Co-opting regulation bypass repair (CRBR) as a gene correction strategy for monogenic diseases

Abstract

With the development of CRISPR/Cas9-mediated gene editing technologies, correction of disease- causing mutations has become possible. However, current gene correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene correction strategy, Co-opting Regulation Bypass Repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end-joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR gene repair results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We demonstrated the potential of CRBR strategy for human gene therapy by rescuing a mouse model of the Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either large deletion or nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we expressed a GFP CDS-terminator cassette that was integrated downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans which paves the way for autologous cell-tissue replacement therapy for gene repair in beta cells.