dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy

Riedmayr, Lisa M.; Hinrichsmeyer, Klara S.; Karguth, Nina; Böhm, Sybille; Splith, Victoria; Michalakis, Stylianos und Becirovic, Elvir (2022): dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. In: Nature Protocols, Bd. 17, Nr. 3: S. 781-818

Abstract

The authors provide a versatile gene therapy approach that is mutation- and gene size-independent, using dCas9-VPR-based transcriptional activation of functionally equivalent genes. They show how to apply this for gene therapy for inherited retinal dystrophies in mice as an example. Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the appropriate counterpart(s) to compensate for the missing gene function. This approach offers key advantages over conventional gene therapies because it is mutation- and gene size-independent. Here, we describe a protocol for the design, execution and evaluation of such gene therapies using dCas9-VPR. We offer guidelines on how to identify functionally equivalent genes, design and clone single guide RNAs and evaluate transcriptional activation in vitro. Moreover, focusing on inherited retinal diseases, we provide a detailed protocol on how to apply this strategy in mice using dual recombinant adeno-associated virus vectors and how to evaluate its functionality and off-target effects in the target tissue. This strategy is in principle applicable to all organisms that possess functionally equivalent genes suitable for transcriptional activation and addresses pivotal unmet needs in gene therapy with high translational potential. The protocol can be completed in 15-20 weeks.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Chemie und Pharmazie > Department für Pharmazie - Zentrum für Pharmaforschung
• Themengebiete: 500 Naturwissenschaften und Mathematik > 540 Chemie
• ISSN: 1754-2189
• Sprache: Englisch
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 360003551
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 118803580