Abstract: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 fibr… Show more
“…AON 3 and 4 enhanced GAA enzymatic activity in myotubes derived from patient 1 (Figure 4E) and patient 2 (Figure S3D) up to 2-fold and was ineffective in myotubes from control 1 (Figure 4F) and control 2 (Figure S3E). These efficacies were similar to those observed in fibroblasts 5 . AON treatment was well tolerated.…”
Section: Resultssupporting
confidence: 82%
“…This suggested the possibility that AONs 3 and 4 may act by inhibiting usage of a natural pseudo exon rather than by repressing a putative intronic splice silencer.Figure 5Blocking of a Natural Pseudo Exon Restores GAA Exon 2 Splicing(A) The splicing silencer in intron 1 is predicted to be the pY-tract of a natural pseudo exon. Alamut was used to predict splice sites around the splicing silencer identified previously 5 . Note that predictions were independent of the IVS1 variant.…”
Section: Resultsmentioning
confidence: 99%
“…Alamut was used to predict splice sites around the splicing silencer identified previously 5 . Note that predictions were independent of the IVS1 variant.…”
Section: Resultsmentioning
confidence: 99%
“…The IVS1 variant is located in the polypyrimidine tract (pY-tract) of GAA exon 2 and weakens the recognition of the splice acceptor site of this exon 13, 15, 16. AONs that restored splicing were identified in a screen for cis -acting splicing silencer elements, and these targeted an element in intron 1, 175 nucleotides upstream of the affected exon 2 5 . It was unclear from this study whether the AON-mediated inhibition of a putative splicing silencer element would be operational in differentiated skeletal muscle cells, which represent the cell type relevant for Pompe disease, and what the underlying mechanism was.…”
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.
“…AON 3 and 4 enhanced GAA enzymatic activity in myotubes derived from patient 1 (Figure 4E) and patient 2 (Figure S3D) up to 2-fold and was ineffective in myotubes from control 1 (Figure 4F) and control 2 (Figure S3E). These efficacies were similar to those observed in fibroblasts 5 . AON treatment was well tolerated.…”
Section: Resultssupporting
confidence: 82%
“…This suggested the possibility that AONs 3 and 4 may act by inhibiting usage of a natural pseudo exon rather than by repressing a putative intronic splice silencer.Figure 5Blocking of a Natural Pseudo Exon Restores GAA Exon 2 Splicing(A) The splicing silencer in intron 1 is predicted to be the pY-tract of a natural pseudo exon. Alamut was used to predict splice sites around the splicing silencer identified previously 5 . Note that predictions were independent of the IVS1 variant.…”
Section: Resultsmentioning
confidence: 99%
“…Alamut was used to predict splice sites around the splicing silencer identified previously 5 . Note that predictions were independent of the IVS1 variant.…”
Section: Resultsmentioning
confidence: 99%
“…The IVS1 variant is located in the polypyrimidine tract (pY-tract) of GAA exon 2 and weakens the recognition of the splice acceptor site of this exon 13, 15, 16. AONs that restored splicing were identified in a screen for cis -acting splicing silencer elements, and these targeted an element in intron 1, 175 nucleotides upstream of the affected exon 2 5 . It was unclear from this study whether the AON-mediated inhibition of a putative splicing silencer element would be operational in differentiated skeletal muscle cells, which represent the cell type relevant for Pompe disease, and what the underlying mechanism was.…”
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.
“…We note that two GAA variants per patients were . This variant leads to partial or complete skipping of exon 2 during GAA pre-mRNA splicing, which has been described previously (Bergsma et al, 2015;Boerkoel et al, 1995;Dardis et al, 2014;Huie, Chen, Brooks, Grix, & Hirschhorn, 1994;van der Wal, Bergsma, Pijnenburg, van der Ploeg, & Pijnappel, 2017;van der Wal, Bergsma, van Gestel et al, 2017). (Figure 3a,b).…”
Section: Analysis Of Gaa Variants Based On Clinical Phenotypesupporting
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.
Immune‐mediated inflammatory diseases (IMIDs) impose an immeasurable burden on individuals and society. While the conventional use of immunosuppressants and disease‐modifying drugs has provided partial relief and control, their inevitable side effects and limited efficacy cast a shadow over finding a cure. Promising nucleic acid drugs have shown the potential to exert precise effects at the molecular level, with different classes of nucleic acids having regulatory functions through varying mechanisms. For the better delivery of nucleic acids, safe and effective viral vectors and non‐viral delivery systems (including liposomes, polymers, etc.) have been intensively explored. Herein, after describing a range of nucleic acid categories and vectors, we focus on the application of therapeutic nucleic acid delivery in various IMIDs, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, asthma, ankylosing spondylitis, systemic lupus erythematosus, and uveitis. Molecules implicated in inflammation and immune dysregulation are abnormally expressed in a series of IMIDs, and their meticulous modulation through nucleic acid therapy results in varying degrees of remission and improvement of these diseases. By synthesizing findings centered on specific molecular targets, this review delivers a systematic elucidation and perspective towards advancing and utilization of nucleic acid therapeutics for managing IMIDs.
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