Article
A Compensatory U1snRNA Partially Rescues FAH
Splicing and Protein Expression in a Splicing-Defective Mouse Model of Tyrosinemia Type I
Dario Balestra
1,* , Daniela Scalet
1, Mattia Ferrarese
1, Silvia Lombardi
1, Nicole Ziliotto
1, Chrystal C. Croes
2,3, Naomi Petersen
2, Piter Bosma
2,3, Federico Riccardi
4, Franco Pagani
4, Mirko Pinotti
1,5and Stan F. J. van de Graaf
2,31
Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy;
scldnl@unife.it (D.S.); frrmtt1@unife.it (M.F.); lmbslv@unife.it (S.L.); zltncl1@unife.it (N.Z.);
pnm@unife.it (M.P.)
2
Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; chrystal.croes@outlook.com (C.C.C.); n.petersen@amc.uva.nl (N.P.);
p.j.bosma@amc.uva.nl (P.B.); k.f.vandegraaf@amc.uva.nl (S.F.J.v.d.G.)
3
Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology and Metabolism, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
4
Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology, 34149 Trieste, Italy; Federico.Riccardi@icgeb.org (F.R.); pagani@icgeb.org (F.P.)
5
LTTA, University of Ferrara, 44121 Ferrara, Italy
* Correspondence: blsdra@unife.it; Tel.: +39-0532-974485
Received: 4 March 2020; Accepted: 18 March 2020; Published: 20 March 2020
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
0splice site (5
0ss) 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 (U1
F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1
F(AAV8-U1
F), but not of U1
wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1
Ftreated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1
Fon 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.
Keywords: FAH; fumarylacetoacetate hydrolase deficiency; Tyrosinemia type I; aberrant splicing;
mouse models; U1snRNA; RNA therapeutics
1. Introduction
Mutations leading to aberrant pre-mRNA splicing account for a significant proportion of human genetic disorders [1] and includes changes at the 5
0or 3
0splice sites (ss) but also within exons. The splicing process is a very complex and finely regulated mechanism with several sequence elements and
Int. J. Mol. Sci. 2020, 21, 2136; doi:10.3390/ijms21062136 www.mdpi.com/journal/ijms