Combining Engineered U1 snRNA and Antisense Oligonucleotides to Improve the Treatment of a BBS1 Splice Site Mutation

Saskia Breuel,Mariann Vorm,Anja U Bräuer,Marta Owczarek-Lipska,John Neidhardt

doi:10.1016/j.omtn.2019.08.014

Saskia Breuel, Mariann Vorm + Show 3 more

Open Access

https://doi.org/10.1016/j.omtn.2019.08.014

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Abstract

Manipulation of pre-mRNA processing is a promising approach toward overcoming disease-causing mutations and treating human diseases. We show that a combined treatment applying two splice-manipulating technologies improves therapeutic efficacies to correct mutation-induced splice defects. Previously, we identified a family affected by retinitis pigmentosa caused by the homozygous BBS1 splice donor site mutation c.479G > A. The mutation leads to both exon 5 skipping and intron 5 retention. We developed a therapeutic approach applying lentivirus-mediated gene delivery of engineered U1 small nuclear RNA (U1), which resulted in increased levels of correctly spliced BBS1. Herein, we show that the therapeutic effect of the engineered U1 efficiently reverted exon skipping but failed to reduce the intron retention. To complement the engineered U1 treatment, we identified four different antisense oligonucleotides (AONs) that block intron 5 retention in BBS1 transcripts. A treatment using engineered U1 in combination with AONs showed the highest therapeutic efficacy and increased the amount of correctly spliced BBS1 transcripts. We did not detect elevated levels of apoptotic cell death in AON-treated cell lines. In conclusion, engineered U1 or AONs provide efficient therapies with complementary effects and can be combined to increase efficacy of therapeutic approaches to correct splice defects.

Highlights

The Bardet-Biedl syndrome (BBS, known as Laurence-MoonBardet-Biedl syndrome) constitutes a rare autosomal recessive disease that affects several organs
We have developed a novel therapeutic approach that applies a combination of antisense oligonucleotides (AONs) and an engineered U1 with the aim to synergistically improve treatment efficacies
We demonstrated that U1 can be engineered to partially correct mutation-induced splice defects in BBS1

Summary

INTRODUCTION

The Bardet-Biedl syndrome (BBS, known as Laurence-MoonBardet-Biedl syndrome) constitutes a rare autosomal recessive disease that affects several organs. The prevalence of BBS is estimated to be 1:100,000 in North America and Europe, but may increase in subpopulations or isolated communities (1:18,000 in Newfoundland, 1:13,500 in Bedouin communities, and 1:4,000 on the Faroe islands).[1,2,3] Heterogeneity exists among BBS genes and until now, 21 genes are known to be associated with BBS.[4] These genes include BBS1–BBS20 and NPHP1 Biallelic mutations in these genes account for approximately 80% of the cases. CRIPSR-Cas, antisense oligonucleotides (AON), or engineered U1 small nuclear RNA (U1) splice factors. All of these technologies have been demonstrated be able to efficiently correct mutationinduced defects of a target gene. We have developed a novel therapeutic approach that applies a combination of AONs (to block the intron 5 retention) and an engineered U1 (to correct the exon 5 skipping) with the aim to synergistically improve treatment efficacies

RESULTS

DISCUSSION

MATERIALS AND METHODS