Genome editing-based read-through of stop codons in cells with retinoblastoma-relevant nonsense mutations in the RB1 gene

Abstract

Purpose : Nonsense mutations are the most prevalent variety of germline and somatic mutations in patients with retinoblastoma. This study aimed to investigate the therapeutic potential of genome editing-based technology in the restoration of RB1 gene transcripts and retinoblastoma protein in cells containing nonsense mutations in the RB1 gene seen in patients with retinoblastoma.

Methods : We prepared human skin fibroblast cells with clinically relevant nonsense mutations in the RB1 gene by genome editing based on activation-induced cytidine deaminases. Treated cells were cultured from single cells and each single cell-derived clone was used for further experiments. Then, the cells were treated with adenine base editors targeting nonsense mutations. The efficacy of genome editing was evaluated by the next-generation sequencing.

Results : We obtained skin fibroblast cells with nonsense mutations in the RB1 gene. From these cells, adenine base editors effectively corrected the mutations.

Conclusions : We expect that this genome editing-based technology can help to recover RB1 gene transcripts and retinoblastoma protein in retinal cells containing nonsense mutations. It might be of therapeutic potential in the treatment of patients with retinoblastoma or people at risk due to germline RB1 mutations.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.