease-causing mutation: c.-32-13T > G 7. The incidence of this variant is higher in Caucasians and identified in ninety percent of the adult-onset Pompe patients 8. This mutation is known to cause complete skipping of exon 2 from most GAA transcripts (Supplementary Fig. S1) 9,10 , and disease onset and severity is modestly correlated with the residual lysosomal acid-α-glucosidase (GAA) activity in those patients 11-13. Generally, less than 1% of

Analysis of alternative mRNA splicing in vemurafenib-resistant melanoma cells

“…It is possible that the here identified AS event in MPRIP impairs this function, meaning that alternatively spliced MPRIP cannot bind and activate MLC phosphatase, thus promoting MLC activity, stress fibre contractility and therapy resistance. It would be interesting to test this hypothesis in future experiments, for example by perturbing MPRIP using RNA-interference or switching the alternative splicing of MPRIP back to normal using splice-switching oligonucleotides [55, 56].…”

A Generic Assay to Detect Aberrant ARSB Splicing and mRNA Degradation for the Molecular Diagnosis of MPS VI

“… 20 , 21 , 43 We and others have shown recently that it is feasible for the lysosomal storage disease Pompe disease to restore GAA expression after aberrant splicing caused by several GAA variants, including c.1552-3C>G, c.1256A>T, c.2190-345A>G, 26 and the common IVS1 variant. 42 , 44 , 45 , 46 However, AONs that blocked the cryptic splice sites of the pseudoexons in ARSB introns 5 and 6 failed to promote canonical ARSB splicing ( Figure S4 ), suggesting a more complex underlying mechanism for ARSB splicing. We speculate that the large sizes of introns 5 and 6 offer too many alternative options for splicing to allow a simple competition model between utilization of canonical splice sites and the cryptic splice sites of the pseudoexons.…”

Splice correction therapies for familial hypercholesterolemic patients with low-density lipoprotein receptor mutations

“…With a plethora of mutations within the LDLR gene, we anticipate that there may be several intra-exonic or intronic mutations that lead to exon skipping – in this instance we may need to include an exon as a therapeutic strategy. We published an ASO mediated therapeutic strategy for the treatment of adult onset Pompe disease patients carrying the common c.-32-13T>G mutation within the acid alpha-glucosidase (GAA) transcript [ 42 ▪ ]. The single mutation resulted in the generation of multiple aberrantly spliced transcripts that caused both cryptic splicing and exon exclusion.…”

“…The single mutation resulted in the generation of multiple aberrantly spliced transcripts that caused both cryptic splicing and exon exclusion. Through splice modulating ASOs, we were able to increase normal transcript splicing and increase GAA activity in multiple patient cell lines [ 42 ▪ ]. This application is similar to FH in the sense that ASOs may provide a viable treatment option for patients who currently have intolerance to their current treatment options.…”