Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

The most common variant causing Pompe disease is c.-32-13T>G (IVS1) in the acid α-glucosidase (GAA) gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs) to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA)-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity

From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: A Pipeline for the Development of Antisense Oligonucleotides

While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect

High Sustained Antibody Titers in Patients with Classic Infantile Pompe Disease Following Immunomodulation at Start of Enzyme Replacement Therapy

Objective: To evaluate whether immunomodulation at start of enzyme replacement therapy induces immune tolerance to recombinant human acid alpha-glucosidase (rhGAA) in patients with classic infantile Pompe disease. Study design: Three patients (1 cross reactive immunologic material negative, 2 cross reactive immunologic material positive) were treated with 4 weekly doses of rituximab, weekly methotrexate, and monthly intravenous immunoglobulin and enzyme replacement therapy at 40 mg/kg/week. Antibody titers were measured using enzyme-linked immunosorbent assay. Neutralizing effects on rhGAA activity and cellular uptake were determined and combined with pharmacokinetic analysis. Clinical efficacy was evaluated by (ventilator-free) survival, reduction in left ventricular mass index, and improvement of motor function. Results: Immunomodulation induced B cell depletion that was accompanied by absence of antibody formation in all 3 patients. Upon cessation of rituximab treatment, all 3 patients showed B cell recovery, which was accompanied by formation of very high sustained antibody titers in 2 patients. Neutralizing effects on infused rhGAA were low to mild/moderate. All patients were alive at study end, learned to walk, and showed (near) normalization of left ventricular mass index. Conclusions: Immunomodulation as recommended in the literature prevented formation of rhGAA antibodies only during B cell depletion but failed to induce immune tolerance in 2 out of 3 patients

GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells

Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA) enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1) GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs) that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC)-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs

Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation

Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene

Extension of the Pompe mutation database by linking disease-associated variants to clinical severity

Pompe disease is an autosomal recessive lysosomal storage disorder caused by disease‐associated variants in the acid alpha‐glucosidase (GAA) gene. The current Pompe mutation database provides a severity rating of GAA variants based on in silico predictions and expression studies. Here, we extended the database with clinical information of reported phenotypes. We added additional in silico predictions for effects on splicing and protein function and for cross reactive immunologic material (CRIM) status, minor allele frequencies, and molecular analyses. We analyzed 867 patients and 562 GAA variants. Based on their combination with a GAA null allele (i.e., complete deficiency of GAA enzyme activity), 49% of the 422 disease‐associated variants could be linked to classic infantile, childhood, or adult phenotypes. Predictions and immunoblot analyses identified 131 CRIM negative and 216 CRIM positive variants. While disease‐associated missense variants were found throughout the GAA protein, they were enriched up to seven‐fold in the catalytic site. Fifteen percent of disease‐associated missense variants were predicted to affect splicing. This should be confirmed using splicing assays. Inclusion of clinical severity rating in the Pompe mutation database provides an invaluable tool for diagnosis, prognosis of disease progression, treatment regimens, and the future development of personalized medicine for Pompe disease

Supine MRI for regional breast radiotherapy: Imaging axillary lymph nodes before and after sentinel-node biopsy

Regional radiotherapy (RT) is increasingly used in breast cancer treatment. Conventionally, computed tomography (CT) is performed for RT planning. Lymph node (LN) target levels are delineated according to anatomical boundaries. Magnetic resonance imaging (MRI) could enable individual LN delineation. The purpose was to evaluate the applicability of MRI for LN detection in supine treatment position, before and after sentinel-node biopsy (SNB). Twenty-three female breast cancer patients (cTis-3N0M0) underwent 1.5 T MRI, before and after SNB, in addition to CT. Endurance for MRI was monitored. Axillary levels were delineated. LNs were identified and delineated on MRI from before and after SNB, and on CT, and compared by Wilcoxon signed-rank tests. LN locations and LN-based volumes were related to axillary delineations and associated volumes. Although postoperative effects were visible, LN numbers on postoperative MRI (median 26 LNs) were highly reproducible compared to preoperative MRI when adding excised sentinel nodes, and higher than on CT (median 11, p < 0.001). LN-based volumes were considerably smaller than respective axillary levels. Supine MRI of LNs is feasible and reproducible before and after SNB. This may lead to more accurate RT target definition compared to CT, with potentially lower toxicity. With the MRI techniques described here, initiation of novel MRI-guided RT strategies aiming at individual LNs could be possible

Update of the Pompe variant database for the prediction of clinical phenotypes: Novel disease-associated variants, common sequence variants, and results from newborn screening

Pompe disease is an inherited disorder caused by disease-associated variants in the acid α-glucosidase gene (GAA). The Pompe disease GAA variant database (http://www.pompevariantdatabase.nl) is a curated, open-source, disease-specific database, and lists disease-associated GAA variants, in silico predictions, and clinical phenotypes reported until 2016. Here, we provide an update to include 226 disease-associated variants that were published until 2020. We also listed 148 common GAA sequence variants that do not cause Pompe disease. GAA variants with unknown severity that were identified only in newborn screening programs were listed as a new feature to indicate the reason why phenotypes were still unknown. Expression studies were performed for common missense variants to predict their severity. The updated Pompe disease GAA variant database now includes 648 disease-associated variants, 26 variants from newborn screening, and 237 variants with unknown severity. Regular updates of the Pompe disease GAA variant database will be required to improve genetic counseling and the study of genotype–phenotype relationships