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

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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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Most cited references 18

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Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

John Zuris, David Thompson, Yilai Shu … (2014)

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

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Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.

Gerald Schwank, Bon-Kyoung Koo, Valentina Sasselli … (2013)

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect. Copyright © 2013 Elsevier Inc. All rights reserved.

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Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

Ayal Hendel, Rasmus O Bak, Joseph Clark … (2015)

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

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Author and article information

Journal

Journal ID (nlm-journal-id): 9604648

Journal ID (pubmed-jr-id): 20305

Journal ID (nlm-ta): Nat Biotechnol

Journal ID (iso-abbrev): Nat. Biotechnol.

Title: Nature biotechnology

ISSN (Print): 1087-0156

ISSN (Electronic): 1546-1696

Publication date Nihms-submitted: 19 January 2017

Publication date (Electronic): 01 February 2016

Publication date (Print): March 2016

Publication date PMC-release: 09 May 2017

Volume: 34

Issue: 3

Pages: 328-333

Affiliations

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

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

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

[4 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

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

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

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

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

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

[10 ]College of Pharmacy, the Ohio State University, Columbus, Ohio, USA

[11 ]Skolkovo Institute of Science and Technology, Skolkovo, Russia

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

[13 ]Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

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

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

Author notes

Correspondence should be addressed to W.X. ( wen.xue@ 123456umassmed.edu ) or D.G.A. ( dgander@ 123456mit.edu )

Article

Manuscript ID: NIHMS843247

DOI: 10.1038/nbt.3471

PMC ID: 5423356

PubMed ID: 26829318

SO-VID: 3d1e585f-635e-4caa-892c-fe8fd9f0e908

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

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Abstract

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Genome Engineering using CRISPR

Most cited references 18

Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

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