An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency

Abstract

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5′ splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies

“…Based on our previous in vitro and in vivo finding that engineered U1snRNA, either compensatory or exon specific (ExSpe) variants, efficiently rescue splicing of OTC exon 4 in the mouse context (Balestra et al 2020a ), we expressed a panel of U1snRNA variants (Fig. 5 A) designed on the human OTC context.…”

“…To create the human OTC minigene (OTC h ), the genomic region of human OTC gene (NG_008471) including the last 535 bp of intron 3, exon 4 (88 bp) and the first 681 bp of intron 4 was amplified from the DNA of a healthy subject using high-fidelity PfuI DNA-Polymerase (ThermoFisher Scientific, Waltham, MA, USA) and primers hOTCF and hOTCR, and subsequently cloned into the pTB (41) by exploiting the Nde I restriction site inserted within primers. The mouse OTC minigene (OTC m ) was available from previous studies (Balestra et al 2020a ). Nucleotide changes were introduced into the human and mouse OTC minigenes by mutagenesis (Balestra et al 2020a ).…”

“…The mouse OTC minigene (OTC m ) was available from previous studies (Balestra et al 2020a ). Nucleotide changes were introduced into the human and mouse OTC minigenes by mutagenesis (Balestra et al 2020a ). Expression vectors for the U1snRNA variants were created as previously reported (Balestra et al 2015 ).…”

“…These differences offer the opportunity for a deep investigation of the functional relevance of species-specific gene variations. Moreover, they could affect correction efficiency of U1snRNA variants designed to force exon 4 inclusion, which we showed to be capable to efficiently rescue OTC expression at both RNA and protein levels in the spf ash mouse model (Balestra et al 2020a ).…”

“…Among molecules acting at RNA levels, engineered variants of the U1snRNA, the RNA component of the spliceosomal U1 ribonucleoprotein (U1snRNP) mediating 5′ss recognition and thus exon definition in the earliest splicing steps (De Conti et al 2013 ), have proven to be effective in modulating splicing for therapeutic purposes. In particular, U1snRNA variants with increased complementarity with the 5′ss of the defective exon (named compensatory U1snRNA), or targeting the downstream intronic sequences (named Exon specific U1snRNA, ExSpeU1), have demonstrated their ability to rescue exon skipping caused by different types of mutations in cellular and animal models of several human diseases (Donadon et al 2018 , 2019 ; Scalet et al 2018 , 2019 ; Yamazaki et al 2018 ; Balestra et al 2019a , 2020a ; Lee et al 2019 ; Donegà et al 2020 ; Martín et al 2021 ).…”