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

Abstract

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

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.…”

“…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. The expression of the splicing-defective human transgene in wild-type mice has been previously exploited to create surrogate models of coagulation factor VII or IX deficiency [7,12], which demonstrated the correction ability of the U1snRNA variants. Only recently it was shown that the delivery of an engineered U1snRNA by adeno-associated virus (AAV) rescues splicing and protein expression and, most importantly, the disease phenotype and survival in a mouse model of spinal muscular atrophy (SMA) [20].…”

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.…”

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

“…ExSpeU1s have the same composition of normal U1 snRNPs but while U1 snRNPs interact with the 5'splice site (5'ss), ExSpeU1s target intronic sequences by means of their engineered 5' tail (Rogalska et al, 2016). It was previously shown that their binding downstream affected exons corrects aberrant splicing in several cellular (Alanis et al, 2012;Dal Mas, Fortugno, et al, 2015;Nizzardo et al, 2015;Tajnik et al, 2016) and mouse models (Balestra et al 2014;Dal Mas, Rogalska, et al 2015;Balestra et al 2016;Rogalska et al 2016;Donadon et al 2018). Strikingly, an ExSpeU1 delivered by Adeno Associated Virus (AAV) resulted to an effective and safe therapy in a Spinal Muscular Atrophy (SMA) mouse model extending the survival from 10 days to˜6 months (Donadon et al, 2019).…”