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

Nature Protocols volume 17, pages 781–818 (2022)Cite this article

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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.

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Fig. 1: dCas9-VPR–mediated transcriptional activation of a functionally equivalent gene in mouse photoreceptors.
Fig. 2: Timeline and workflow of experiments.
Fig. 3: AAV tropism for different tissues.
Fig. 4: Constructs required for in vitro and in vivo experiments.
Fig. 5: Anticipated results for a transactivation gene therapy in vitro and in vivo.

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Data availability

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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Acknowledgements

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.).

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  1. These authors contributed equally: Lisa M. Riedmayr, Klara S. Hinrichsmeyer.

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  1. Department of Pharmacy–Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany

    Lisa M. Riedmayr, Klara S. Hinrichsmeyer, Nina Karguth, Sybille Böhm, Victoria Splith & Elvir Becirovic

  2. Department of Ophthalmology, Ludwig-Maximilians-Universität München, Munich, Germany

    Stylianos Michalakis

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Contributions

S.B., V.S., L.M.R. and K.S.H. designed and performed the experiments. L.M.R, K.S.H., N.K. and E.B. wrote the manuscript with contributions from S.M.

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Correspondence to Elvir Becirovic.

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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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Böhm, S. et al. Sci. Adv. 6, eaba5614 (2020): https://doi.org/10.1126/sciadv.aba5614

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Supplementary Table 1

Human IRD-linked genes and potential functionally equivalent counterparts

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Statistical source data for Fig. 5a–c.

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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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