CRISPR/Enhanced-Deletion Cas9-mediated allele-specific targeting of the rhodopsingene: a novel mutation-independent approach to treat RHO-linked autosomal dominantRetinitis pigmentosa

点击翻译按钮获取中文摘要

About 10%-30% of autosomal dominant retinitis pigmentosa (adRP) cases are caused by mutations in the rhodopsin (RHO) gene, encoding the visual pigment in rod photoreceptors. More than 150 different RHO dominant mutations have been functionally characterized, and shown to act via dominant negative effect or gain of function mechanisms. Therefore, silencing or selective disruption of the mutant allele is the fundamental rational for genetic therapies in RHO-linked adRP. CRISPR/Cas9-based genome editing offers a powerful approach to generate permanent therapeutic modification at the DNA level, thereby avoiding several concerns (e.g. retinotoxicity due to overexpression or ectopic mis-expression) raised on the ‘knockdown and replacement’ strategy for the treatment of RHO-linked adRP propagated by some groups. Other research efforts (including antisense oligonucleotide-based and CRISPR/Cas9-based technologies) are focused on selective disruption of certain mutant alleles, and thus do not adequately address the allelic heterogeneity of RHO mutations. This project pursues a novel alternatively approach - SEDIMS-X (selective disruption of the mutant allele through targeting common SNPs by Cas9-exo) - which is expected to overcome such hurdles and limitations. SEDIMS-X applies CRISPR/Cas9 technology to selectively disrupt mutant alleles by targeting few common SNPs rather than the individual disease-causing mutation. SEDIMS-X employs novel high-fidelity Cas9-exoexo fusion variants able to enhance disruptive indels from single gRNA-directed double-strand breaks. Based on encouraging preliminary results, this project further explores in-depth the applicability and utilization of SEDIMS-X on adRP-linked RHO mutations towards clinical translation. To this end, we will apply a stringent workflow to design and select effective Cas9-exo/gRNA compounds through the establishment of a high-throughput cell-based reporter system, validate their allele specificity in human dermal fibroblasts, and analyze the efficacy and functional outcome on transcript and protein level in patient-derived rod photoreceptor precursor cells. Moreover, we will carefully assess potential off-target activity of this novel approach. We expect that SEDIMS-X will extend and complement and extend the existing repertoire of preclinical, experimental therapeutic strategies for patients with RHO-linked adRP by overcoming some of the current limitations.