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

Abstract

The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5′ splice site (5′ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects. Here, we challenged the U1snRNA in the FAH5961SB mouse model of hepatic fumarylacetoacetate hydrolase (FAH) deficiency (Hereditary Tyrosinemia type I, HT1) due to the FAH c.706G>A splicing mutation. Through minigene expression studies we selected a compensatory U1snRNA (U1F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1F (AAV8-U1F), but not of U1wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1F treated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1F on the splicing profile and overall gene expression, thus pointing toward gene specificity. These data provide early in vivo proof-of-principle of the correction potential of compensatory U1snRNAs in HTI and encourage further optimization on a therapeutic perspective, and translation to other splicing-defective forms of metabolic diseases.

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

“…The transduction efficiency of the AAV8-U1 has been evaluated by two complementary approaches. In particular, the immune-histochemical analysis revealed that the Green Fluorescent Protein (GFP) staining, a marker of viral transduction, was comparable among mice irrespectively of the AAV8-U1 injected and not homogeneously distributed, with large variations in the number of GFP-positive hepatocytes across liver sections ( Figure S1A ), as previously observed by us [ 22 , 29 ] and others [ 30 , 31 ]. This finding was consistent with the AAV gene copy number that appeared to be comparable among experimental groups ( Figure S1B ).…”

“…The AAV8-coding plasmid was created to harbors the coding cassettes for both the U1snRNA (wild type or engineered one) and GFP genes under the control of the natural or Phosphoglycerate kinase 1 (PGK) promoters, respectively. An adenovirus-free transient transfection method [ 35 ] was exploited to generate deno-associated virus serotype 8 (AAV8), and a vector genome titer was determined, as previously described [ 22 ].…”

“…Importantly, different studies demonstrated that unique U1snRNAs targeting intronic sequences downstream of a defective exon (Exon specific U1snRNA, ExSpeU1) can rescue several exon-skipping mutations at 5′ss, 3′ss or within the exon, and this was proven both in vitro and in vivo [ 17 , 18 , 19 , 20 , 21 ]. Very recently, we have provided the early proof of principle of efficacy in vivo of U1snRNA variants for another metabolic disorder, hereditary tyrosinemia type I [ 22 ]. In this animal model, the delivery of a compensatory U1snRNA partially rescued the causative splicing mutation at both RNA and protein levels and resulted in a slightly prolonged mice survival.…”

Rescue of common exon‐skipping mutations in cystic fibrosis with modified U1 snRNAs

“…The positive effect we observed here on different CFTR mutations, along with those previously reported for other diseases in several cellular and mouse models (Dario Balestra et al, 2020; Dal Mas et al, 2015a; Donadon et al, 2018; Tajnik et al, 2016), indicates a general applicability and translatability of the ExSpeU1 approach in rescuing exon skipping defects. Even if our results show a clear rescue potential of the ExSpeU1 approach in CF, the therapeutic window is restricted to those splicing defects that do not disrupt the invariant AG and GT dinucleotides of the splice site.…”

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

“…Similarly, a pharmacological approach to correct splicing requires high-throughput screening platforms and may lack of specificity (Berg et al, 2019;Giuliano et al, 2018;Liang et al, 2017;Merkert et al, 2019;Pereira et al, 2019). The positive effect we observed here on different CFTR mutations, along with those previously reported for other diseases in several cellular and mouse models (Dario Balestra et al, 2020;Dal Mas, Fortugno, et al, 2015;Donadon et al, 2018;Tajnik et al, 2016), indicates a general applicability and translatability of the ExSpeU1 approach in rescuing exon skipping defects. Even if our results show a clear rescue potential of the ExSpeU1 approach in CF, the therapeutic window is restricted to those splicing defects that do not disrupt the invariant AG and GT dinucleotides of the splice site.…”