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Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy

Nature Biomedical Engineering volume 8, pages 118–131 (2024)Cite this article

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Abstract

Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.

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Fig. 1: Development of adenine base editing to correct SMN2 exon 7 C6T.
Fig. 2: SMN2 C6T editing in fibroblasts derived from patients with SMA.
Fig. 3: Analysis of SMN2 C6T base editing specificity.
Fig. 4: AAV-mediated delivery of base editors for in vivo SMN2 C6T editing.

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Data availability

Plasmids from this study have been made available through Addgene (https://www.addgene.org/browse/article/28233786/). Primary datasets are available in Supplementary Tables 1, 2, 6 and 7. Sequencing datasets were deposited with the NCBI Sequence Read Archive (PRJNA1014715). The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We acknowledge the following individuals: C. Tou (ddPCR), E. King and D. Ramos (AAV and mouse tissue extraction discussions), J. Ferreira da Silva and L. Hille (data analysis), L. Ma and N. Kim (cloning), R. Silverstein (coding and data analysis), H. Stutzman (gRNA cloning) and E. Eichelberger, R. Spellman and W. das Neves (cell culture). We acknowledge M. Mabuchi and G. B. Robb for providing purified SpRY and SpRY-HF1 protein (generated as described previously96). This work was supported by a Charles A. King Trust Postdoctoral Research Fellowship, Bank of America, N.A., Co-Trustees (C.R.R.A.); a James L. and Elisabeth C. Gamble Endowed Fund for Neuroscience Research/Mass General Neuroscience Transformative Scholar Award (C.R.R.A.); an MGH Physician/Scientist Development Award (C.R.R.A.); an MGH Executive Committee on Research Fund for Medical Discovery Fundamental Research Fellowship Award (K.A.C.); a Frederick Banting and Charles Best Canadian Institutes of Health Research Doctoral Research Award (A.R.); a St. Jude Children’s Research Hospital Collaborative Research Consortium on Novel Gene Therapies for Sickle Cell Disease (S.Q.T.); a Muscular Dystrophy Association grant 575466 (R.K.); a Muscular Dystrophy Canada grant (R.K.); a Canadian Institutes of Health Research grant PJT-156379 (R.K.); an MGH Innovation Discovery Grant (to C.R.R.A. and B.P.K.); an MGH Executive Committee on Research Howard M. Goodman Fellowship (B.P.K.); and National Institutes of Health grants R01DC017117 (C.A.M.), U01AI157189 (S.Q.T.), U01AI176471 (S.Q.T.), P01HL142494 (B.P.K.) and DP2CA281401 (B.P.K.). Some aspects of schematics in Fig. 4 and Supplementary Figs. 20 and 21 were adapted from vector art provided by Servier Medical Art by Servier, which is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0).

Author information

Authors and Affiliations

  1. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Christiano R. R. Alves, Leillani L. Ha, Kathleen A. Christie, Roman M. Doll, Kathryn J. Swoboda & Benjamin P. Kleinstiver

  2. Department of Neurology, Massachusetts General Hospital, Boston, MA, USA

    Christiano R. R. Alves & Kathryn J. Swoboda

  3. Department of Neurology, Harvard Medical School, Boston, MA, USA

    Christiano R. R. Alves & Kathryn J. Swoboda

  4. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Leillani L. Ha, Kathleen A. Christie, Roman M. Doll & Benjamin P. Kleinstiver

  5. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Rebecca Yaworski, Emma R. Sutton, Aoife Reilly, Ariane Beauvais & Rashmi Kothary

  6. Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

    Rebecca Yaworski, Emma R. Sutton, Aoife Reilly, Ariane Beauvais & Rashmi Kothary

  7. Centre for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario, Canada

    Rebecca Yaworski, Emma R. Sutton, Aoife Reilly, Ariane Beauvais & Rashmi Kothary

  8. Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Cicera R. Lazzarotto & Shengdar Q. Tsai

  9. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Kathleen A. Christie & Benjamin P. Kleinstiver

  10. Molecular Biosciences/Cancer Biology Program, Heidelberg University and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Roman M. Doll

  11. Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, USA

    Demitri de la Cruz & Casey A. Maguire

  12. Program in Neuroscience, Harvard Medical School, Boston, MA, USA

    Demitri de la Cruz & Casey A. Maguire

  13. Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada

    Rashmi Kothary

Authors
  1. Christiano R. R. Alves
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  2. Leillani L. Ha
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  3. Rebecca Yaworski
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  4. Emma R. Sutton
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  5. Cicera R. Lazzarotto
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  6. Kathleen A. Christie
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  7. Aoife Reilly
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  8. Ariane Beauvais
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  9. Roman M. Doll
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  10. Demitri de la Cruz
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  11. Casey A. Maguire
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  12. Kathryn J. Swoboda
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  13. Shengdar Q. Tsai
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  14. Rashmi Kothary
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  15. Benjamin P. Kleinstiver
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Contributions

C.R.R.A. and B.P.K. conceived of and designed the study. All authors designed, performed or supervised experiments, and/or analysed data. C.R.R.A., L.L.H., K.A.C. and R.M.D. performed cell culture, molecular and biochemical experiments. D.d.l.C and C.A.M. produced AAVs. R.Y., E.R.S., A.B., A.R. and R.K. conducted the in vivo experiments with mice and analysed data. C.R.L. and S.Q.T. designed and performed CHANGE-seq experiments. K.J.S. collected primary human fibroblasts and clinical data. C.R.R.A. and B.P.K. wrote the manuscript with contributions and/or revisions from all authors.

Corresponding authors

Correspondence to Christiano R. R. Alves or Benjamin P. Kleinstiver.

Ethics declarations

Competing interests

C.R.R.A., K.A.C., K.J.S. and B.P.K. are inventors on a patent application filed by Mass General Brigham (MGB) that describes genome engineering technologies to treat SMA. S.Q.T. and C.R.L are co-inventors on a patent application describing the CHANGE-seq method. S.Q.T. is a member of the scientific advisory board of Kromatid, Twelve Bio and Prime Medicine. C.A.M. has a financial interest in Sphere Gene Therapeutics, Chameleon Biosciences and Skylark Bio, companies developing gene therapy platforms. C.A.M.’s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. C.A.M. has a filed patent application with claims involving the AAV-F capsid. B.P.K. is an inventor on additional patents or patent applications filed by MGB that describe genome engineering technologies. B.P.K. is a consultant for EcoR1 capital and is on the scientific advisory board of Acrigen Biosciences, Life Edit Therapeutics and Prime Medicine. S.Q.T. and B.P.K. have financial interests in Prime Medicine, a company developing therapeutic CRISPR–Cas technologies for gene editing. B.P.K.‘s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. The other authors declare no competing interests.

Peer review

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Supplementary information

Supplementary Information

Supplementary notes, figures and references.

Reporting Summary

Peer Review File

Supplementary Table 1

CHANGE-seq data summary.

Supplementary Table 2

Off-target sequencing, and results of statistical tests.

Supplementary Table 3

gRNA target sites.

Supplementary Table 4

Plasmids.

Supplementary Table 5

Oligonucleotides and probes.

Supplementary Table 6

Base-editor data.

Supplementary Table 7

gnomAD output for SMN2.

Supplementary Table 8

Primary datasets (SI).

Source data

Figs. 1–4 and Supplementary figures

Source data for all figures and supplementary figures.

Fig. 2

Unprocessed western blots for Fig. 2e.

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Alves, C.R.R., Ha, L.L., Yaworski, R. et al. Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy. Nat. Biomed. Eng 8, 118–131 (2024). https://doi.org/10.1038/s41551-023-01132-z

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