Gene Therapeutic Approach Using Mutation-adapted U1 snRNA to Correct a RPGR Splice Defect in Patient-derived Cells

Glaus, Esther; Schmid, F; Costa, Romain Da; Berger, Wolfgang; Neidhardt, John

doi:10.1038/mt.2011.7

Cited by 59 publications

(58 citation statements)

References 46 publications

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“…6 Fibroblast cell cultures were established. 14 Fibroblasts from the BC family were not available but venous blood for RNA from patient IV3 was available.…”

Section: Materials and Methods Subjectsmentioning

confidence: 99%

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome

et al. 2012

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Wagner syndrome (WS) is an autosomal dominant vitreoretinopathy affecting various ocular features and is caused by mutations in the canonical splice sites of the VCAN gene, which encodes the large chondroitin sulfate proteoglycan, versican. We report the identification of novel splice acceptor and donor-site mutations (c.4004 À1G4C and c.9265 þ 2T4A) in two large WS families from France and the United Kingdom. To characterize their pathogenic mechanisms we performed qRT-PCR experiments on RNA from patient-derived tissues (venous blood and skin fibroblasts). We also analyzed RNA from the original Swiss family reported by Wagner (who has the previously reported c.9265 þ 1G4A mutation). All three mutations resulted in a quantitative increase of transcript variants lacking exons 7 and/or 8. However, the magnitude of the increase varied between tissues and mutations. We discuss altered balance of VCAN splice variants in combination with reduction in glycosaminoglycan protein modifications as possible pathogenic mechanisms.

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“…6 Fibroblast cell cultures were established. 14 Fibroblasts from the BC family were not available but venous blood for RNA from patient IV3 was available.…”

Section: Materials and Methods Subjectsmentioning

confidence: 99%

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome

et al. 2012

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“…Differently, our and other groups extensively exploited the physiological role of the U1snRNA to promote exon inclusion in the presence of exon-skipping mutations, 5,6,7,8,9,10,11,12,13,14,15,16,17 a relevant cause of severe forms of human genetic disease. 18,19,20 The first generation of engineered U1snRNA had a modified 5′ tail with increased complementarity to defective 5′ss, and were shown to rescue exon inclusion in several cellular 5,6,7,8,9,10,11,12,13,14,15,16,21 and also in vivo 17 disease models.…”

Section: Introductionmentioning

confidence: 99%

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

Balestra

Scalet

Pagani

et al. 2016

Molecular Therapy - Nucleic Acids

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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.

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“…We and others showed that an increase in complementary of the U1 with the mutated SD corrects splice defects following virus-mediated treatment of patient-derived cell lines (Hartmann et al, 2010;Glaus et al, 2011;Schmid et al, 2011). The efficacy of this approach was tested for mutations at SD position -1, + 1, or + 3.…”

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confidence: 99%

A Gene Therapeutic Approach to Correct Splice Defects with Modified U1 and U6 snRNPs

et al. 2013

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Splicing is an essential cellular process to generate mature transcripts from pre-mRNA. It requires the splice factor U1 small nuclear ribonucleoprotein (U1), which promotes exon recognition by base-pairing interaction with the splice donor site (SD). After U1 dissociation, exon recognition is maintained by U6 small nuclear ribonucleoproteins (U6). It has been shown that SD mutations lower the binding affinity of U1 and cause splice defects in about 10% of patients with monogenetic diseases. U1 isoforms specifically designed to bind the mutated SD with increased affinity can correct these splice defects. We investigated the applicability of this gene therapeutic approach for different mutated SD positions. A minigene-based splicing assay was established to study a typical SD derived from the gene BBS1. We found that mutations at seven SD positions caused splice defects. In four cases, mutationadapted U1 isoforms completely corrected these splice defects. Partial correction was found for splice defects induced by the mutation at SD position +5. The limited therapeutic efficacy at this position was alleviated by applying a combined treatment with mutation-adapted U1 and U6. The sequence complementarity between U6 and three SD positions (+4, +5,and +6) was relevant for the outcome of the therapy. Between 30 and 100% of the normal transcripts can be restored. The treatment significantly decreased both exon skipping and intron retention. Massive missplicing of off-target transcripts was not detected. Our study helps to assess the therapeutic efficacy of mutation-adapted U snRNAs in gene therapy and illustrates their strong potential to correct splice defects, which cause many different inherited conditions. AbstractSplicing is an essential cellular process to generate mature transcripts from pre-mRNA. It requires the splice factor U1 small nuclear ribonucleoprotein (U1), which promotes exon recognition by base-pairing interaction with the splice donor site (SD). After U1 dissociation, exon recognition is maintained by U6 small nuclear ribonucleoproteins (U6). It has been shown that SD mutations lower the binding affinity of U1 and cause splice defects in about 10% of patients with monogenetic diseases. U1 isoforms specifically designed to bind the mutated SD with increased affinity can correct these splice defects. We investigated the applicability of this gene therapeutic approach for different mutated SD positions. A minigene-based splicing assay was established to study a typical SD derived from the gene BBS1. We found that mutations at seven SD positions caused splice defects. In four cases, mutation-adapted U1 isoforms completely corrected these splice defects. Partial correction was found for splice defects induced by the mutation at SD position + 5. The limited therapeutic efficacy at this position was alleviated by applying a combined treatment with mutation-adapted U1 and U6. The sequence complementarity between U6 and three SD positions ( + 4, + 5,and + 6) was relevant for the outcome of the ther...

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Gene Therapeutic Approach Using Mutation-adapted U1 snRNA to Correct a RPGR Splice Defect in Patient-derived Cells

Cited by 59 publications

References 46 publications

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome

Novel VCAN mutations and evidence for unbalanced alternative splicing in the pathogenesis of Wagner syndrome

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

A Gene Therapeutic Approach to Correct Splice Defects with Modified U1 and U6 snRNPs

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