Improvement of SMN2 Pre-mRNA Processing Mediated by Exon-Specific U1 Small Nuclear RNA

Abstract

Exon-specific U1 snRNAs (ExSpe U1s) are modified U1 snRNAs that interact with intronic sequences downstream of the 5' splice site (ss) by complementarity. This process restores exon skipping caused by different types of mutation. We have investigated the molecular mechanism and activity of these molecules in spinal muscular atrophy (SMA), a genetic neuromuscular disease where a silent exonic transition on the survival motor neuron 2 (SMN2) leads to exon 7 (E7) skipping. By using different cellular models, we show that a single chromosome-integrated copy of ExSpe U1 induced a significant correction of endogenous SMN2 E7 splicing and resulted in the restoration of the corresponding SMN protein levels. Interestingly, the analysis of pre-mRNA transcript abundance and decay showed that ExSpe U1s promote E7 inclusion and stabilizes the SMN pre-mRNA intermediate. This selective effect on pre-mRNA stability resulted in higher levels of SMN mRNAs in comparison with those after treatment with an antisense oligonucleotide (AON) that targets corresponding intronic sequences. In mice harboring the SMN2 transgene, AAV-mediated delivery of ExSpe U1 increased E7 inclusion in brain, heart, liver, kidney, and skeletal muscle. The positive effect of ExSpe U1s on SMN pre-mRNA processing highlights their therapeutic potential in SMA and in other pathologies caused by exon-skipping mutations.

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

“…The U1snRNA-based technology acts at post-transcriptional levels thus maintaining the transcriptional gene regulation in physiologic tissues, and takes advantage of a very small therapeutic transgene (~600 bp) that can be packaged in virtually any viral vector successfully exploited for gene therapy purposes. 34 Furthermore, the ExSpeU1 expression is driven by its strong ubiquitous promoter, and in the model of Spinal Muscular Atrophy a single ExSpeU1 copy was shown to trigger an appreciable rescue, 25,28 which could lead to the use of low doses of viral vector to achieve a therapeutic impact.…”

“…However, these U1snRNAs are often tailored on the disease-causing mutation and have the intrinsic risk of off-target effects by recognizing the partially conserved donor splice site 22 in other splicing units. For this reason, we recently developed a second-generation U1snRNAs, named Exon Specific U1snRNAs (ExSpeU1s), that are designed to recognize intronic, often poorly conserved and thus gene-specific, regions downstream of the affected exon, and shown to efficiently rescue exon-skipping in different disease models, 23,24,25,26,27,28 Of great importance, in cellular cultures, a unique ExSpeU1 was able to rescue normal splicing from different type of exon-skipping mutations either at the 5′ss or acceptor splice site (3′ss), or within the exon. 23,29 Although these gene-specificity and activity ExSpeU1 features would significantly extend the applicability of a single therapeutic molecule to panels of mutations and thus cohorts of patients, a key issue when addressing the numerous diseases with heterogeneous mutational patterns (www.hgmd.cf.ac.uk), this effect of ExSpeU1s in vivo has not been investigated yet.…”

Understanding molecular mechanisms in propionic acidemia and investigated therapeutic strategies

“…Two different approaches have been used with success to modulate the splicing process in PA, adapted U1 snRNA overexpression to overcome 5' splice site mutations [75,76] and the use of antisense oligonucleotides to suppress splicing at specific splice sites [77,78]. Both strategies have been assayed in PA patients' fibroblasts [79,80].…”

“…Further studies are necessary to investigate if the adapted U1 snRNA also binds more stably to other 5'splice sites of key genes inhibiting the entry Understanding molecular mechanisms in PA and investigated therapeutic strategies Expert Opinion on Orphan Drugs (2015) 3 (12) of other spliceosomal components due to delayed release of the U1 snRNP, resulting in unforeseen side effects precluding a therapeutical defect. Using an exon-specific U1 snRNA may also provide an alternative approach for 5' splice site mutations, as described for several genes [75,76,83].…”

A deep learning approach to identify new gene targets of a novel therapeutic for human splicing disorders

“…New therapeutic approaches aimed at correction of pre-mRNA splicing defects, including antisense oligonucleotides, splicing modulator compounds (SMCs), and modified exon-specific U1 small nuclear RNA, have shown significant promise in many diseases [27][28][29][30][31][32][33][34][35] . SMCs are attractive because they can be optimized for broad tissue distribution and are orally administered 27,36,37 .…”