fellow Yonsei University Seoul, Seoul-t'ukpyolsi, Republic of Korea
Abstract: This study aims to establish a CRISPR/Cas9-based treatment for GGC repeat expansion diseases. Specifically, we focused on Neuronal Intranuclear Inclusion Disease (NIID) and Fragile X-associated tremor/ataxia syndrome (FXTAS). The approach involves deriving iPSCs from the blood of NIID or FXTAS patients and developing a CRISPR/Cas9 system to correct the GGC repeat expansions. The corrected iPSCs were differentiated into neurons to restore normal neuronal function, demonstrating the therapeutic potential of this system. Blood samples were collected from NIID and FXTAS patients, PBMCs were isolated, and GGC repeat expansions were confirmed via RT-PCR. iPSCs were established using Sendai virus to introduce Yamanaka factors, and pluripotency, GGC repeat expansions, and methylation were verified. A one-vector CRISPR/Cas9 system was constructed under the CAG promoter. This system included sgRNA, Cas9 (with or without cytosine base editors), and eGFP. Both dCas9 and nCas9 versions were developed, and APOBEC3A/B/H cytosine base editors were incorporated for cytosine-to-thymine conversion. The nCas9-A3A system edited cytosines within the GGC repeats, converting GGC to GGT. This process disrupted the abnormal mRNA structure and normalized cellular function. Corrected iPSCs were successfully differentiated into neurons, and phenotypic changes were assessed using disease-specific markers. This CRISPR/Cas9 system effectively targeted GGC expansions in NIID and FXTAS. The approach has potential applications in other GGC repeat disorders and broader trinucleotide repeat diseases. These include Myotonic dystrophy type 1, Spinocerebellar ataxia, Huntington’s disease, and Friedrich’s ataxia. Furthermore, the system could be applied in vivo for gene therapies in preclinical and clinical trials.
