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.
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Figure 5 shows example data that were obtained with this protocol. Additional data related to this protocol can be found in the original paper12 or may be requested from the authors. Source data are provided with this paper.
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We thank B. Noack, J. Koch and K. Skokann for their excellent technical support. We also thank M. Al-Ubaidi for the gift of the 661W cells. Furthermore, we thank the entire Biel laboratory for support and advice. This work was supported by the Deutsche Forschungsgemeinschaft, SPP2127 (to E.B. and S.M), and by the German Research Foundation Grants SFB 870 B05 (to S.M.).
E.B., S.M., S.B., V.S. and L.M.R. are authors on a patent application related to this work (no. EP19198830, filed 23 September 2019). The other authors declare no competing interests.
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Key reference using this protocol
Böhm, S. et al. Sci. Adv. 6, eaba5614 (2020): https://doi.org/10.1126/sciadv.aba5614
Supplementary Table 1
Human IRD-linked genes and potential functionally equivalent counterparts
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Riedmayr, L.M., Hinrichsmeyer, K.S., Karguth, N. et al. dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nat Protoc 17, 781–818 (2022). https://doi.org/10.1038/s41596-021-00666-3
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