In utero CRISPR-mediated therapeutic editing of metabolic genes

Abstract

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector–mediated delivery of CRISPR–Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.

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Fig. 1: In utero base editing of the Pcsk9 gene.
Fig. 2: Functional effects of in utero Pcsk9 base editing and comparison to postnatal editing.
Fig. 3: In utero base editing of Hpd in the Fah–/– mouse model.
Fig. 4: In utero Hpd base editing improves liver function and rescues the lethal phenotype of Fah–/– mice.

Data availability

The data that support the findings of this study are available within the paper and its supplementary information files. DNA sequencing data have been deposited on the NCBI Sequence Read Archive under accession SRP155635.

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Acknowledgements

This work was supported by grants T32-HL007843 (A.C.W.), R01-HL118744 and R01-HL126875 (K.M.) from the National Institutes of Health; grant UL1-TR001878 from the National Center for Advancing Translational Sciences of the National Institutes of Health and the Institute for Translational Medicine and Therapeutics at the University of Pennsylvania (W.H.P. and K.M.); grant GE-16-001-IU from the University of Pennsylvania Orphan Disease Center (W.H.P.); the Winkelman Family Fund in Cardiovascular Innovation (K.M.); the Burroughs Wellcome Fund Career Award for Medical Scientists (R.J.); and generous family gifts to the Children’s Hospital of Philadelphia (CHOP) (W.H.P.). We thank A. Weilerstein and L. Ma for their help with animal care, the Translational Core Laboratory at CHOP for assistance with liver function tests, and A. Radu, the Pathology Core at CHOP and the Histology Core at the Cardiovascular Institute at the University of Pennsylvania for their assistance with histology.

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Contributions

A.C.R., J.D.S., A.C.C., and H.A.H. performed the experiments and acquired and analyzed the data. A.C.R. was the lead individual on the tyrosinemia experiments, J.D.S. was the lead individual on the Pcsk9 experiments, and A.C.C. did all initial in vitro work and continued as an instrumental contributor to both the Pcsk9 and tyrosinemia projects. N.J.A. and B.E.C. provided technical help, performed the prenatal injections, and assisted in acquiring the data. H.L. provided technical help, performed the in vitro ELISA assays, and acquired/analyzed the data. P.Z. provided technical help and designed plasmids. K.S. and W.L. provided technical help, performed the qRT-PCR experiments, and assisted in acquiring the data. L.L. performed critical technical help. D.A. and W.Z. designed the initial R26mTmG/+ screening studies. R.J. provided critical technical help and the histologic analyses. E.M.M. designed the original R26mTmG/+ screening studies and provided critical experimental guidance. K.M. and W.H.P. designed all the experiments, oversaw the performance of all the experiments, analyzed the data, and wrote the paper with support from A.C.R., J.D.S., and A.C.C.

Corresponding authors

Correspondence to Kiran Musunuru or William H. Peranteau.

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The authors declare no competing interests.

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Supplementary information

41591_2018_184_MOESM3_ESM.mp4

In utero vitelline vein injection of an E16 fetus with dilute trypan blue

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1–5

Reporting Summary

Supplementary Video 1

In utero vitelline vein injection of an E16 fetus with dilute trypan blue

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Rossidis, A.C., Stratigis, J.D., Chadwick, A.C. et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat Med 24, 1513–1518 (2018). https://doi.org/10.1038/s41591-018-0184-6

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