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In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency

Nature Biotechnology volume 33, pages 584–586 (2015)Cite this article

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Genome editing using a variety of different nucleases holds great potential to knock out or repair disease-causing genes. An ideal nuclease delivery vehicle is short-lived, does not integrate into the genome and can enter target cells efficiently. These requirements have not yet been achieved simultaneously by any nuclease delivery vector. We and others have used modified mRNA, which is nonintegrating and provides a transient pulse of protein expression, as an alternative to traditional viral vectors1,2,3,4,5. This approach allowed us to deliver therapeutic proteins in mouse models of surfactant protein B (SP-B) deficiency3 and experimental asthma4. Here we apply nuclease-encoding, chemically modified (nec) mRNA to deliver site-specific nucleases in a well-established transgenic mouse model of SP-B deficiency6, in which SP-B cDNA is under the control of a tetracycline-inducible promoter7. Administration of doxycycline drives SP-B expression at levels similar to those in wild-type mice (Supplementary Fig. 1), whereas cessation of doxycycline leads to phenotypic changes similar to those of the human disease, including thickened alveolar walls, heavy cellular infiltration, increased macrophages and neutrophils, interstitial edema, augmented cytokines in the lavage, a decline in lung function and fatal respiratory distress leading to death within days8,9. We inserted a constitutive CAG promoter immediately upstream of the SP-B cDNA to allow doxycycline-independent expression and prolonged life in treated mice.

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Figure 1: nec-mRNA cleaves the SP-B cassette, induces HDR in vitro and is expressed in lung cells in vivo.
Figure 2: Rescue of SP-B deficient mice by in vivo gene manipulation.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG KO 4258/2-1, to M.S.D.K and L.E.M.), fortüne grant (no. 1980-0-0, to M.S.D.K.), by the European Respiratory Society (Maurizio Vignola Award, to M.S.D.K.), in part by the Mukoviszidose e.V. (S03/12 to M.S.D.K.), in part by the Deutsche Forschungsgemeinschaft Emmy Noether Programme (HA 5274/3-1, to D. H.), in part by the Interfaculty Center for Pharmacogenomics and Drug Research (ICEPHA Twinning Grant 22-0-0 to S.B.-H.) and in part by DFG (to B.N., S.B.-H. and M.G. (970/8.1)). Site-specific nucleases were kindly provided by Sangamo BioSciences. Thank you to D. Fraude for excellent illustration support. Primary antibody against SP-B was kindly provided by M.G.

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Author notes

  1. Azita J Mahiny, Alexander Dewerth and Lauren E Mays: These authors contributed equally to the work.

Authors and Affiliations

  1. Department of Pediatrics I–Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy, University of Tübingen, Tübingen, Germany

    Azita J Mahiny, Alexander Dewerth, Lauren E Mays, Mohammed Alkhaled, Jennifer Rottenberger, Darina M Brosch, Philipp Reautschnig, Pacharapan Surapolchai, Franziska Zeyer, Melanie Carevic, Martina Bakele, Rupert Handgretinger, Dominik Hartl & Michael S D Kormann

  2. Department of Pharmacology and Experimental Therapy, University of Tübingen, Tübingen, Germany

    Benedikt Mothes, Bernd Nürnberg & Sandra Beer-Hammer

  3. ICePha, University of Tübingen, Tübingen, Germany

    Benedikt Mothes & Matthias Schwab

  4. Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Saarbrücken, Germany

    Emad Malaeksefat, Brigitta Loretz & Claus-Michael Lehr

  5. Department of Pediatric Pulmonology, Dr. von Hauner Children's Hospital, Ludwig Maximilians University, Munich, Germany

    Andrea Schams & Matthias Griese

  6. Member of the German Center for Lung Research (DZL), Munich, Germany

    Andrea Schams & Matthias Griese

  7. Department of Clinical Pharmacology, University of Tübingen, Tübingen, Germany

    Matthias Schwab

  8. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany

    Matthias Schwab

  9. Department of Pharmacy, Saarland University, Saarbrücken, Germany

    Claus-Michael Lehr

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  1. Azita J Mahiny
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Contributions

M.S.D.K. designed research, and together with A.J.M. and A.D., carried out experiments and analyzed data. B.M. performed the isolated lung experiments, and together with S.B.-H., participated in the interpretation and analysis of the data, with S.B.-H. and B.N. providing administrative and technical support. M.A., E.M., B.L., L.E.M., J.R., D.M.B., P.R., P.S., F.Z., A.S., M.C. and M.B. conceived and performed experiments. M.G., M.S., R.H., D.H. and C.-M.L. provided materials. M.S.D.K. and L.E.M. wrote the manuscript. All authors discussed results and implications, commented on or edited parts of the manuscript.

Corresponding authors

Correspondence to Claus-Michael Lehr or Michael S D Kormann.

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Competing interests

M.S.D.K. and L.E.M. are listed as co-inventors on a patent application related to this work. M.S.D.K. is an inventor on a patent licensed to the biopharmaceutical company Ethris GmbH.

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Supplementary Figures 1–22, Supplementary Sequences and Supplementary Discussion (PDF 48690 kb)

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Mahiny, A., Dewerth, A., Mays, L. et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat Biotechnol 33, 584–586 (2015). https://doi.org/10.1038/nbt.3241

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