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Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Nature Biotechnology volume 34, pages 328–333 (2016)Cite this article

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Abstract

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

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Figure 1: In vitro delivery of Cas9 mRNA mediates efficient genome editing in cells.
Figure 2: In vivo delivery of Cas9 mRNA and AAV-HDR template cures type I tyrosinemia mice.
Figure 3: In vivo delivery of Cas9 mRNA and AAV corrects Fah mutation.

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Acknowledgements

We thank M. Grompe, S. Levine, T. Jacks, P. Sharp, E. Sontheimer, C. Mello, P. Zamore, M. Moore, T. Flotte, T. Tammela, F. Sanchez-Rivera, T. Papagiannakopoulos, D. Wang, J. Moore and A. Vegas for discussions and for sharing reagents, S. Hough for technical assistance and K. Cormier for histology. This work is supported by grants from the National Institutes of Health (NIH), 5R00CA169512 and Worcester Foundation (to W.X.). H.Y. is supported by Skoltech Center and 5-U54-CA151884-04 (NIH Centers for Cancer Nanotechnology Excellence and the Harvard-MIT Center of Cancer Nanotechnology Excellence). Y.D. acknowledges support from the National Institute of Biomedical Imaging and Bioengineering for his postdoctoral fellowship 1F32EB017625. V.K. acknowledges support from the Russian scientific fund, grant number 14-34–00017. This work is supported in part by Cancer Center Support (core) grant P30-CA14051 from the NIH. We thank the Swanson Biotechnology Center for technical support. We thank C. Wang at Boston Children's Hospital Viral Core for AAV prep (supported by core grant 5P30EY012196-17). The authors acknowledge the service to the MIT community of the late S. Collier.

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Authors and Affiliations

  1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Hao Yin, Joseph R Dorkin, Qiongqiong Wu, Junghoon Yang, Sneha Suresh, Stephen Walsh, Roman L Bogorad, Robert Langer & Daniel G Anderson

  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Chun-Qing Song, Angela Park, Aizhan Bizhanova & Wen Xue

  3. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Chun-Qing Song, Lihua J Zhu & Wen Xue

  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Joseph R Dorkin

  5. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Lihua J Zhu, Ankit Gupta, Mehmet F Bolukbasi, Scot A Wolfe & Wen Xue

  6. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Lihua J Zhu & Zhiping Weng

  7. Department of Bioinformatics, School of Life Science and Technology, Tongji University, Shanghai, P.R. China

    Yingxiang Li

  8. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Mehmet F Bolukbasi & Scot A Wolfe

  9. Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Guangping Gao

  10. College of Pharmacy, the Ohio State University, Columbus, Ohio, USA

    Yizhou Dong

  11. Skolkovo Institute of Science and Technology, Skolkovo, Russia

    Victor Koteliansky

  12. Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, Russia

    Victor Koteliansky

  13. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Robert Langer & Daniel G Anderson

  14. Harvard-MIT Division of Health Sciences & Technology, Cambridge, Massachusetts, USA

    Robert Langer & Daniel G Anderson

  15. Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Robert Langer & Daniel G Anderson

Authors
  1. Hao Yin
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Contributions

H.Y., W.X. and D.G.A. designed the study. H.Y. and W.X. directed the project. H.Y., C.-Q.S., J.R.D., L.J.Z., Y.L., Q.W., J.Y., S.S., A.B., A.G., M.F.B., A.P., S.W. and R.L.B. performed experiments and analyzed data. G.G., Z.W., Y.D., V.K., S.A.W. and R.L. provided reagents and conceptual advice. H.Y., W.X. and D.G.A. wrote the manuscript with comments from all authors.

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Correspondence to Wen Xue or Daniel G Anderson.

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Competing interests

D.G.A., H.Y., J.R.K. and W.X. have applied for patents on the subject matter of this paper. D.G.A. is a scientific co-founder of CRISPR Therapeutics.

Integrated supplementary information

Supplementary Figure 1 Cas9 mRNA nanoparticles characterization.

(a) nano.Cas9 formulation scheme. Cas9 mRNA was mixed with C12-200, DOPE, Cholesterol, C14PEG2000 and arachidonic acid in a microfluidic chamber. (b) nano.Cas9 structure is characterized by cryo-TEM. Scale bar indicates 100nm. (c) Average diameter of nano.Cas9 was measured by dynamic light scattering. The size of nano.Cas9 (d) and the polydispersity index (PDI) (e) were measured 0, 7, 11 or 18 days after formulation and storage at 4˚C.

Supplementary Figure 2 The expression of proteins in mouse liver after mRNA nanoparticles treatment.

(a) C57bl/6 mice were i.v. injected with nanoparticles encapsulated with β-gal (b and c) or Cas9 mRNA (nano.Cas9, d and e), and livers taken. (b) The expression of β-gal protein was measured in liver lysate at 14 hours after injection. (c) The activity of β-gal in liver sections was determined by salmon-gal assay. Scale bar indicates 200 µm. (d) The expression of Cas9 protein was measured in liver lysate 14 hours after injection. 50μg negative control samples mixed with 10, 1 or 0.1ng Cas9 protein served as positive controls. β-actin served as a loading control in (b) and (d). (e) The Cas9 mRNA level in liver was determined by qRT-PCR at 4, 14, and 24 hours after injection (n=3 mice).

Supplementary Figure 3 Cas9 mRNA nanoparticles are well tolerated.

C57/Bl6 mice were treated with 2mg/kg nano.Cas9, and histology (a), the levels of liver damage markers (b) and plasma cytokines (c) were determined after 24 hours. Scale bar indicates 50μM. (n=4 mice).

Supplementary Figure 4 The time course of sgRNA expression in mouse liver.

Mice were injected with AAV-HDR and livers taken at 0, 3, 7 and 14 days after injection. qRT-PCR was performed to determine sgRNA expression in liver. The expression levels were normalized to Day 3 (n = 4 mice).

Supplementary Figure 5 A PCR approach proves substitution of the correct sequence.

(a) Design of the PCR primers. Blue arrow indicates the reverse PCR primer, which is outside the repair template. The sequence of the forward primer is presented, and “G” and “CC” in the corrected sequence are highlighted. (b) Genomic DNA of the liver tissue was extracted, and PCR was performed using the primers in (a). The predicted size of PCR product is 1.02kb. A representative sample from each group is shown (n = 3 mice). (c) The PCR product from (b) was cloned to a TA cloning vector and Sanger sequenced. The corrected “G” and “CC” are highlighted.

Supplementary Figure 6 Viral delivery of Cas9 does not increase HDR rate compared to mRNA delivery.

(a) Design of AAV-HDR template. Four “G” point mutations resulting in stabilization of β-Catenin are highlighted. Ad.Cas9 is an adenovirus expressing Cas9. (b) β-Catenin IHC. AAV-HDR-Ctnnb1 alone serves as a control. Arrows denote β-Catenin positive hepatocytes. (c-d) The Ctnnb1 locus in the liver total DNA of Ad.Cas9+ AAV-HDR-Ctnnb1 treated mice (n=2) were deep sequenced to measure indels. (e) β-Catenin positive hepatocytes were counted to determine the percentage of HDR. P < 0.01 (n = 3 mice). mRNA delivery of Cas9 yields higher rate of HDR for Fah (>6%).

Supplementary Figure 7 Cas9 mRNA delivery has minimal off-target effects at assayed sites in vivo.

(a) Top ranking off-target sites (OT1, OT3 and OT4) for sgFah and the predicted score (Hsu et al, 2013). Mismatch bases are in red. Score for the wildtype sgFah.2 targets the mutant Fah which has one mismatch with wildtype Fah (wt Fah). (b) Indel frequency is low and is comparable between control mouse and nano-Cas9+ AAV-HDR mouse. OT1, OT3 and OT4 regions were PCR amplified from mouse liver genomic DNA and analyzed by deep sequencing. (c) Surveyor assay did not detect indels at OT1, OT3 and OT4. Predicted size of uncut and cut bands are indicated.

Supplementary Figure 8 Indel rate measured by deep sequencing for GUIDE-Seq off-target sites.

OT1 is the strongest off-target sites identified by GUIDE-Seq. GOT1-11 are additional genomic sites that displayed GUIDE-Seq oligonucleotide insertions. (a) Mouse Hepa1-6 liver cells transfected with pX330.sgFah.2. #1 and #2 are replicates. (b) Mouse livers treated with nano.Cas9 and AAV-HDR (treated) or control treated (control). Fah is the on-target site. See Table S9 for details.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–6 and Supplementary Sequences (PDF 3895 kb)

Supplementary Table 7

sgRNA2 GUIDE-seq +&- strand peaks (PDF 40329 kb)

Supplementary Table 8

sgRNA2 GUIDE-seq merged peaks (PDF 75 kb)

Supplementary Table 9

Deep sequencing of off-target sites (PDF 192 kb)

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Yin, H., Song, CQ., Dorkin, J. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34, 328–333 (2016). https://doi.org/10.1038/nbt.3471

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