Purchase this article with an account.
Clara Men, Kinga Maria Bujakowska, Emily Place, Qin Liu, Eric A Pierce; Sequence-specific suppression of alleles causing dominantly inherited retinal degenerations using the RNA-guided nuclease Cas9. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1086.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Dominant mutations account for a substantial proportion of inherited retinal degenerations (IRDs). Dominant mutations may cause disease through haploinsufficiency or dominant negative/gain-of-function (GOF) effects. Gene supplementation has shown promise for use in rescuing haploinsufficiencies, but this approach cannot be extended to GOF mutations. This project proposes a potential strategy to treat diseases caused by GOF mutations by specifically suppressing the function of the allele at the genetic level. Recent advances in genome editing technologies have made this approach possible. In particular, the Cas9 nuclease system may allow for specific mutagenesis of GOF alleles.
Patients with autosomal dominant IRD were seen at MEEI and consented to the study. After taking into account the likelihood of allele pathogenicity, we identified six targets for genome editing therapy, including mutations in the RHO, BEST1, IMPDH1, SNRNP200, PRPH2, and PRPF8 genes. A literature search also supported the potential gain-of-function or dominant negative mechanisms of the identified alleles. Cas9 guide RNAs were tested for allele-specific targeting in COS-7 cells with pEGxxFP plasmids containing either the mutated allele target sequence or the wildtype sequence of the identified genes. The efficiency of Cas9 cleavage was quantified by FACS. The rate of allele-specific targeting by Cas9 was also assessed in patient-derived and control fibroblasts by the Surveyor assay.
In COS-7 cells transfected with constructed pEGxxFP plasmids, preliminary results for the BEST1 and PRPH2 genes showed up to a 2.5-fold increase in cutting efficiency in cells transfected with plasmids containing the mutant allele compared to those with the wildtype allele in the presence of Cas9 guide RNAs with perfect complementarity to the mutant sequence, while the SNRNP200 gene showed a 1.5-fold increase. This suggests that the Cas9 system could be used to specifically target mutated alleles with single base-pair mutations. Data from the other genes are in progress. Experiments testing allele-specific targeting by Cas9 guide RNAs in patient fibroblasts will also be presented.
The RNA-guided Cas9 nuclease is a potentially useful tool for targeted therapy in IRDs. Genome editing could provide long-term suppression of gain-of-function alleles to treat dominant disease.
This PDF is available to Subscribers Only