Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation

Abstract

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.

Clinical and genetic findings in TRPM1‐related congenital stationary night blindness

“…amenable to anti-sense oligonucleotide and CRISPR-Cas9 approaches (Garanto et al 2016;Garanto et al 2018;Maeder et al 2019;Panagiotopoulos et al 2020;Tomkiewicz et al 2021).…”

Antisense oligonucleotides targeting exon 11 are able to partially rescue the Neurofibromatosis Type 2 phenotype in vitro

“…It has only been possible through CRISPR/Cas technology by the induction of single nucleotide substitutions [46,47]. Allele-specific approaches have been successfully tested for intronic variants through splice modulation ASOs that mask the erroneous signalling and given their intronic location and distance to splicing regulatory regions, proper protein synthesis can be recovered [48][49][50][51][52].…”

“…The most described effect of these molecules targeting intron/exon boundary is exon skipping. It has only been possible through CRISPR/Cas technology by the induction of single nucleotide substitutions 62,63 Allele-specific approaches have been successfully tested for intronic variants through splice modulation ASOs that mask the erroneous signalling and given their intronic location and distance to splicing regulatory regions, proper protein synthesis can be recovered 44,[64][65][66][67] . Few works have applied ESSENCE (exon-specific splicing enhancement by small chimeric effectors) method to rescue disease-associated exon skipping and modulate alternative splicing 33,68,69 .…”

Toward the Treatment of Inherited Diseases of the Retina Using CRISPR-Based Gene Editing

“…The first of the steric block ASO IRD trials, targeting exon 13 of USH2A is currently preparing for phase III trial with early limited release of promising preclinical data and early clinical data [NCT03780257] (115). More concerns for the development of CRISPR-Cas9 trials is that ASO trials appear to be targeting the same loci as the early CRISPR-Cas9 trials (26,116). This not only means that there is direct competition for treatment but may also limit the pool of patients required to complete larger phase III studies in a timely manner for these rare diseases.…”