Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model

Abstract

Familial dysautonomia (FD) is a rare genetic disease with no treatment, caused by an intronic point mutation (c.2204+6T>C) that negatively affects the definition of exon 20 in the elongator complex protein 1 gene (ELP1 also known as IKBKAP). This substitution modifies the 5′ splice site and, in combination with regulatory splicing factors, induces different levels of exon 20 skipping, in various tissues. Here, we evaluated the therapeutic potential of a novel class of U1 snRNA molecules, exon-specific U1s (ExSpeU1s), in correcting ELP1 exon 20 recognition. Lentivirus-mediated expression of ELP1-ExSpeU1 in FD fibroblasts improved ELP1 splicing and protein levels. We next focused on a transgenic mouse model that recapitulates the same tissue-specific mis-splicing seen in FD patients. Intraperitoneal delivery of ELP1-ExSpeU1s-adeno-associated virus particles successfully increased the production of full-length human ELP1 transcript and protein. This splice-switching class of molecules is the first to specifically correct the ELP1 exon 20 splicing defect. Our data provide proof of principle of ExSpeU1s-adeno-associated virus particles as a novel therapeutic strategy for FD.

First Replication of the Involvement of OTUD6B in Intellectual Disability Syndrome With Seizures and Dysmorphic Features

“…We identified two novel heterozygous OTUD6B splicing mutations (c.324+1G>C and c.405+1G>A) in an Italian child with an intellectual disability syndrome, reinforcing the concept that mutations in genes encoding enzymes involved in regulating post-translational modifications play an important role in intellectual disability and related syndromes ( Bustos et al, 2018 ). The qualitative and quantitative analysis of the consequences of the identified mutations on splicing was instrumental to understand the contribution of the two alleles to disease pathogenesis, an essential prerequisite to set up future personalized therapies (i.e., small molecules, modified snRNAs, or antisense oligonucleotides) ( Slaugenhaupt et al, 2004 ; Hammond and Wood, 2011 ; Donadon et al, 2018 ) with the aim to directly correct the molecular defects. The application of exome-sequencing once more revealed to be a straightforward approach to the genetic diagnosis in a case with a complex phenotype partially overlapping with different syndromes and suggests that, in case of suspected Rubinstein–Taybi syndrome with negative results for mutations in CREBBP and EP300 genes, the screening of OTUD6B should be performed and its associated syndrome (intellectual developmental disorder with dysmorphic facies, seizures, and distal limb anomalies; IDDFSDA, OMIM#617452) should be included as a differential diagnosis.…”

A Compensatory U1snRNA Partially Rescues FAH Splicing and Protein Expression in a Splicing-Defective Mouse Model of Tyrosinemia Type I

“…Among them, the use of the small nuclear ribonucleoprotein U1 (U1snRNP) that, in the earliest splicing step, plays a key role in the exon definition by mediating the recognition of the 5 ss through base pair complementarity with its RNA component (U1snRNA) [3]. Variants of the U1snRNA with increased complementarity with the 5 ss of the defective exon (compensatory U1snRNA), or targeting the downstream intronic sequences (Exon specific U1snRNA, ExSpeU1), have shown the ability to rescue mRNA splicing in the presence of disease-causing mutations at 5 ss, 3 ss or within exons [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. While the correction effect has been clearly shown in several cellular models of human disease the evaluation of their therapeutic potential requires investigations in animal models harboring the disease-causing splicing mutations, which are very rare.…”

Disease-causing variants of the conserved +2T of 5′ splice sites can be rescued by engineered U1snRNAs

“…On the basis of the frequency and relevance of these nucleotide changes and on their mechanism, we and others have devised a correction approach based on variants of the U1snRNA designed to restore complementarity with the defective 5′ss (compensatory U1snRNAs; Pinotti et al., ) or to target downstream intronic regions (exon‐specific U1snRNAs; ExSpeU1; Alanis et al., ). For different human genetic disorders, in both cellular (Balestra et al., ; Dal Mas et al., ; Glaus, Schmid, Da Costa, Berger, & Neidhardt, ; Scalet et al., ; Schmid et al., ; Tajnik et al., ; van der Woerd et al., ) and animal (Balestra et al., ; Balestra et al., ; Dal Mas, Rogalska, Bussani, & Pagani, ; Donadon et al., ; Rogalska et al., ) models, the engineered U1snRNAs were shown to be effective on variants at 5′ss but also within the exon or at the 3′ss. However, these approaches failed to rescue changes at the highly conserved nucleotides +1G and +2T of the 5′ss (Alanis et al., ; Cavallari et al., ), which are the most represented (Buratti et al., ; Krawczak et al., ) and severe ones, and commonly considered to be virtually null.…”