In vivo prime editing of a metabolic liver disease in mice

Author:

Böck Desirée1ORCID,Rothgangl Tanja1ORCID,Villiger Lukas1ORCID,Schmidheini Lukas12ORCID,Matsushita Mai2ORCID,Mathis Nicolas1ORCID,Ioannidi Eleonora1,Rimann Nicole3,Grisch-Chan Hiu Man3ORCID,Kreutzer Susanne45ORCID,Kontarakis Zacharias45ORCID,Kopf Manfred2ORCID,Thöny Beat367ORCID,Schwank Gerald1ORCID

Affiliation:

1. Institute of Pharmacology and Toxicology, University of Zurich, 8057 Zürich, Switzerland.

2. Institute of Molecular Health Sciences, ETH Zürich, 8093 Zürich, Switzerland.

3. Division of Metabolism and Children’s Research Center, University Children’s Hospital Zurich, 8032 Zürich, Switzerland.

4. Genome Engineering and Measurement Laboratory (GEML), ETH Zürich, 8093 Zürich, Switzerland.

5. Functional Genomics Center Zurich, ETH Zürich/University of Zurich, 8057 Zürich, Switzerland.

6. Zurich Center for Integrative Human Physiology, 8006 Zürich, Switzerland.

7. Neuroscience Center Zurich, 8057 Zürich, Switzerland.

Abstract

Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Sp Cas9 prime editor (PE) lacking the RNaseH domain (PE2 Δ RnH ) and an intein-split construct (PE2 p.1153) for adeno-associated virus–mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2 Δ RnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah enu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 14 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

General Medicine

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