Characterising splicing defects of ABCA4 variants within exons 13-50 in patient-derived fibroblasts.

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.

Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1.

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.

Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing.

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.

Stargardt disease and progress in therapeutic strategies.

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.

Expression phenotypes suggest that Der participates in a specific, high affinity interaction with membranes.

The GTPase Der is universally conserved in bacteria and is structurally unique as it consists of two GTP-binding domains in tandem (G-domain 1 and G-domain 2) whereas all the other GTPases posses a single GTPase domain. In order to assess the function of Der we have fractionated whole cell lysates containing over expressed Der. This analysis indicated that Der was present in sucrose gradient fractions containing membrane proteins. The interaction with the membrane fraction was specific for Der, since the related GTPase, Era, did not form the membrane complex. In addition, three independent criteria suggested a high affinity interaction; (1) the interaction can be detected under partially denaturing conditions using a gel electrophoresis co-migration assay, (2) the interaction survived 16 h sucrose gradient centrifugation, and (3) the complex could be efficiently reconstituted from purified components. Microscopic examination of cells containing over expressed Der showed that the cell wall structure was disrupted at both cell poles. This phenotype required Der domain three since domain deletion mutations showed no affect on cell wall structure. Surprisingly point mutations that ablate nucleotide binding of either GTP binding domain result in a defect in cell wall structure at only a single cell pole. The data reported here were considered together with results presented previously to suggest that Der may engage in a functional cyclic interaction between ribosomes and the membrane in Escherichia coli.

Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies.

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.

Generation of two induced pluripotent stem cell lines from a patient with Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene.

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.

Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease.

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.

Phenotype-genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion-insertion variant causing a splicing defect.

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.

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries.

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.