Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro.

Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.

Broad variation in phenotypes for common GAA genotypes in Pompe disease.

Patients with the common c.-32-13T > G/null GAA genotype have a broad variation in age at symptom onset, ranging from early childhood to late adulthood. Phenotypic variation for other common GAA genotypes remains largely unexplored. Here, we analyzed variation in age at symptom onset for the most common GAA genotypes using the updated and extended Pompe GAA variant database. Patients with the c.2647-7G > A/null genotype invariably presented symptoms at adulthood, while the c.-32-13T > G/null, c.546G > T/null, c.1076-22T > G/null, c.2238G > C/null, and c.2173C > T/null genotypes led to presentations from early childhood up to late adulthood. The c.1309C > T/null genotype was associated with onset at early to late childhood. Symptom onset shifted toward higher ages in homozygous patients. These findings indicate that a broad variation in symptom onset occurs for various common GAA genotypes, suggesting the presence of modifying factors. We identified three new compound heterozygous c.-32-13T > G/null patients who carried the genetic modifier c.510C > T and who showed symptom onset at childhood. While c.510C > T acted by lowering GAA enzyme activity, other putative genetic modifiers did not at the group level, suggesting that these act in trans on processes downstream of GAA enzyme activity.

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.

Opportunities and challenges for antisense oligonucleotide therapies.

Antisense oligonucleotide (AON) therapies involve short strands of modified nucleotides that target RNA in a sequence-specific manner, inducing targeted protein knockdown or restoration. Currently, 10 AON therapies have been approved in the United States and Europe. Nucleotides are chemically modified to protect AONs from degradation, enhance bioavailability and increase RNA affinity. Whereas single stranded AONs can efficiently be delivered systemically, delivery of double stranded AONs requires capsulation in lipid nanoparticles or binding to a conjugate as the uptake enhancing backbone is hidden in this conformation. With improved chemistry, delivery vehicles and conjugates, doses can be lowered, thereby reducing the risk and occurrence of side effects. AONs can be used to knockdown or restore levels of protein. Knockdown can be achieved by single stranded or double stranded AONs binding the RNA transcript and activating RNaseH-mediated and RISC-mediated degradation respectively. Transcript binding by AONs can also prevent translation, hence reducing protein levels. For protein restoration, single stranded AONs are used to modulate pre-mRNA splicing and either include or skip an exon to restore protein production. Intervening at a genetic level, AONs provide therapeutic options for inherited metabolic diseases as well. This review provides an overview of the different AON approaches, with a focus on AONs developed for inborn errors of metabolism.

A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease.

DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.

Enzymatic diagnosis of Pompe disease: lessons from 28 years of experience.

Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

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

Identification and characterization of disease-associated variants in monogenic disorders is an important aspect of diagnosis, genetic counseling, prediction of disease severity, and development of therapy. However, the effects of disease-associated variants on pre-mRNA splicing and mRNA degradation are difficult to predict and often missed. Here we present a generic assay for unbiased identification and quantification of arylsulfatase B (ARSB) mRNA for molecular diagnosis of patients with mucopolysaccharidosis VI (MPS VI). We found that healthy control individuals have inefficient ARSB splicing because of natural skipping of exon 5 and inclusion of two pseudoexons in introns 5 and 6. Analyses of 12 MPS VI patients with 10 different genotypes resulted in identification of a 151-bp intron inclusion caused by the c.1142+2T>C variant and detection of low ARSB expression from alleles with the c.629A>G variant. A special case showed skipping of exon 4 and low ARSB expression. Although no disease-associated DNA variant could be identified in this patient, the molecular diagnosis could be made based on RNA. These results highlight the relevance of RNA-based analyses to establish a molecular diagnosis of MPS VI. We speculate that inefficient natural splicing of ARSB may be a target for therapy based on promotion of canonical splicing.

Novel GAA Variants and Mosaicism in Pompe Disease Identified by Extended Analyses of Patients with an Incomplete DNA Diagnosis.

Pompe disease is a metabolic disorder caused by a deficiency of the glycogen-hydrolyzing lysosomal enzyme acid α-glucosidase (GAA), which leads to progressive muscle wasting. This autosomal-recessive disorder is the result of disease-associated variants located in the GAA gene. In the present study, we performed extended molecular diagnostic analysis to identify novel disease-associated variants in six suspected Pompe patients from four different families for which conventional diagnostic assays were insufficient. Additional assays, such as a generic-splicing assay, minigene analysis, SNP array analysis, and targeted Sanger sequencing, allowed the identification of an exonic deletion, a promoter deletion, and a novel splicing variant located in the 5' UTR. Furthermore, we describe the diagnostic process for an infantile patient with an atypical phenotype, consisting of left ventricular hypertrophy but no signs of muscle weakness or motor problems. This led to the identification of a genetic mosaicism for a very severe GAA variant caused by a segmental uniparental isodisomy (UPD). With this study, we aim to emphasize the need for additional analyses to detect new disease-associated GAA variants and non-Mendelian genotypes in Pompe disease where conventional DNA diagnostic assays are insufficient.

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.

A genetic modifier of symptom onset in Pompe disease.

BACKGROUND: Neonatal screening for Pompe disease is complicated by difficulties in predicting symptom onset in patients with the common c.-32-13T>G (IVS1) variant/null (i.e. fully deleterious) acid α-glucosidase (GAA) genotype. This splicing variant occurs in 90% of Caucasian late onset patients, and is associated with a broad range of symptom onset. METHODS: We analyzed a cohort of 143 compound heterozygous and 10 homozygous IVS1 patients, and we assessed ages at symptom onset, the presence of cis-acting single nucleotide variants (SNVs), and performed splicing analysis and enzyme activity assays. FINDINGS: In compound heterozygous IVS1 patients, the synonymous variant c.510C>T was uniquely present on the IVS1 allele in 9/33 (27%) patients with childhood onset, but was absent from 110 patients with onset in adulthood. GAA enzyme activity was lower in fibroblasts from patients who contained c.510C>T than it was in patients without c.510C>T. By reducing the extent of leaky wild-type splicing, c.510C>T modulated aberrant splicing caused by the IVS1 variant. The deleterious effect of c.510C>T was also found in muscle cells, the main target cells in Pompe disease. In homozygous IVS1 patients, the c.510C>T variant was absent in 4/4 (100%) asymptomatic individuals and present in 3/6 (50%) symptomatic patients. In cells from homozygous IVS1 patients, c.510C>T caused reduced leaky wild-type splicing. INTERPRETATION: c.510C>T is a genetic modifier in compound heterozygous and homozygous IVS1 patients. This finding is important for neonatal screening programs for Pompe disease. FUND: This work was funded by grants from Sophia Children's Hospital Foundation (SSWO, grant S17-32) and Metakids (2016-063).

Alternative Splicing in Genetic Diseases: Improved Diagnosis and Novel Treatment Options.

Alternative splicing is an important mechanism to regulate gene expression and to expand the repertoire of gene products in order to accommodate an increase in complexity of multicellular organisms. It needs to be precisely regulated, which is achieved via RNA structure, splicing factors, transcriptional regulation, and chromatin. Changes in any of these factors can lead to disease. These may include the core spliceosome, splicing enhancer/repressor sequences and their interacting proteins, the speed of transcription by RNA polymerase II, and histone modifications. While the basic principle of splicing is well understood, it is still very difficult to predict splicing outcome, due to the multiple levels of regulation. Current molecular diagnostics mainly uses Sanger sequencing of exons, or next-generation sequencing of gene panels or the whole exome. Functional analysis of potential splicing variants is scarce, and intronic variants are often not considered. This likely results in underestimation of the percentage of splicing variants. Understanding how sequence variants may affect splicing is not only crucial for confirmation of diagnosis and for genetic counseling, but also for the development of novel treatment options. These include small molecules, transsplicing, antisense oligonucleotides, and gene therapy. Here we review the current state of molecular mechanisms of splicing regulation and how deregulation can lead to human disease, diagnostics to detect splicing variants, and novel treatment options based on splicing correction.

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.

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.

Characterization and dynamics of pericentromere-associated domains in mice.

Despite recent progress in genome topology knowledge, the role of repeats, which make up the majority of mammalian genomes, remains elusive. Satellite repeats are highly abundant sequences that cluster around centromeres, attract pericentromeric heterochromatin, and aggregate into nuclear chromocenters. These nuclear landmark structures are assumed to form a repressive compartment in the nucleus to which genes are recruited for silencing. We have designed a strategy for genome-wide identification of pericentromere-associated domains (PADs) in different mouse cell types. The ∼1000 PADs and non-PADs have similar chromatin states in embryonic stem cells, but during lineage commitment, chromocenters progressively associate with constitutively inactive genomic regions at the nuclear periphery. This suggests that PADs are not actively recruited to chromocenters, but that chromocenters are themselves attracted to inactive chromatin compartments. However, we also found that experimentally induced proximity of an active locus to chromocenters was sufficient to cause gene repression. Collectively, our data suggest that rather than driving nuclear organization, pericentromeric satellite repeats mostly co-segregate with inactive genomic regions into nuclear compartments where they can contribute to stable maintenance of the repressed status of proximal chromosomal regions.

Identification and characterization of aberrant GAA pre-mRNA splicing in pompe disease using a generic approach.

Identification of pathogenic variants in monogenic diseases is an important aspect of diagnosis, genetic counseling, and prediction of disease severity. Pathogenic mechanisms involved include changes in gene expression, RNA processing, and protein translation. Variants affecting pre-mRNA splicing are difficult to predict due to the complex mechanism of splicing regulation. A generic approach to systematically detect and characterize effects of sequence variants on splicing would improve current diagnostic practice. Here, it is shown that such approach is feasible by combining flanking exon RT-PCR, sequence analysis of PCR products, and exon-internal quantitative RT-PCR for all coding exons. Application of this approach to one novel and six previously published variants in the acid-alpha glucosidase (GAA) gene causing Pompe disease enabled detection of a total of 11 novel splicing events. Aberrant splicing included cryptic splice-site usage, intron retention, and exon skipping. Importantly, the extent of leaky wild-type splicing correlated with disease onset and severity. These results indicate that this approach enables sensitive detection and in-depth characterization of variants affecting splicing, many of which are still unrecognized or poorly understood. The approach is generic and should be adaptable for application to other monogenic diseases to aid in improved diagnostics.

A central role for TFIID in the pluripotent transcription circuitry.

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.