Development of tailored CRISPR tools for gene therapy applications: treatment of autosomal dominant retinitis pigmentosa

The CRISPR-Cas technology transformed the field of genome editing by allowing the easy and permanent modification of a target DNA sequence. Despite great improvements, the currently available gene editing toolbox still presents significant issues regarding specificity, deliverability and genomic target coverage. The aim of my thesis is to expand the number of available CRISPR enzymes by mining the human microbiome through Alia’s proprietary discovery platform. Novel nucleases with improved characteristics for deliverability and PAM requirements have been identified. The diverse characteristics of the newly discovered effectors allows to screen and select the best nuclease to design novel targeting strategies for the treatment of specific diseases. In this work I selected the best novel nucleases to target the mutated RHO allele in autosomal dominant retinitis pigmentosa (adRP). Retinitis pigmentosa (RP) is a common inherited retinal disorder that affects 1 every 4000 individuals. One-third of the RP cases are autosomal dominant (adRP) with gain-of-function mutations, with RHO being the most frequently mutated gene (20% to 30% of patients). Here, a new allele-specific and mutation-independent editing strategy was designed for the treatment of adRP exploiting the simultaneous targeting of a high-frequency non-pathogenic single nucleotide polymorphism (SNP) located in the 5’UTR of RHO (rs7984 SNP) and an intronic region of the gene resulting in the formation of large deletion and inversion events that selectively inactivate the mutated RHO allele. By screening the panel of novel nucleases for their ability to efficiently produce the desired edit in the RHO gene, I identified ATN122 as the best candidate. I tested several combinations of sgRNAs, targeting the rs7984A SNP and the intron, and identified two pairs of guides which produced more than double the amount of relevant editing events over the SpCas9 benchmark. Moreover, ATN122 showed high genomic specificity, combined with higher allele specificity for the SNP rs7984A compared to SpCas9. Finally, the best guide pairs candidates have been tested in mouse models for activity and allele specificity. Subretinal injection of a single adeno-associated viral vector (AAV) expressing both the two sgRNAs and ATN122 resulted in an average editing efficiency 3 on the whole tissue close to 20%, while maintaining allele specificity (non-target allele editing < 3.5%). Notably, allele specific editing strongly correlated with a significant decrease in mutant RHO mRNA levels (approximately 30% on the whole retina). Overall, I was able to expand the CRISPR toolbox with new and active nucleases to be employed for the tailored treatment of a specific disease and developed a new strategy for the treatment of RHO-adRP.