New publication: Novel gene therapy approach using CRISPR and engineered U1 to target splicing defects in Stargardt disease

Prof Raymond Wong and his team have tested the use of CRISPR/Cas and engineered U1 technologies to reduce splicing defects of ABCA4 in retinal cells. This study demonstrate the potential of using RNA-targeting genetic technologies as a novel gene approach to treat Stargardt disease. This research involves a multi-disciplinary team of collaborators from Australia, Sweden, Hong Kong, Taiwan, China and USA.

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

Roxanne Hsiang-Chi Liou, Daniel Urrutia-Cabrera, Chia-Fei Liu,  Sihua Wu, Ida Maria Westin, Irina Golovleva, Guei-Sheung Liu, Satheesh Kumar, Samuel McLenachan, Fred Kuanfu Chen, Fei-Ting Hsu, Chien-Ling Huang, Tom Edwards, Keith R Martin, Albert Wu Cheng, Raymond C.B. Wong

Molecular Therapy -Nucleic Acids, accepted 2025.11.18. (IF = 10.18)

Abstract

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 ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 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 ABCA4 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 ABCA4 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 ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 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 ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

Read the publication here.