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

• Published in: Molecular therapy 2021-11, Vol.29 (11), p.3274-3292
• Main Authors: Hu, Jingjie, Bourne, Rebecca A., McGrath, Barbara C., Lin, Alice, Pei, Zifei, Cavener, Douglas R.
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; CRISPR-Cas Systems; CRISPR/Cas9; diabetes; eIF-2 Kinase - genetics; Female; gene editing; Gene Editing - methods; Gene Expression; Gene Knockdown Techniques; Gene Order; gene repair; Gene Targeting; Genes, Reporter; genetic diseases; Genetic Diseases, Inborn - genetics; Genetic Diseases, Inborn - therapy; Genetic Markers; Genetic Therapy - methods; Genetic Vectors - genetics; Humans; Male; Mice; Mutation; mutations; Original; RNA, Guide, CRISPR-Cas Systems
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2021.04.017

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 results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans, which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair. [Display omitted] Co-opting regulation bypass repair (CRBR) can rescue a wide array of gene mutations while maintaining the normal genomic context and regulatory functions. It holds the potential for being an effective and powerful new tool for the reversal of monogenic diseases using gene therapy.