Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing

Nature Biomedical Engineering volume 5, pages 169–178 (2021)Cite this article

Subjects

A Publisher Correction to this article was published on 29 October 2020

This article has been updated

Abstract

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: In vitro validation of Rpe65 mutation correction by the ABE.
Fig. 2: Subretinal delivery of ABE corrects the mutation and restores RPE65 expression in rd12 mice.
Fig. 3: Restoration of a visual cycle and retinal function in rd12 mice after base editing.
Fig. 4: Base editing restores neuronal activity of the primary visual cortex (V1) in response to visual stimuli.
Fig. 5: V1 neurons in A5-treated mice show selectivity to stimulus parameters.

Similar content being viewed by others

Data availability

The main data supporting the results of this study are available within the paper and its Supplementary Information. The deep-sequencing data are available from the Sequence Read Archive under accession number PRJNA644016.

Change history

References

  1. Cremers, F. P., van den Hurk, J. A. & den Hollander, A. I. Molecular genetics of Leber congenital amaurosis. Hum. Mol. Genet. 11, 1169–1176 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Den Hollander, A. I., Roepman, R., Koenekoop, R. K. & Cremers, F. P. M. Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog. Retin. Eye Res. 27, 391–419 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Miraldi Utz, V., Coussa, R. G., Antaki, F. & Traboulsi, E. I. Gene therapy for RPE65-related retinal disease. Ophthalmic Genet. 39, 671–677 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Maguire, A. M. et al. Efficacy, safety, and durability of voretigene neparvovec-rzyl in RPE65 mutation-associated inherited retinal dystrophy: results of phase 1 and 3 trials. Ophthalmology 126, 1273–1285 (2019).

    Article  PubMed  Google Scholar 

  5. Bainbridge, J. W. et al. Long-term effect of gene therapy on Leber’s congenital amaurosis. N. Engl. J. Med. 372, 1887–1897 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Jacobson, S. G. et al. Improvement and decline in vision with gene therapy in childhood blindness. N. Engl. J. Med. 372, 1920–1926 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cideciyan, A. V. et al. Human retinal gene therapy for Leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement. Proc. Natl Acad. Sci. USA 110, E517–E525 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Gardiner, K. L. et al. Long-term structural outcomes of late-stage RPE65 gene therapy. Mol. Ther. 28, 266–278 (2020).

    Article  CAS  PubMed  Google Scholar 

  9. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Chapman, J. R., Taylor, M. R. & Boulton, S. J. Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47, 497–510 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Rossidis, A. C. et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat. Med. 24, 1513–1518 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Osborn, M. J. et al. Base editor correction of COL7A1 in recessive dystrophic epidermolysis bullosa patient-derived fibroblasts and iPSCs. J. Invest. Dermatol. 140, 338–347 (2020).

    Article  CAS  PubMed  Google Scholar 

  20. Ryu, S.-M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).

    Article  CAS  PubMed  Google Scholar 

  21. Pang, J. J. et al. Retinal degeneration 12 (rd12): a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA). Mol. Vis. 11, 152–162 (2005).

    CAS  PubMed  Google Scholar 

  22. Zhong, Z. et al. Seven novel variants expand the spectrum of RPE65-related Leber congenital amaurosis in the Chinese population. Mol. Vis. 25, 204–214 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Redmond, T. M. et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat. Genet. 20, 344–351 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Jo, D. H. et al. CRISPR–Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis. Sci. Adv. 5, eaax1210 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhang, Y. et al. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci. Rep. 4, 5405 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Puppo, A. et al. Retinal transduction profiles by high-capacity viral vectors. Gene Ther. 21, 855–865 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Xin, H., Wan, T. & Ping, Y. Off-targeting of base editors: BE3 but not ABE induces substantial off-target single nucleotide variants. Signal Transduct. Target. Ther. 4, 9 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Kiser, P. D., Golczak, M. & Palczewski, K. Chemistry of the retinoid (visual) cycle. Chem. Rev. 114, 194–232 (2014).

    Article  CAS  PubMed  Google Scholar 

  36. Benchorin, G., Calton, M. A., Beaulieu, M. O. & Vollrath, D. Assessment of murine retinal function by electroretinography. Bio Protoc. 7, e2218 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Leinonen, H. & Tanila, H. Vision in laboratory rodents—tools to measure it and implications for behavioral research. Behav. Brain Res. 352, 172–182 (2018).

    Article  PubMed  Google Scholar 

  38. Hofbauer, A. & Dräger, U. C. Depth segregation of retinal ganglion cells projecting to mouse superior colliculus. J. Comp. Neurol. 234, 465–474 (1985).

    Article  CAS  PubMed  Google Scholar 

  39. Wang, L., Sarnaik, R., Rangarajan, K., Liu, X. & Cang, J. Visual receptive field properties of neurons in the superficial superior colliculus of the mouse. J. Neurosci. 30, 16573–16584 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kretschmer, F., Sajgo, S., Kretschmer, V. & Badea, T. C. A system to measure the optokinetic and optomotor response in mice. J. Neurosci. Methods 256, 91–105 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lee, C. et al. CRISPR-pass: gene rescue of nonsense mutations using adenine base editors. Mol. Ther. 27, 1364–1371 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Acland, G. M. et al. Gene therapy restores vision in a canine model of childhood blindness. Nat. Genet. 28, 92–95 (2001).

    CAS  PubMed  Google Scholar 

  45. Acland, G. M. et al. Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol. Ther. 12, 1072–1082 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Jacobson, S. G. et al. Safety of recombinant adeno-associated virus type 2–RPE65 vector delivered by ocular subretinal injection. Mol. Ther. 13, 1074–1084 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Chelstowska, S., Widjaja-Adhi, M. A. K., Silvaroli, J. A. & Golczak, M. Impact of LCA-associated E14L LRAT mutation on protein stability and retinoid homeostasis. Biochemistry 56, 4489–4499 (2017).

    Article  CAS  PubMed  Google Scholar 

  48. Kitamura, T. et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp. Hematol. 31, 1007–1014 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR–Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Walter, D. M. et al. Systematic in vivo inactivation of chromatin-regulating enzymes identifies Setd2 as a potent tumor suppressor in lung adenocarcinoma. Cancer Res. 77, 1719–1729 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Golczak, M., Kiser, P. D., Lodowski, D. T., Maeda, A. & Palczewski, K. Importance of membrane structural integrity for RPE65 retinoid isomerization activity. J. Biol. Chem. 285, 9667–9682 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Foik, A. T. et al. Retinal origin of electrically evoked potentials in response to transcorneal alternating current stimulation in the rat. Invest. Ophthalmol. Vis. Sci. 56, 1711–1718 (2015).

    Article  PubMed  Google Scholar 

  53. Brainard, D. H. The psychophysics toolbox. Spat. Vis. 10, 433–436 (1997).

    Article  CAS  PubMed  Google Scholar 

  54. Foik, A. T. et al. Detailed visual cortical responses generated by retinal sheet transplants in rats with severe retinal degeneration. J. Neurosci. 38, 10709–10724 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Daruwalla, H. Hashimoto, A. Lewin and A. Browne for technical assistance and providing materials. We are also grateful to members of the Palczewski laboratory for helpful comments regarding this study. K.P. is the Leopold Chair of Ophthalmology at the Gavin Herbert Eye Institute, Department of Ophthalmology, University of California, Irvine. This research was supported in part by grants to K.P. from the National Institutes of Health (NIH) (nos. EY009339, EY027283, EY025451 and EY019312) and the Research to Prevent Blindness Stein Innovation Award. S.S. was supported by NIH grant nos. F30EY029136, T32GM007250 and T32EY024236. E.H.C. was supported by NIH grant nos. T32GM007250 and T32GM008803. H.L. was supported by Fight for Sight, the Eye and Tissue Bank Foundation (Finland), The Finnish Cultural Foundation and the Orion Research Foundation. G.A.N. was supported as a Howard Hughes Medical Institute fellow of the Helen Hay Whitney Foundation. P.D.K. was supported by the US Department of Veterans Affairs (I01BX004939). We also acknowledge support from a Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology, University of California, Irvine.

Author information

Author notes

  1. These authors contributed equally: Susie Suh, Elliot H. Choi.

Authors and Affiliations

  1. Gavin Herbert Eye Institute, Department of Ophthalmology, University of California, Irvine, Irvine, CA, USA

    Susie Suh, Elliot H. Choi, Henri Leinonen, Zhiqian Dong & Krzysztof Palczewski

  2. Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA

    Susie Suh & Elliot H. Choi

  3. Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA

    Andrzej T. Foik & David C. Lyon

  4. International Centre for Translational Eye Research, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland

    Andrzej T. Foik

  5. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Gregory A. Newby, Wei-Hsi Yeh & David R. Liu

  6. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Gregory A. Newby, Wei-Hsi Yeh & David R. Liu

  7. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Gregory A. Newby, Wei-Hsi Yeh & David R. Liu

  8. Program in Speech and Hearing Bioscience and Technology, Harvard Medical School, Boston, MA, USA

    Wei-Hsi Yeh

  9. Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA

    Philip D. Kiser & Krzysztof Palczewski

  10. Research Service, VA Long Beach Healthcare System, Long Beach, CA, USA

    Philip D. Kiser

  11. Department of Chemistry, University of California, Irvine, Irvine, CA, USA

    Krzysztof Palczewski

Authors
  1. Susie Suh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elliot H. Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henri Leinonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrzej T. Foik
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gregory A. Newby
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wei-Hsi Yeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhiqian Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Philip D. Kiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David C. Lyon
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. David R. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Krzysztof Palczewski
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.S., E.H.C. and K.P. conceived of the strategy and designed the experiments. S.S. and E.H.C. designed, performed and analysed the in vitro experiments. S.S., E.H.C., H.L., A.T.F., D.C.L., Z.D. and P.D.K. designed, performed and analysed the in vivo experiments. G.A.N., W.-H.Y. and D.R.L. performed and analysed the off-target activity. S.S., E.H.C. and K.P. wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Susie Suh or Krzysztof Palczewski.

Ethics declarations

Competing interests

D.R.L. is a consultant and co-founder of Beam Therapeutics, Prime Medicine, Editas Medicine and Pairwise Plants, companies that use genome editing.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–9, Tables 1–4 and references.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Suh, S., Choi, E.H., Leinonen, H. et al. Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing. Nat Biomed Eng 5, 169–178 (2021). https://doi.org/10.1038/s41551-020-00632-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-020-00632-6

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing