Alternative RNA splicing defects in pediatric cancers: new insights in tumorigenesis and potential therapeutic vulnerabilities.

BACKGROUND: Compared with adult cancers, pediatric cancers are uniquely characterized by a genomically stable landscape and lower tumor mutational burden. Alternative splicing, however, a global cellular process that produces different messenger RNA/protein isoforms from a single messenger RNA transcript, has been increasingly implicated in the development of pediatric cancers. DESIGN: We review the current literature on the role of alternative splicing in adult cancer, cancer predisposition syndromes, and pediatric cancers. We also describe multiple splice variants identified in adult cancers and confirmed through comprehensive genomic profiling in our institutional cohort of rare, refractory, and relapsed pediatric and adolescent young adult cancer patients. Finally, we summarize the contributions of alternative splicing events to neoantigens and chemoresistance and prospects for splicing-based therapies. RESULTS: Published dysregulated splicing events can be categorized as exon inclusion, exon exclusion, splicing factor up-regulation, or splice site alterations. We observe these phenomena in cancer predisposition syndromes (Lynch syndrome, Li-Fraumeni syndrome, CHEK2) and pediatric leukemia (B-cell acute lymphoblastic leukemia), sarcomas (Ewing sarcoma, rhabdomyosarcoma, osteosarcoma), retinoblastoma, Wilms' tumor, and neuroblastoma. Within our institutional cohort, we demonstrate splice variants in key regulatory genes (CHEK2, TP53, PIK3R1, MDM2, KDM6A, NF1) that resulted in exon exclusion or splice site alterations, which were predicted to impact functional protein expression and promote tumorigenesis. Differentially spliced isoforms and splicing proteins also impact neoantigen creation and treatment resistance, such as imatinib or glucocorticoid regimens. Additionally, splice-altering strategies with the potential to change the therapeutic landscape of pediatric cancers include antisense oligonucleotides, adeno-associated virus gene transfers, and small molecule inhibitors. CONCLUSIONS: Alternative splicing plays a critical role in the formation and growth of pediatric cancers, and our institutional cohort confirms and highlights the broad spectrum of affected genes in a variety of cancers. Further studies that elucidate the mechanisms of disease-inducing splicing events will contribute toward the development of novel therapeutics.

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing.

Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10-intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5'ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10-15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3' arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5'ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5' arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5'ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

Estimating the prevalence of functional exonic splice regulatory information.

In addition to coding information, human exons contain sequences necessary for correct splicing. These elements are known to be under purifying selection and their disruption can cause disease. However, the density of functional exonic splicing information remains profoundly uncertain. Several groups have experimentally investigated how mutations at different exonic positions affect splicing. They have found splice information to be distributed widely in exons, with one estimate putting the proportion of splicing-relevant nucleotides at >90%. These results suggest that splicing could place a major pressure on exon evolution. However, analyses of sequence conservation have concluded that the need to preserve splice regulatory signals only slightly constrains exon evolution, with a resulting decrease in the average human rate of synonymous evolution of only 1-4%. Why do these two lines of research come to such different conclusions? Among other reasons, we suggest that the methods are measuring different things: one assays the density of sites that affect splicing, the other the density of sites whose effects on splicing are visible to selection. In addition, the experimental methods typically consider short exons, thereby enriching for nucleotides close to the splice junction, such sites being enriched for splice-control elements. By contrast, in part owing to correction for nucleotide composition biases and to the assumption that constraint only operates on exon ends, the conservation-based methods can be overly conservative.

Splicing therapy for neuromuscular disease.

Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision.

Pairwise Engineering of Tandemly Aligned Self-Splicing Group I Introns for Analysis and Control of Their Alternative Splicing.

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated.

Developing Therapeutic Splice-Correcting Antisense Oligomers for Adult-Onset Pompe Disease with c.-32-13T>G Mutation.

The mutation c.-32-13T>G in the GAA gene impacts normal exon 2 splicing and is found in two-thirds of late-onset Pompe disease cases. We have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients carrying this common mutation. We performed in silico analysis of the GAA gene transcript for potential splicing silencers and designed oligomers targeting motifs predicted to enhance exon 2 retention in the mature mRNA. Two patient-derived fibroblasts were obtained from Coriell Institute for Medical Research, and seven fibroblast strains from unrelated patients were supplied by Westmead Hospital in Sydney, Australia. Both fibroblasts and forced-myogenic cells were treated with optimized phosphorodiamidate morpholino oligomers supplied by Sarepta Therapeutics. Total RNA and protein were extracted from the cells after incubation with phosphorodiamidate morpholino oligomers, and RT-PCR and RT-qPCR were performed to confirm exon 2 inclusion is enhanced. Acid α-glucosidase activity and expression levels were also assessed to confirm therapeutic potential.

PQBP1 regulates striatum development through balancing striatal progenitor proliferation and differentiation.

The balance between cell proliferation and differentiation is essential for maintaining the neural progenitor pool and brain development. Although the mechanisms underlying cell proliferation and differentiation at the transcriptional level have been studied intensively, post-transcriptional regulation of cell proliferation and differentiation remains largely unclear. Here, we show that deletion of the alternative splicing regulator PQBP1 in striatal progenitors results in defective striatal development due to impaired neurogenesis of spiny projection neurons (SPNs). Pqbp1-deficient striatal progenitors exhibit declined proliferation and increased differentiation, resulting in a reduced striatal progenitor pool. We further reveal that PQBP1 associates with components in splicing machinery. The alternative splicing profiles identify that PQBP1 promotes the exon 9 inclusion of Numb, a variant that mediates progenitor proliferation. These findings identify PQBP1 as a regulator in balancing striatal progenitor proliferation and differentiation and provide alternative insights into the pathogenic mechanisms underlying Renpenning syndrome.

Design of Bifunctional Antisense Oligonucleotides for Exon Inclusion.

Bifunctional antisense oligonucleotide (AON) is a specially designed AON to regulate pre-messenger RNA (pre-mRNA) splicing of a target gene. It is composed of two domains. The antisense domain contains sequences complementary to the target gene. The tail domain includes RNA sequences that recruit RNA binding proteins which may act positively or negatively in pre-mRNA splicing. This approach can be designed as targeted oligonucleotide enhancers of splicing, named TOES, for exon inclusion; or as targeted oligonucleotide silencers of splicing, named TOSS, for exon skipping. Here, we provide detailed methods for the design of TOES for exon inclusion, using SMN2 exon 7 splicing as an example. A number of annealing sites and the tail sequences previously published are listed. We also present methodology of assessing the effects of TOES on exon inclusion in fibroblasts cultured from a SMA patient. The effects of TOES on SMN2 exon 7 splicing were validated at RNA level by PCR and quantitative real-time PCR, and at protein level by western blotting.

U7 snRNA, a Small RNA with a Big Impact in Gene Therapy.

The uridine-rich 7 (U7) small nuclear RNA (snRNA) is a component of a small nuclear ribonucleoprotein (snRNP) complex. U7 snRNA naturally contains an antisense sequence that identifies histone premessenger RNAs (pre-mRNAs) and is involved in their 3' end processing. By altering this antisense sequence, researchers have turned U7 snRNA into a versatile tool for targeting pre-mRNAs and modifying splicing. Encapsulating a modified U7 snRNA into a viral vector such as adeno-associated virus (also referred as vectorized exon skipping/inclusion, or VES/VEI) enables the delivery of this highly efficacious splicing modulator into a range of cell lines, primary cells, and tissues. In addition, and in contrast to antisense oligonucleotides, viral delivery of U7 snRNA enables long-term expression of antisense sequences in the nucleus as part of a stable snRNP complex. As a result, VES/VEI has emerged as a promising therapeutic platform for treating a large variety of human diseases caused by errors in pre-mRNA splicing or its regulation. Here we provide an overview of U7 snRNA's natural function and its applications in gene therapy.

Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3' Splice-Site Selection.

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3' splice sites (3'ss). Both proteins preferentially bind uridine-rich sequences upstream of 3'ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3'ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3'ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3'ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

Functional characterization and phenotypic spectrum of three recurrent disease-causing deep intronic variants of the CFTR gene.

BACKGROUND: The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown. METHODS: Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected. RESULTS: Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency. CONCLUSION: These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies.

Invention and Early History of Exon Skipping and Splice Modulation.

Since its discovery in 1977, much has been known about RNA splicing and how it plays a central role in human development, function, and, notably, disease. Defects in RNA splicing account for at least 10% of all genetic disorders, with the number expected to increase as more information is uncovered on the contribution of noncoding genomic regions to disease. Splice modulation through the use of antisense oligonucleotides (AOs) has emerged as a promising avenue for the treatment of these disorders. In fact, two splice-switching AOs have recently obtained approval from the US Food and Drug Administration: eteplirsen (Exondys 51) for Duchenne muscular dystrophy, and nusinersen (Spinraza) for spinal muscular atrophy. These work by exon skipping and exon inclusion, respectively. In this chapter, we discuss the early development of AO-based splice modulation therapy-its invention, first applications, and its evolution into the approach we are now familiar with. We give a more extensive history of exon skipping in particular, as it is the splice modulation approach given the most focus in this book.

Morpholino-Mediated Exon Inclusion for SMA.

The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both preclinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to a recently approved drug. AONs with phosphorodiamidate morpholino oligomer (PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to a recent conditional approval for one DMD PMO drug. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex).

Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by a mutation in SMN1 that stops production of SMN (survival of motor neuron) protein. Insufficient levels of SMN results in the loss of motor neurons, which causes muscle weakness, respiratory distress, and paralysis. A nearly identical gene (SMN2) contains a C-to-T transition which excludes exon 7 from 90% of the mature mRNA transcripts, leading to unstable proteins which are targeted for degradation. Although SMN2 cannot fully compensate for a loss of SMN1 due to only 10% functional mRNA produced, the discovery of the intronic splicing silencer (ISS-N1) opened a doorway for therapy. By blocking its function with antisense oligonucleotides manipulated for high specificity and efficiency, exon 7 can be included to produce full-length mRNA, which then compensates for the loss of SMN1. Nusinersen (Spinraza), the first FDA-approved antisense oligonucleotide drug targeting SMA, was designed based on this concept and clinical studies have demonstrated a dramatic improvement in patients. Novel chemistries including phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNAs), as well as peptide conjugates such as Pip that facilitate accurate targeting to the central nervous system, are explored to increase the efficiency of exon 7 inclusion in the appropriate tissues to ameliorate the SMA phenotype. Due to the rapid advancement of treatments for SMA following the discovery of ISS-N1, the future of SMA treatment is highly promising.

Nusinersen in the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is one of the most common genetic causes of infantile death arising due to mutations in the SMN1 gene and the subsequent loss of motor neurons. With the discovery of the intronic splicing silencer N1 (ISS-N1) as a potential target for antisense therapy, several antisense oligonucleotides (ASOs) are being developed to include exon 7 in the final mRNA transcript of the SMN2 gene and thereby increasing the production of spinal motor neuron (SMN) proteins. Nusinersen (spinraza), a modified 2'-O-methoxyethyl (MOE) antisense oligonucleotide is the first drug to be approved by Food and Drug Agency (FDA) in December of 2016. Here we briefly review the pharmacological relevance of the drug, clinical trials, toxicity, and future directions following the approval of nusinersen.

In Vitro Evaluation of Antisense-Mediated Exon Inclusion for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA), the most common gentic cause of infantile death caused by mutations in the SMN1 gene, presents a unique case in the field of splice modulation therapy, where a gene (or lack of) is responsible for causing the disease phenotype but treatment is not focused around it. Antisense therapy targeting SMN2 which leads to SMN protein expression has been at the forefront of research when it comes to developing a feasible therapy for treating SMA. Recent FDA approval of an antisense-based drug with the 2'-methoxyethoxy (2'MOE) chemistry, called nusinersen (Spinraza), brought antisense drugs into the spotlight. The 2'MOE, although effective, has weaknesses such as the inability to cross the blood-brain barrier and the high cost of treatment. This propelled the research community to investigate new chemistries of antisense oligonucleotides (ASOs) that may be better in both treatment and cost efficiency. Here we describe two types of ASOs, phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNA)-DNA mixmers, being investigated as potential treatments for SMA, and methods used to test their efficacy, including quantitative RT-PCR, Western blotting, and immunofluorescence staining to detect SMN in nuclear gems/Cajal bodies, in type I SMA patient fibroblast cell lines.

Systemic and ICV Injections of Antisense Oligos into SMA Mice and Evaluation.

Spinal muscular atrophy (SMA) is the most common genetic cause of infantile death caused by mutations in the SMN1 gene. Nusinersen (Spinraza), an antisense therapy-based drug with the 2'-methoxyethoxy (2'MOE) chemistry approved by the FDA in 2016, brought antisense drugs into the spotlight. Antisense-mediated exon inclusion targeting SMN2 leads to SMN protein expression. Although effective, 2'MOE has weaknesses such as the inability to cross the blood-brain barrier and the high cost of treatment. To investigate new chemistries of antisense oligonucleotides (ASOs), SMA mouse models can serve as an important source. Here we describe methods to test the efficacy of ASOs, such as phosphorodiamidate morpholino oligomers (PMOs), in a severe SMA mouse model.

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.