Header

UZH-Logo

Maintenance Infos

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; Becirovic, Elvir (2022). dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nature Protocols, 17(3):781-818.

Abstract

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.

Abstract

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.

Statistics

Citations

Dimensions.ai Metrics
8 citations in Web of Science®
8 citations in Scopus®
Google Scholar™

Altmetrics

Additional indexing

Item Type:Journal Article, refereed, further contribution
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Ophthalmology Clinic
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Life Sciences > General Biochemistry, Genetics and Molecular Biology
Language:English
Date:March 2022
Deposited On:17 Nov 2022 08:32
Last Modified:27 Feb 2024 02:49
Publisher:Nature Publishing Group
ISSN:1750-2799
OA Status:Closed
Publisher DOI:https://doi.org/10.1038/s41596-021-00666-3
PubMed ID:35132255
Full text not available from this repository.