Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the <i>ABCA4</i> gene. The splicing variants c.5461-10T>C and c.4773+3A>G in <i>ABCA4</i> cause the skipping of exon 39-40 and exon 33-34, respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these <i>ABCA4</i> splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C, and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing <i>ABCA4</i> variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced <i>ABCA4</i> c.5461-10T>C variants and up to 78% reduction of the <i>ABCA4</i> c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced <i>ABCA4</i> c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for <i>ABCA4</i>, providing an important step to advance the development of gene therapy to treat STGD1.