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The pre-mRNA splicing process is an essential aspect of gene expression and function and plays a substantial role in the complexity of higher eukaryotes. The development of antisense oligonucleotides (AOs) to harness the splicing process and manipulate it to treat various inherited and acquired diseases has been boosted by its flexibility and customisation capability. As the amount of research in this space increases, certain aspects need to be considered, in particular, how nonsequential splicing of pre-mRNA can impact AO-mediated splicing manipulation. In this chapter, we reviewed literature discussing intron removal order and several examples of disease-causing mutations impacted by this phenomenon. We also compared two strategies used to study intron removal order and the occasions that they are best suited. Finally, we discuss how nonsequential splicing could facilitate or impede the development of splice-manipulating AOs and aspects to consider when analysing AO effectiveness.

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T > C; 5603A > T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.

Usher syndrome (USH) is the most common form of deaf-blindness, with an estimated prevalence of 4.4 to 16.6 per 100,000 people worldwide. The most common form of USH is type IIA (USH2A), which is caused by homozygous or compound heterozygous mutations in the USH2A gene and accounts for around half of all USH cases. USH2A patients show moderate to severe hearing loss from birth, with diagnosis of retinitis pigmentosa in the second decade of life and variable vestibular involvement. Although hearing aids or cochlear implants can provide some mitigation of hearing deficits, there are currently no treatments aimed at preventing or restoring vision loss in USH2A patients. In this review, we first provide an overview of the molecular biology of the USH2A gene and its protein isoforms, which include a transmembrane protein (TM usherin) and an extracellular protein (EC usherin). The role of these proteins in the inner ear and retina and their impact on the pathogenesis of USH2A is discussed. We review animal cell-derived and patient cell-derived models currently used in USH2A research and conclude with an overview of potential treatment strategies currently in preclinical development and clinical trials.

Oligonucleotides and nucleic acid analogues that alter gene expression are now showing therapeutic promise in human disease. Whilst the modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, such modifications may also confer unexpected physicochemical and biological properties. Gapmer mixed-modified and DNA oligonucleotides on a phosphorothioate backbone can bind non-specifically to intracellular proteins to form a variety of toxic inclusions, driven by the phosphorothioate linkages, but also influenced by the oligonucleotide sequence. Recently, the non-antisense or other off-target effects of 2′ O- fully modified phosphorothioate linkage oligonucleotides are becoming better understood. Here, we report chemistry-specific effects of oligonucleotides composed of modified or unmodified bases, with phosphorothioate linkages, on subnuclear organelles and show altered distribution of nuclear proteins, the appearance of highly stable and strikingly structured nuclear inclusions, and disturbed RNA processing in primary human fibroblasts and other cultured cells. Phosphodiester, phosphorodiamidate morpholino oligomers, and annealed complimentary phosphorothioate oligomer duplexes elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of transfected fibroblasts exhibiting such disruption. Our data add to the growing body of evidence of off-target effects of some phosphorothioate nucleic acid drugs in primary cells and suggest alternative approaches to mitigate these effects.

Purpose : Stargardt disease (STGD1, OMIM: 248200) is mainly caused by missense, frameshifting or nonsense mutations in the ATP-binding cassette transporter gene, ABCA4. However, sequence variants that alter splicing are also pathogenic. Herein, we describe an in vitro investigation of aberrant splicing in ABCA4 variants detected in a STGD1 cohort using patient-derived fibroblast-based assay. In addition, retinal pigment epithelium (RPE) cells differentiated from patient-derived induced pluripotent stem cells (iPSC) were used to further validate such splicing errors. Methods : A cohort of 68 patients clinically diagnosed with STGD1 were recruited in this study. Genomic DNA obtained from recruited STGD1 patients was analysed by a commercial Stargardt/Macular dystrophy screening panel, targeting all exons of ABCA4 and flanking intronic regions, as well as already-known deep-intronic variants of ABCA4. Fibroblasts were propagated from 68 patients, total RNA was extracted and ABCA4 transcript structure was analysed by RT-PCR. The iPSCs reprogrammed from 2 patients carrying heterozygous c.[5461-10T>C;5603A>T] alleles were differentiated into RPE cells and the ABCA4 transcripts re-examined by RT-PCR. Results : A total of 73 unique ABCA4 alleles were identified. Biallelic ABCA4 variants were detected in 66 patients (66/68, 97.06%) and 2 patients (2/68, 2.94%) had a single ABCA4 variant detected. Only exons 13-50 of ABCA4 could be readily amplified from fibroblast RNA. In this region, 9 out of 55 (16.36%) variants, carried by 19 patients (28%), resulted in aberrant splicing. The most prevalent splice variant, c.5461-10T>C, is complexed with c.5603A>T and carried heterozygously by 7 patients (10%). This variant results in mature ABCA4 mRNA transcripts missing exon 39, or exons 39 and 40. The splicing defect was also evident in patient-derived iPSC-RPE cells. Conclusions : Patient-derived fibroblasts are useful for identifying ABCA4 splicing variants affecting exons 13-50. The

Background: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy. Materials and Methods: A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials.Results: STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the diverse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed. Conclusions: STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations.

Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

Stargardt disease (STGD1) is the most common inherited retinal dystrophy and ABCA4 c.546-–10 T>C is the most commonly reported splice mutation. Here, we generated and characterized two induced pluripotent stem cell (iPSC) lines from a STGD1 patient with compound heterozygous mutations in ABCA4 (c.[5461-10 T > C;5603A > T];[4163 T > C;455G > A]). Episomal vectors containing OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed to conduct the reprogramming of patient-derived fibroblasts. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency markers and showed trilineage differentiation potential. These lines can provide a powerful platform for further investigating the pathophysiological consequences of mutations in ABCA4.

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

Purpose of review Antisense oligomers (ASOs) have been available for decades: however, only recently have these molecules been applied clinically. This review aims to discuss the possible development of antisense-mediated splice correction therapies as precision medicines for familial hypercholesterolemic patients carrying mutations that compromise normal splicing of the low-density lipoprotein receptor (LDLR) gene transcript. Recent findings Three antisense drugs are currently being assessed in ongoing clinical trials for dyslipidemias, aiming to lower the plasma concentrations of lipoproteins that lead to end-organ damage, principally coronary artery disease. Although a handful of drugs may be applicable to many patients with familial hypercholesterolemia (FH), mutation-specific personalised antisense drugs may be even more effective in selected patients. Currently, there is no therapy that effectively addresses mutations in the LDLR, the major cause of FH. Many mutations in the LDLR that disrupt normal pre-mRNA processing could be applicable to splice correction therapy to restore receptor activity. Summary Precision medicine could provide long-term economic and social benefits if they can be implemented effectively and sustainably. Many mutations found in the LDLR gene could be amendable to therapeutic splice correction and we should consider developing a therapeutic ASO platform for these mutations.

Following publication of the original article [1], the authors would like to correct a formula from “T > C” to “C > T” in two paragraphs...

Purpose : Stargardt disease (STGD1, OMIM: 248200) is mainly caused by missense, frameshifting or nonsense mutations in the ATP-binding cassette transporter gene, ABCA4. However, sequence variants that alter splicing are also pathogenic. Herein, we describe an in vitro investigation of aberrant splicing in ABCA4 variants detected in a STGD1 cohort using patient-derived fibroblast-based assay. In addition, retinal pigment epithelium (RPE) cells differentiated from patient-derived induced pluripotent stem cells (iPSC) were used to further validate such splicing errors. Methods : A cohort of 68 patients clinically diagnosed with STGD1 were recruited in this study. Genomic DNA obtained from recruited STGD1 patients was analysed by a commercial Stargardt/Macular dystrophy screening panel, targeting all exons of ABCA4 and flanking intronic regions, as well as already-known deep-intronic variants of ABCA4. Fibroblasts were propagated from 68 patients, total RNA was extracted and ABCA4 transcript structure was analysed by RT-PCR. The iPSCs reprogrammed from 2 patients carrying heterozygous c.[5461-10T>C;5603A>T] alleles were differentiated into RPE cells and the ABCA4 transcripts re-examined by RT-PCR. Results : A total of 73 unique ABCA4 alleles were identified. Biallelic ABCA4 variants were detected in 66 patients (66/68, 97.06%) and 2 patients (2/68, 2.94%) had a single ABCA4 variant detected. Only exons 13-50 of ABCA4 could be readily amplified from fibroblast RNA. In this region, 9 out of 55 (16.36%) variants, carried by 19 patients (28%), resulted in aberrant splicing. The most prevalent splice variant, c.5461-10T>C, is complexed with c.5603A>T and carried heterozygously by 7 patients (10%). This variant results in mature ABCA4 mRNA transcripts missing exon 39, or exons 39 and 40. The splicing defect was also evident in patient-derived iPSC-RPE cells. Conclusions : Patient-derived fibroblasts are useful for identifying ABCA4 splicing variants affecting exons 13-50. The

Polyglutamine (polyQ) ataxias are a heterogenous group of neurological disorders all caused by an expanded CAG trinucleotide repeat located in the coding region of each unique causative gene. To date, polyQ ataxias encompass six disorders: spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 and account for a larger group of disorders simply known as polyglutamine disorders, which also includes Huntington’s disease. These diseases are typically characterised by progressive ataxia, speech and swallowing difficulties, lack of coordination and gait, and are unfortunately fatal in nature, with the exception of SCA6. All the polyQ spinocerebellar ataxias have a hallmark feature of neuronal aggregations and share many common pathogenic mechanisms, such as mitochondrial dysfunction, impaired proteasomal function, and autophagy impairment. Currently, therapeutic options are limited, with no available treatments that slow or halt disease progression. Here, we discuss the common molecular and clinical presentations of polyQ spinocerebellar ataxias. We will also discuss the promising antisense oligonucleotide therapeutics being developed as treatments for these devastating diseases. With recent advancements and therapeutic approvals of various antisense therapies, it is envisioned that some of the studies reviewed may progress into clinical trials and beyond.

Background: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy. Materials and Methods: A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials.Results: STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the diverse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed. Conclusions: STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations.

Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

Stargardt disease (STGD1) is the most common inherited retinal dystrophy and ABCA4 c.546-–10 T>C is the most commonly reported splice mutation. Here, we generated and characterized two induced pluripotent stem cell (iPSC) lines from a STGD1 patient with compound heterozygous mutations in ABCA4 (c.[5461-10 T > C;5603A > T];[4163 T > C;455G > A]). Episomal vectors containing OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed to conduct the reprogramming of patient-derived fibroblasts. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency markers and showed trilineage differentiation potential. These lines can provide a powerful platform for further investigating the pathophysiological consequences of mutations in ABCA4.

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-α-glucosidase 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.

Pompe disease, or glycogen storage disease II is a rare, progressive disease leading to skeletal muscle weakness due to deficiency of the acid α-1,4-glucosidase enzyme (GAA). The severity of disease and observed time of onset is subject to the various combinations of heterozygous GAA alleles. Here we have characterized two novel mutations: c.2074C>T and c.1910_1918del, and a previously reported c.1082C>G mutation of uncertain clinical significance. These mutations were found in three unrelated patients with adult-onset Pompe disease carrying the common c.-32-13T>G mutation. The c.2074 C>T nonsense mutation has obvious consequences on GAA expression but the c.1910_1918del (deletion of 3 amino acids) and c.1082C>G missense variants are more subtle DNA changes with catastrophic consequences on GAA activity. Molecular and clinical analyses from the three patients corresponded with the anticipated pathogenicity of each mutation.

The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2′-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.

Parkin-type autosomal recessive juvenile-onset Parkinson’s disease is caused by mutations in the PRKN gene and accounts for 50% of all autosomal recessive Parkinsonism cases. Parkin is a neuroprotective protein that has dual functions as an E3 ligase in the ubiquitin–proteasome system and as a transcriptional repressor of p53. While genomic deletions of PRKN exon 3 disrupt the mRNA reading frame and result in the loss of functional parkin protein, deletions of both exon 3 and 4 maintain the reading frame and are associated with a later onset, milder disease progression, indicating this particular isoform retains some function. Here, we describe in vitro evaluation of antisense oligomers that restore functional parkin expression in cells derived from a Parkinson’s patient carrying a heterozygous PRKN exon 3 deletion, by inducing exon 4 skipping to correct the reading frame. We show that the induced PRKN transcript is translated into a shorter but semi-functional parkin isoform able to be recruited to depolarised mitochondria, and also transcriptionally represses p53 expression. These results support the potential use of antisense oligomers as a disease-modifying treatment for selected pathogenic PRKN mutations.

Background Deletion–insertion (delins) variants in the retina‐specific ATP‐binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data. Methods Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient‐derived fibroblasts was analyzed by RT‐PCR to evaluate splicing behavior of the ABCA4 variants. Results Affected members carrying the novel c.6031_6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early‐onset cone‐rod dystrophy to late‐onset macular dystrophy. This variant resulted in a 56‐nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C‐terminus would be truncated from the ABCA4 protein. Conclusion This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient‐derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment.

The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2′-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.

Parkin-type autosomal recessive juvenile-onset Parkinson’s disease is caused by mutations in the PRKN gene and accounts for 50% of all autosomal recessive Parkinsonism cases. Parkin is a neuroprotective protein that has dual functions as an E3 ligase in the ubiquitin–proteasome system and as a transcriptional repressor of p53. While genomic deletions of PRKN exon 3 disrupt the mRNA reading frame and result in the loss of functional parkin protein, deletions of both exon 3 and 4 maintain the reading frame and are associated with a later onset, milder disease progression, indicating this particular isoform retains some function. Here, we describe in vitro evaluation of antisense oligomers that restore functional parkin expression in cells derived from a Parkinson’s patient carrying a heterozygous PRKN exon 3 deletion, by inducing exon 4 skipping to correct the reading frame. We show that the induced PRKN transcript is translated into a shorter but semi-functional parkin isoform able to be recruited to depolarised mitochondria, and also transcriptionally represses p53 expression. These results support the potential use of antisense oligomers as a disease-modifying treatment for selected pathogenic PRKN mutations.

Background Deletion–insertion (delins) variants in the retina‐specific ATP‐binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data. Methods Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient‐derived fibroblasts was analyzed by RT‐PCR to evaluate splicing behavior of the ABCA4 variants. Results Affected members carrying the novel c.6031_6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early‐onset cone‐rod dystrophy to late‐onset macular dystrophy. This variant resulted in a 56‐nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C‐terminus would be truncated from the ABCA4 protein. Conclusion This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient‐derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment.

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2 '-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2 '-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5 ' region of exon 51. These results indicated that locked nucleic acid/2 '-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone.

With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-NucleofectorTM or Neon. electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000TM for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000TM or Lipofectamine 2000. for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-NucleofectorTM X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2 '-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2 '-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5 ' region of exon 51. These results indicated that locked nucleic acid/2 '-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone.

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5′ end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2′-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-NucleofectorTM or Neon. electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000TM for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000TM or Lipofectamine 2000. for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-NucleofectorTM X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

A deficiency of the enzyme acid alpha‐glucosidase leads to Pompe disease, also known as glycogen storage disease II (GSDII). It is an autosomal recessive disorder, and affected individuals are unable to degrade glycogen stored within lysosomes, leading to an accumulation of glycogen in tissue. Clinically, GSDII may manifest with a broad spectrum of severity, ranging from severe/infantile to a milder late onset adult form. The most common GAA mutation associated with the latter form is IVS1‐13T>G, found in over two thirds of adult onset GSDII patients. The consequence of this mutation is production of a non-functional GAA transcript because exon 2 is excluded during pre-RNA splicing. Current enzyme replacement therapy for Pompe disease has drawbacks, including immune reaction and poor delivery to target tissues. Therefore an efficient alternative therapy is needed. We sought to restore inclusion of GAA exon 2 using splice switching antisense oligomers (AO) directed at splice silencer elements to promote recognition and retention of exon 2 in the mature GAA mRNA, thereby restoring enzyme function. Screen of the best AO sequences that promote exon 2 inclusion in GAA transcript was carried out in fibroblasts derived from adult Pompe patients. Phosphorodiamidate morpholino oligomers (PMOs) were transfected into patient cells and RT‐PCR and acid‐alpha‐glucosidase enzyme activitywere performed. Several PMOs showed an increase in full‐length amplicon and increase inGAAactivity. These results show thatPMOmediatedmodification of GAA transcript could have therapeutic potential for a large portion of adult onset Pompe patients.

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine disorders. Although SCA3 is pathogenically heterogeneous, the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. There is currently no cure, nor effective treatment strategy for affected individuals. SCA3 is caused by an expanded polyglutamine tract found in ataxin‐3, resulting in conformational changes that lead to toxic gain of function. This expanded glutamine tract is located at the 5' end of the penultimate exon (exon 10) of the ATXN3 gene (14q32.1). This study aims to use antisense oligonucleotide (AO) mediated exon skipping to develop a potential therapeutic strategy for the treatment of SCA3. Preliminary in vitro data in healthy cells show that it is possible to create a novel, internally truncated protein, missing the CAG repeat contained in ATXN3 and still maintain many normal functions. Ongoing research is to be conducted in patient cells to determine whether the CAG expansion will affect the efficacy of the AOs to product a truncated protein. This research shows proof of concept that the removal of exon 10 alone, although out of frame, is possible. Alongside this, another strategy may be to disrupt normal ATXN3 expression by targeting exons which disrupt the reading frame and thereby down‐regulate protein expression. We hypothesize that reducing the normal and mutant protein may alleviate symptoms and/or delay onset or slow progression of the disease.

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine disorders. Although SCA3 is pathogenically heterogeneous, the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. There is currently no cure, nor effective treatment strategy for affected individuals. SCA3 is caused by an expanded polyglutamine tract found in ataxin‐3, resulting in conformational changes that lead to toxic gain of function. This expanded glutamine tract is located at the 5' end of the penultimate exon (exon 10) of the ATXN3 gene (14q32.1). This study aims to use antisense oligonucleotide (AO) mediated exon skipping to develop a potential therapeutic strategy for the treatment of SCA3. Preliminary in vitro data in healthy cells show that it is possible to create a novel, internally truncated protein, missing the CAG repeat contained in ATXN3 and still maintain many normal functions. Ongoing research is to be conducted in patient cells to determine whether the CAG expansion will affect the efficacy of the AOs to product a truncated protein. This research shows proof of concept that the removal of exon 10 alone, although out of frame, is possible. Alongside this, another strategy may be to disrupt normal ATXN3 expression by targeting exons which disrupt the reading frame and thereby down‐regulate protein expression. We hypothesize that reducing the normal and mutant protein may alleviate symptoms and/or delay onset or slow progression of the disease.

A deficiency of the enzyme acid alpha‐glucosidase leads to Pompe disease, also known as glycogen storage disease II (GSDII). It is an autosomal recessive disorder, and affected individuals are unable to degrade glycogen stored within lysosomes, leading to an accumulation of glycogen in tissue. Clinically, GSDII may manifest with a broad spectrum of severity, ranging from severe/infantile to a milder late onset adult form. The most common GAA mutation associated with the latter form is IVS1‐13T>G, found in over two thirds of adult onset GSDII patients. The consequence of this mutation is production of a non-functional GAA transcript because exon 2 is excluded during pre-RNA splicing. Current enzyme replacement therapy for Pompe disease has drawbacks, including immune reaction and poor delivery to target tissues. Therefore an efficient alternative therapy is needed. We sought to restore inclusion of GAA exon 2 using splice switching antisense oligomers (AO) directed at splice silencer elements to promote recognition and retention of exon 2 in the mature GAA mRNA, thereby restoring enzyme function. Screen of the best AO sequences that promote exon 2 inclusion in GAA transcript was carried out in fibroblasts derived from adult Pompe patients. Phosphorodiamidate morpholino oligomers (PMOs) were transfected into patient cells and RT‐PCR and acid‐alpha‐glucosidase enzyme activitywere performed. Several PMOs showed an increase in full‐length amplicon and increase inGAAactivity. These results show thatPMOmediatedmodification of GAA transcript could have therapeutic potential for a large portion of adult onset Pompe patients.

Integrin alpha-4 (ITGA4) is a validated therapeutic target for multiple sclerosis (MS) and Natalizumab, an antibody targeting ITGA4 is currently approved for treating MS. However, there are severe side effects related to this therapy. In this study, we report the development of a novel DNAzyme that can efficiently cleave the ITGA4 transcript. We designed a range of DNAzyme candidates across various exons of ITGA4. RNV143, a 30mer arm-loop-arm type DNAzyme efficiently cleaved 84% of the ITGA4 mRNA in human primary fibroblasts. RNV143 was then systematically modified by increasing the arm lengths on both sides of the DNAzymes by one, two and three nucleotides each, and incorporating chemical modifications such as inverted-dT, phosphorothioate backbone and LNA-nucleotides. Increasing the arm length of DNAzyme RNV143 did not improve the efficiency however, an inverted-dT modification provided the most resistance to 3′ → 5′ exonuclease compared to other modifications tested. Our results show that RNV143A could be a potential therapeutic nucleic acid drug molecule towards the treatment for MS.

Integrin alpha-4 (ITGA4) is a validated therapeutic target for multiple sclerosis (MS) and Natalizumab, an antibody targeting ITGA4 is currently approved for treating MS. However, there are severe side effects related to this therapy. In this study, we report the development of a novel DNAzyme that can efficiently cleave the ITGA4 transcript. We designed a range of DNAzyme candidates across various exons of ITGA4. RNV143, a 30mer arm-loop-arm type DNAzyme efficiently cleaved 84% of the ITGA4 mRNA in human primary fibroblasts. RNV143 was then systematically modified by increasing the arm lengths on both sides of the DNAzymes by one, two and three nucleotides each, and incorporating chemical modifications such as inverted-dT, phosphorothioate backbone and LNA-nucleotides. Increasing the arm length of DNAzyme RNV143 did not improve the efficiency however, an inverted-dT modification provided the most resistance to 3′ → 5′ exonuclease compared to other modifications tested. Our results show that RNV143A could be a potential therapeutic nucleic acid drug molecule towards the treatment for MS.

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

Fibronectin, encoded by FN gene, is a glycosylated protein found on cell surfaces, in extracellular matrix and plasma. It is involved in many events, including cell adhesion, migration, wound healing and blood coagulation. Alternative splicing of the FN1 transcript produces up to 20 transcript variants, which are tissue and developmentally dependent and also regulated by physiological and pathological conditions. It has been shown that the presence of a fibronectin isoform containing an extra domain A (EDA+‐FN), normally absent from the circulation, may be an indicator of chronic inflammation and also promotes ischemia/reperfusion brain injury through a TLR4‐dependent mechanism. We reduced the levels of the EDA+‐FN isoform using splice switching antisense oligonucleotides (AO). AOs, comprising 2'‐O‐methyl modified bases on a phosphorothioate backbone, were designed to exclude the EDA domain in exon 33 from the FN1 transcript. The ratio of EDA+‐FN to EDA‐‐FN spliceoforms was analysed 24 h after the fibroblasts were transfected with AOs as cationic lipoplexes. The most efficient AO targeting exon 33 was able to induce an approximately 70% reduction in EDA+‐FN transcript in treated cells. Splice switching AOs are emerging as powerful tools in therapeutic alternative splicing. The exon skipping AO identified in this study will be further investigated for therapeutic potential in pathological conditions caused by elevated level of the EDA+‐FN isoform.

Fibronectin, encoded by FN gene, is a glycosylated protein found on cell surfaces, in extracellular matrix and plasma. It is involved in many events, including cell adhesion, migration, wound healing and blood coagulation. Alternative splicing of the FN1 transcript produces up to 20 transcript variants, which are tissue and developmentally dependent and also regulated by physiological and pathological conditions. It has been shown that the presence of a fibronectin isoform containing an extra domain A (EDA+‐FN), normally absent from the circulation, may be an indicator of chronic inflammation and also promotes ischemia/reperfusion brain injury through a TLR4‐dependent mechanism. We reduced the levels of the EDA+‐FN isoform using splice switching antisense oligonucleotides (AO). AOs, comprising 2'‐O‐methyl modified bases on a phosphorothioate backbone, were designed to exclude the EDA domain in exon 33 from the FN1 transcript. The ratio of EDA+‐FN to EDA‐‐FN spliceoforms was analysed 24 h after the fibroblasts were transfected with AOs as cationic lipoplexes. The most efficient AO targeting exon 33 was able to induce an approximately 70% reduction in EDA+‐FN transcript in treated cells. Splice switching AOs are emerging as powerful tools in therapeutic alternative splicing. The exon skipping AO identified in this study will be further investigated for therapeutic potential in pathological conditions caused by elevated level of the EDA+‐FN isoform.