Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice

Christiano R R Alves^{1

2

3}, Sabyasachi Das¹, Vijai Krishnan¹, Leillani L Ha^{1

4}, Lauren R Fox^{1

4}, Hannah E Stutzman^{1

4}, Claire E Shamber¹, Pazhanichamy Kalailingam¹, Siobhan McCarthy¹, Christian L Lino Cardenas^{5

6}, Claire E Fong¹, Takahiko Imai⁷, Sunayana Mitra¹, Shuqi Yun^{1

4}, Rachael K Wood⁸, Friederike M C Benning^{9

10}, Kangsan Roh^{5

6}, Joseph Lawton^{5

6}, Nahye Kim^{1

4

11}, Rachel A Silverstein^{1

4

12}, Joana Ferreira da Silva^{1

4

11}, Demitri de la Cruz^{2

3}, Rashmi Richa^{5

6}, Jun Xie¹³, Heather L Gray-Edwards¹³, Rajeev Malhotra^{5

6}, David Y Chung^{2

3

7

14}, Luke H Chao^{9

10}, Shengdar Q Tsai⁸, Casey A Maguire^{2

3}, Mark E Lindsay^#^{15

16}, Benjamin P Kleinstiver^#^{17

18

19}, Patricia L Musolino^#^{20

21

22

23}

Affiliations

¹ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
² Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
³ Department of Neurology, Harvard Medical School, Boston, MA, USA.
⁴ Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁵ Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
⁶ Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
⁷ Neurovascular Research Unit, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
⁸ Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
⁹ Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
¹⁰ Department of Genetics, Harvard Medical School, Boston, MA, USA.
¹¹ Department of Pathology, Harvard Medical School, Boston, MA, USA.
¹² Biological and Biomedical Sciences Program, Harvard University, Boston, MA, USA.
¹³ Department of Genetics and Cellular Medicine, UMass Chan Medical School, Worcester, MA, USA.
¹⁴ Division of Neurocritical Care, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
¹⁵ Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Lindsay.Mark@mgh.harvard.edu.
¹⁶ Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Lindsay.Mark@mgh.harvard.edu.
¹⁷ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA. bkleinstiver@mgh.harvard.edu.
¹⁸ Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. bkleinstiver@mgh.harvard.edu.
¹⁹ Department of Pathology, Harvard Medical School, Boston, MA, USA. bkleinstiver@mgh.harvard.edu.
²⁰ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA. pmusolino@mgh.harvard.edu.
²¹ Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. pmusolino@mgh.harvard.edu.
²² Department of Neurology, Harvard Medical School, Boston, MA, USA. pmusolino@mgh.harvard.edu.
²³ Division of Neurocritical Care, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. pmusolino@mgh.harvard.edu.

^# Contributed equally.

PMID: 40935887
DOI: 10.1038/s41551-025-01499-1

Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice

Christiano R R Alves et al. Nat Biomed Eng. 2025.

. 2025 Sep 11.

doi: 10.1038/s41551-025-01499-1. Online ahead of print.

Authors

Affiliations

¹ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
² Department of Neurology, Massachusetts General Hospital, Boston, MA, USA.
³ Department of Neurology, Harvard Medical School, Boston, MA, USA.
⁴ Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁵ Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
⁶ Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
⁷ Neurovascular Research Unit, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.
⁸ Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
⁹ Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
¹⁰ Department of Genetics, Harvard Medical School, Boston, MA, USA.
¹¹ Department of Pathology, Harvard Medical School, Boston, MA, USA.
¹² Biological and Biomedical Sciences Program, Harvard University, Boston, MA, USA.
¹³ Department of Genetics and Cellular Medicine, UMass Chan Medical School, Worcester, MA, USA.
¹⁴ Division of Neurocritical Care, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
¹⁵ Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Lindsay.Mark@mgh.harvard.edu.
¹⁶ Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Lindsay.Mark@mgh.harvard.edu.
¹⁷ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA. bkleinstiver@mgh.harvard.edu.
¹⁸ Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. bkleinstiver@mgh.harvard.edu.
¹⁹ Department of Pathology, Harvard Medical School, Boston, MA, USA. bkleinstiver@mgh.harvard.edu.
²⁰ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA. pmusolino@mgh.harvard.edu.
²¹ Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. pmusolino@mgh.harvard.edu.
²² Department of Neurology, Harvard Medical School, Boston, MA, USA. pmusolino@mgh.harvard.edu.
²³ Division of Neurocritical Care, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. pmusolino@mgh.harvard.edu.

^# Contributed equally.

PMID: 40935887
DOI: 10.1038/s41551-025-01499-1

Abstract

Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.

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Conflict of interest statement

Competing interests: C.L.L.C., R.M., C.A.M., D.Y.C., B.P.K., M.E.L. and P.L.M. are inventors on a patent application filed by MGB that describes the development of genome-editing technologies to treat MSMDS. C.R.R.A., R.A.S., J.F.d.S. and B.P.K. are inventors on additional patents or patent applications filed by MGB that describe genome engineering technologies. C.R.R.A. is a consultant for Biogen and Ilios Therapeutics. S.Q.T. is an inventor on a patent covering CHANGE-seq. S.Q.T. is a member of the scientific advisory board of Prime Medicine and Ensoma. R.M., D.Y.C., C.A.M., B.P.K., M.E.L. and P.L.M. received sponsored research support from Angea Biotherapeutics, a company developing gene therapies for vasculopathies. R.M. receives research funding from Amgen, serves as a consultant for Pharmacosmos, Myokardia/BMS, Renovacor, Epizon Pharma and Third Pole, and performs speaker bureaus through Vox Media, all of which are unrelated to the current work. C.A.M. has financial interests in Chameleon Biosciences, Skylark Bio and Sphere Gene Therapeutics, companies developing adeno-associated virus vector technologies for gene therapy applications; C.A.M. performs paid consulting work for all three companies. C.A.M.’s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. B.P.K. is a consultant for EcoR1 capital, Novartis Venture Fund, Foresite Labs and Jumble Therapeutics, and is on the scientific advisory boards of Acrigen Biosciences, Life Edit Therapeutics and Prime Medicine. B.P.K. has a financial interest 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.

Update of

In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor.
Alves CRR, Das S, Krishnan V, Ha LL, Fox LR, Stutzman HE, Shamber CE, Kalailingam P, McCarthy S, Lino Cardenas CL, Fong CE, Imai T, Mitra S, Yun S, Wood RK, Benning FMC, Lawton J, Kim N, Silverstein RA, da Silva JF, de la Cruz D, Richa R, Malhotra R, Chung DY, Chao LH, Tsai SQ, Maguire CA, Lindsay ME, Kleinstiver BP, Musolino PL. Alves CRR, et al. bioRxiv [Preprint]. 2024 Nov 11:2024.11.11.621817. doi: 10.1101/2024.11.11.621817. bioRxiv. 2024. Update in: Nat Biomed Eng. 2025 Sep 11. doi: 10.1038/s41551-025-01499-1. PMID: 39605323 Free PMC article. Updated. Preprint.

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Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice

Affiliations

Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

References

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials

Miscellaneous