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In vivo expansion of gene-targeted hepatocytes through transient inhibition of an essential gene
Editor’s summary
Homology-directed repair–based gene editing mechanisms can be difficult to apply to terminally differentiated tissues because of their relative lack of cellular replication. De Giorgi et al. developed a sequential transgene plus siRNA approach to increase the expansion of correctly gene-edited hepatocytes in adult mice without provoking liver toxicity or permanently altering liver metabolism during a 1-year follow-up. When combined with a high-protein diet to increase selection pressure, the system gene corrected about a quarter of all hepatocytes per mouse, which could potentially provide enough targeted cell expansion to treat certain liver conditions. This study supports further investigation of this hepatocyte gene editing system. —Catherine Charneski
Abstract
Homology-directed repair (HDR)–based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested Repair Drive, a platform technology for selectively expanding HDR-corrected hepatocytes in adult mice in vivo. Repair Drive involves transient conditioning of the liver by knocking down an essential gene, fumarylacetoacetate hydrolase (Fah), and delivering an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive increased the percentage of correctly targeted hepatocytes in healthy wild-type mice up to 25%, which resulted in a fivefold increased expression of a therapeutic transgene, human factor IX (FIX). Repair Drive was well tolerated and did not induce toxicity or tumorigenesis during a 1-year follow-up. This approach may broaden the range of liver diseases that can be treated with somatic genome editing.
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REFERENCES AND NOTES
1
J. D. Gillmore, E. Gane, J. Taubel, J. Kao, M. Fontana, M. L. Maitland, J. Seitzer, D. O’Connell, K. R. Walsh, K. Wood, J. Phillips, Y. Xu, A. Amaral, A. P. Boyd, J. E. Cehelsky, M. D. McKee, A. Schiermeier, O. Harari, A. Murphy, C. A. Kyratsous, B. Zambrowicz, R. Soltys, D. E. Gutstein, J. Leonard, L. Sepp-Lorenzino, D. Lebwohl, CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021).
2
R. G. Lee, A. M. Mazzola, M. C. Braun, C. Platt, S. B. Vafai, S. Kathiresan, E. Rohde, A. M. Bellinger, A. V. Khera, Efficacy and safety of an investigational single-course CRISPR base-editing therapy targeting PCSK9 in nonhuman primate and mouse models. Circulation 147, 242–253 (2023).
3
N. M. Gaudelli, A. C. Komor, H. A. Rees, M. S. Packer, A. H. Badran, D. I. Bryson, D. R. Liu, Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
4
A. C. Komor, Y. B. Kim, M. S. Packer, J. A. Zuris, D. R. Liu, Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
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Information & Authors
Information
Published In
Science Translational Medicine
Volume 17 | Issue 785
February 2025
February 2025
Copyright
Copyright © 2025 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
This is an article distributed under the terms of the Science Journals Default License.
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Submission history
Received: 19 August 2023
Resubmitted: 29 July 2024
Accepted: 17 January 2025
Acknowledgments
Funding: This work was supported by the US National Institutes of Health grants U42OD026645, DK124477, and HL132840 to W.R.L.; UG3 HL151545 to G.B. and W.R.L.; R01HL152314 to G.B.; DK115461 to K.-D.B.; and HL169511, HL171574, and HL118761 to J.F.M.; the Vivian L. Smith Foundation to J.F.M.; and the American Heart Association 19POST34430092 to M.D.G. This work was supported in part by grant #PNC24-260845 to M.D.G. from the American Association for the Study of Liver Diseases Foundation. This work was also supported by the Texas Digestive Diseases Morphology Core (P30DK56338), the Integrated Microscopy Core (DK56338 and CA125123), and the Human Genome Sequencing Center (1S10OD028587) and with assistance from the Baylor College of Medicine Gene Vector Core.
Author contributions: M.D.G., A.H., M.A.C., C.J.W., A.M.D., and M.N.F. performed in vivo experiments. S.H.P., M.C., and L.S. performed sequencing and bioinformatics analysis of genome editing. A.C., S.H., T.C., S.L., K.W., P.M., J.Q., M.K.S., I.Z., and V.J. designed, tested, and supplied siRNA reagents. M.C.L. performed sectioning, histology, and imaging. K.R.P. assessed pathology of liver biopsies. R.G.L., J.K., and J.F.M. performed and interpreted snRNA-seq. J.H. performed tyrosine measurement. C.B. provided assistance with measuring antihuman FIX antibodies. M.D.G., S.H.P., A.C., K.-D.B., C.B., J.F.M., V.J., G.B., and W.R.L. provided critical intellectual input to the project. W.R.L. conceptualized the Repair Drive strategy. M.D.G., S.H.P., A.C., V.J., G.B., and W.R.L. drafted the manuscript, which was edited and approved by all authors.
Competing interests: W.R.L., A.H., K.-D.B., M.D.G., and M.N.F. have filed a patent application for the Repair Drive technology and its application to gene therapy: WO2021108269 (“Selective expansion of gene-targeted cells”). J.F.M. is a cofounder and owns shares in YAP Therapeutics. The other authors declare that they have no competing interests.
Data and materials availability: All data associated with this study are in the paper or the Supplementary Materials. Primary data used to generate graphs are in data file S1. PacBio and Illumina sequencing data (Figs. 5 and 6 and figs. S29 to S32 and S35 to S38) are accessible at the Sequence Read Archive (SRA) under accession number PRJNA954356. Source code and analysis scripts for GISA-seq are accessible at Zenodo (https://doi.org/10.5281/zenodo.13824659). snRNA-seq Illumina sequencing data (fig. S7) are accessible at the Gene Expression Omnibus (GEO) database under accession number GSE277798.
Authors
Funding Information
National Institutes of Health: U42OD026645
National Institutes of Health: DK124477
National Institutes of Health: HL132840
National Institutes of Health: UG3 HL151545
National Institutes of Health: R01HL152314
National Institutes of Health: DK115461
National Institutes of Health: P30DK56338
National Institutes of Health: DK56338
National Institutes of Health: CA125123
National Institutes of Health: 1S10OD028587
American Heart Association: 19POST34430092
American Association for the Study of Liver Diseases: PNC24-260845
National Institutes of Health: HL169511
National Institutes of Health: HL171574
National Institutes of Health: HL118761
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