CRISPR-based VEGF suppression using paired guide RNAs for treatment of choroidal neovascularization

Molecular Therapy: Nucleic Acids

Glenn Yiu¹  Brian Dang¹  Ratheesh K. Meleppat¹  Tzu-Ni Sin¹  Therlinder Lo¹  Robert J. Zawadzki¹  Sook Hyun Chung¹  Daniella Lent-Schochet¹  Taylor Ngo¹ 

[1] Department of Ophthalmology and Vision Science, UC Davis Health, UC Davis Eye Center, University of California, Davis, Davis, CA 95616, USA;

关键词: MT: RNA/DNA Editing;    ocular gene therapy;    AAV;    CRISPR;    AMD;    neovascular AMD;   

DOI  :  

来源: DOAJ

【 摘 要 】

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic disruption of vascular endothelial growth factor A (Vegfa) with a single gRNA suppresses choroidal neovascularization (CNV) in preclinical studies, offering the prospect of long-term anti-angiogenesis therapy for neovascular age-related macular degeneration (AMD). Genome editing using CRISPR-CRISPR-associated endonucleases (Cas9) with multiple guide RNAs (gRNAs) can enhance gene-ablation efficacy by augmenting insertion-deletion (indel) mutations with gene truncations but may also increase the risk of off-target effects. In this study, we compare the effectiveness of adeno-associated virus (AAV)-mediated CRISPR-Cas9 systems using single versus paired gRNAs to target two different loci in the Vegfa gene that are conserved in human, rhesus macaque, and mouse. Paired gRNAs increased Vegfa gene-ablation rates in human cells in vitro but did not enhance VEGF suppression in mouse eyes in vivo. Genome editing using paired gRNAs also showed a similar degree of CNV suppression compared with single-gRNA systems. Unbiased genome-wide analysis using genome-wide unbiased identification of double-stranded breaks (DSBs) enabled by sequencing (GUIDE-seq) revealed weak off-target activity arising from the second gRNA. These findings suggest that in vivo CRISPR-Cas9 genome editing using two gRNAs may increase gene ablation but also the potential risk of off-target mutations, while the functional benefit of targeting an additional locus in the Vegfa gene as treatment for neovascular retinal conditions is unclear.

【 授权许可】

Unknown