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Elvir Becirovic, Lisa Maria Riedmayr, Martin Biel; CRISPR/Cas9 and antisense oligonucleotides for therapy of a deep intronic CLRN1 splice mutation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5334.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in CLRN1 cause Usher syndrome type III (USH3). USH3 is characterized by progressive and variable hearing impairment, retinitis pigmentosa (RP) and vestibular dysfunction. Due to the lack of appropriate animal models and treatment strategies, no efficient therapy for RP in USH patients exists so far. Here, focusing on the recently identified deep intronic c.254-649T>G splice mutation in CLRN1, we aim at establishing two therapy approaches for USH3: CRISPR/Cas9 and antisense oligonucleotides (AONs). c.254-649T>G is a founder mutation in the Saudi Arabian population and generates an aberrant exon. We evaluated the therapeutic potential of CRISPR/Cas9 and AONs in HEK293 and in human retinal pigment epithelial (hRPE) cells.
To cut out the c.254-649T>G mutation on genomic level using the CRISPR/Cas9 approach we designed two CLRN1 gRNAs flanking the mutation. HEK293 cells were transiently transfected with the spCas9/CLRN1 gRNA cassette. hRPE cells were transduced with two separate adeno-associated viral (AAV) vectors expressing the CLRN1 gRNAs and spCas9, respectively, using the 7m8 capsids. Gene editing efficiencies were tested by ratiometric analysis of the PCR bands from genomic DNA.For splice correction on mRNA level, HEK293 cells transiently transfected with the CLRN1 minigenes were treated with two different AONs (AON1 and AON2, respectively) binding to different positions of the aberrant exon. Splice correction efficiencies of the single AONs were determined by ratiometric intensity analysis of the corresponding RT-PCR bands.
The CRISPR/Cas9 approach led to 27.4 ± 0.8 % vs 19.7 ± 1.2 % gene editing efficiencies in HEK293 compared to hRPE cells. However, due to the presence of non-transfected and/or non-transduced HEK293 and hRPE cells, the factual gene editing efficiencies are expected to be higher.Treatment with AON1 could not correct the c.254-649T>G mRNA splicing. By contrast, AON2 treatment could correct approx. 70 % of the mutant mRNA splicing.
We show that both approaches are suitable for treatment of the c.254-649T>G mutation. Further experiments are necessary to evaluate and quantify the single approaches in other cell lines and/or in appropriate retinal cultures. In case of success, this study will pave the way for the treatment of other deep intronic splice mutations in the retina.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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