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Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy most frequently associated with OPA1 mutations. Most variants result in haploinsufficiency, and patient cells express roughly half of the normal levels of OPA1 protein. OPA1 is a mitochondrial GTPase that is essential for normal mitochondrial function. We identified and characterized STK-002, an antisense oligonucleotide (ASO) designed to prevent the incorporation of a naturally occurring alternatively spliced nonproductive exon in OPA1 . STK-002 dose dependently reduced the inclusion of this exon, and increased OPA1 protein in human cells, including ADOA patient-derived fibroblasts. ADOA patient cells manifest reduced mitochondrial respiration, and treatment with STK-002 improved the parameters of mitochondrial respiratory function in these cells. Since STK-002 increases OPA1 through the wild-type allele, we assessed retinal OPA1 in wild-type cynomolgus monkeys and rabbits after intravitreal administration of STK-002 or a rabbit-specific surrogate. Increased OPA1 protein was produced in retinal tissue in both species at 4 weeks after ASO injection and persisted in monkeys at 8 weeks. STK-002 and enhanced OPA1 immunofluorescence were visualized in retinal ganglion cells of cynomolgus monkeys treated with the ASO. Cumulatively, these data support the progression of STK-002 toward the clinic as the first potential disease-modifying treatment for ADOA.

Single-stranded oligonucleotides have been explored as a therapeutic modality for more than 20 years. Only during the last 5 years have single-stranded oligonucleotides become a modality of choice in the fields of precision medicine and targeted therapeutics. Recently, there have been a number of development efforts involving this modality that have led to treatments for genetic diseases that were once untreatable. This review highlights key applications of single-stranded oligonucleotides that function in a sequence-dependent manner when applied to modulate precursor (pre-)mRNA splicing, gene expression, and immune pathways. These applications have been used to address diseases that range from neurological to muscular to metabolic, as well as to develop vaccines. The wide range of applications denotes the versatility of single-stranded oligonucleotides as a robust therapeutic platform. The focus of this review is centered on approved single-stranded oligonucleotide therapies and the evolution of oligonucleotide therapeutics into novel applications currently in clinical development., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dravet syndrome (DS) is an intractable developmental and epileptic encephalopathy caused largely by de novo variants in the SCN1A gene, resulting in haploinsufficiency of the voltage-gated sodium channel α subunit Na V 1.1. Here, we used Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, nonproductive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human cell lines, as well as in mouse brain. We show that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy (SUDEP) in the F1:129S- Scn1a +/- × C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and Na V 1.1 protein was confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

The splicing factor SRSF1 promotes nonsense-mediated mRNA decay (NMD), a quality control mechanism that degrades mRNAs with premature termination codons (PTCs). Here we show that transcript-bound SRSF1 increases the binding of NMD factor UPF1 to mRNAs while in, or associated with, the nucleus, bypassing UPF2 recruitment and promoting NMD. SRSF1 promotes NMD when positioned downstream of a PTC, which resembles the mode of action of exon junction complex (EJC) and NMD factors. Moreover, splicing and/or EJC deposition increase the effect of SRSF1 on NMD. Lastly, SRSF1 enhances NMD of PTC-containing endogenous transcripts that result from various events. Our findings reveal an alternative mechanism for UPF1 recruitment, uncovering an additional connection between splicing and NMD. SRSF1's role in the mRNA's journey from splicing to decay has broad implications for gene expression regulation and genetic diseases., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that is thought to exacerbate the phenotype of certain pathogenic nonsense mutations by preventing the expression of semi-functional proteins. NMD also limits the efficacy of read-through compound (RTC)-based therapies. Here, we report a gene-specific method of NMD inhibition using antisense oligonucleotides (ASOs) and combine this approach with an RTC to effectively restore the expression of full-length protein from a nonsense-mutant allele.

To identify human intronic sequences associated with 5' splice site recognition, we performed a systematic search for motifs enriched in introns downstream of both constitutive and alternative cassette exons. Significant enrichment was observed for U-rich motifs within 100 nucleotides downstream of 5' splice sites of both classes of exons, with the highest enrichment between positions +6 and +30. Exons adjacent to U-rich intronic motifs contain lower frequencies of exonic splicing enhancers and higher frequencies of exonic splicing silencers, compared with exons not followed by U-rich intronic motifs. These findings motivated us to explore the possibility of a widespread role for U-rich motifs in promoting exon inclusion. Since cytotoxic granule-associated RNA binding protein (TIA1) and TIA1-like 1 (TIAL1; also known as TIAR) were previously shown in vitro to bind to U-rich motifs downstream of 5' splice sites, and to facilitate 5' splice site recognition in vitro and in vivo, we investigated whether these factors function more generally in the regulation of splicing of exons followed by U-rich intronic motifs. Simultaneous knockdown of TIA1 and TIAL1 resulted in increased skipping of 36/41 (88%) of alternatively spliced exons associated with U-rich motifs, but did not affect 32/33 (97%) alternatively spliced exons that are not associated with U-rich motifs. The increase in exon skipping correlated with the proximity of the first U-rich motif and the overall "U-richness" of the adjacent intronic region. The majority of the alternative splicing events regulated by TIA1/TIAL1 are conserved in mouse, and the corresponding genes are associated with diverse cellular functions. Based on our results, we estimate that approximately 15% of alternative cassette exons are regulated by TIA1/TIAL1 via U-rich intronic elements.

We conducted clinical and genetic analyses of 52 cystic fibrosis (CF) patients in Uruguay, which is about half of the known affected individuals in the country. A relatively high proportion had a mild presentation, characterized by pancreatic sufficiency (28%), a strong pulmonary component (97%), and borderline sweat electrolyte measurements (25%). Mutational analysis of CF chromosomes demonstrated a relatively low incidence of the DeltaF508 allele (40%) and a large number of other cystic fibrosis conductance regulator mutations, with an overall detection rate of about 71%. Fifteen different mutations were detected in our patients: DeltaF508, G542X, R1162X, G85E, N1303K, R334W, R75Q, R74W, D1270N, W1282X, DeltaI507, 2789+5G-->A, R1066C, -816C/T, R553X, as well as RNA splicing variant IVS8-5T. This group of Uruguayan CF patients has some characteristics in common with other populations of similar origin (Hispanics), as well as some unique characteristics.

Polish CF patients were screened extensively for mutations in the CFTR gene. Screening data demonstrated a high heterogeneity of CFTR mutations in the Polish population. Total 30 different mutations were characterised in 24 exons or introns of the gene. Among them, six mutations have been reported for the first time and submitted to the CF Genetic Analysis Consortium. In addition, 15 different polymorphisms were found, including three new ones. The screening resulted in 9% increase of the detection rate of CFTR alleles in the tested population. Frequencies of two of the identified mutations (CFTRdele2,3 and 2184insA) are relatively high (2.6% and 1%, respectively) and justify their inclusion into routinely screened mutations in genetic testing of Polish CF population.