Co-opting Regulation Bypass Repair of Genetic Diseases

Penn State University Technology Overview
Penn State Inventors have developed a novel CRISPR-mediated gene correction strategy, named Co-opting Regulation Bypass Repair or “CRBR”, that maintains endogenous regulatory regions but bypasses a spectrum of genetic defects. As seen in , CRBR utilizes homologous independent targeted insertion, part of the NHEJ repair pathway, to insert a cassette that contains the full gene coding sequence plus a stop codon downstream of the target gene’s promoter. Expression of the CRBR cassette, controlled by the target gene’s native promoter and regulatory elements, is equal to that of the wild type gene. Because the CRBR cassette contains the entirety of the target gene coding sequence, the cassette can be used to rescue any coding sequence mutation as well as splice-site mutations. Due to use of the NHEJ pathway, CRBR can be utilized in both mitotic and post-mitotic cells/tissues.
Further Details
Stage of Development
To demonstrate the utility of CRBR, the inventors targeted two genes that are important for pancreatic beta-cell function and glucose homeostasis: eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) and insulin. Patients with rare autosomal disease, Wolcott-Rallison syndrome, exhibit a large deletion or a nonsense mutation within the PERK gene.
Using CRBR, the inventors successfully rescued two independent Perk knockout alleles in mice. All phenotypic anomalies typical of Perk KO were absent in CRBR rescue mice. The inventors further demonstrated the potential of CRBR for human gene therapy by integrating a GFP cassette downstream of the human insulin gene in human cadaver islets. The inventors observed GFP expression under the control of the endogenous insulin promoter in a large percentage of pancreatic beta cells.
Together, initial experiments confirm that CRBR may be especially effective in addressing and correcting genes that have multiple disease-causing variations. Experiments also confirm that CRBR may have utility in autologous cell therapy.

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