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Results for “Modified U7 small nuclear RNA”

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PubMed2024

Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro.

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.

RNA, Small Nuclear↗

PubMed2011

Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping.

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame. We demonstrate restoration of cardiac dystrophin expression at 13 months confirmed by reverse transcription-PCR (RT-PCR) and immunoblot as well as membrane localization by immunohistochemistry. This was accompanied by improved cardiac function as assessed by cardiac magnetic resonance imaging (MRI). Percutaneous transendocardial delivery of rAAV6 expressing a modified U7 exon skipping construct is a safe, effective method for restoration of dystrophin expression and improvement of cardiac function in the GRMD canine and may be easily translatable to human DMD patients.

Alternative Splicing↗

PubMed2025

Clinically relevant pseudoexons of the GALNS gene and their antisense-based correction.

BACKGROUND: Biallelic pathogenic variants in the GALNS gene lead to Mucopolysaccharidosis Type IVA (MPS IVA), a rare lysosomal storage disorder. GALNS encodes the enzyme N-acetylgalactosamine-6-sulfatase, whose deficiency causes accumulation of glycosaminoglycans and leads to a broad spectrum of clinical manifestations primarily affecting the osteoarticular system. Several studies have shown that, in 10%-15% of patients with the biochemical phenotype of MPS IVA, standard molecular genetic testing fails to identify one or both causative variants in the GALNS gene. METHODS: We performed an in-depth investigation of GALNS' splicing, with a special focus on deep-intronic mutations that lead to activation of pseudoexons (PEs). Using bioinformatic tools, we analyzed all deep-intronic variants in GALNS available in public databases and subjected the most relevant ones to in vitro analyses using minigenes. RESULTS: We characterized eight PE-activating variants, one of which (c.121-210C > T) represents a recurrent pathogenic variant which has long been hidden behind the mask of a polymorphic variant. In addition, we demonstrate that GALNS' splicing can produce a diverse range of mRNA isoforms containing so-called wild-type PEs, which are present at low levels as part of non-productive splicing, and weak canonical exons which are prone to skipping. We show that PE-activating variants cluster within wild-type PEs, highlighting the need for closer scrutiny of these regions during genetic testing. Finally, we applied modified U7 small nuclear RNAs and circular RNAs to efficiently block the identified PEs and pave the way for personalized antisense-based therapy for MPS IVA patients. CONCLUSION: The results of this study expand the understanding of GALNS gene splicing, indicating hotspots for splicing mutations. The presented data not only help to increase the diagnostic yield for MPS IVA but also unveil new therapeutic approaches for a number of MPS IVA patients.

Humans↗