Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

Samantha L Ginn; Anais K Amaya; Sophia H Y Liao; Erhua Zhu; Sharon C Cunningham; Michael Lee; Claus V Hallwirth; Grant J Logan; Szun S Tay; Anthony J Cesare; Hilda A Pickett; Markus Grompe; Kimberley Dilworth; Leszek Lisowski; Ian E Alexander

doi:10.1016/j.jhepr.2019.100065

Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

JHEP Rep. 2019 Dec 27;2(1):100065. doi: 10.1016/j.jhepr.2019.100065. eCollection 2020 Feb.

Authors

Samantha L Ginn¹, Anais K Amaya¹, Sophia H Y Liao¹, Erhua Zhu¹, Sharon C Cunningham¹, Michael Lee², Claus V Hallwirth¹, Grant J Logan¹, Szun S Tay¹, Anthony J Cesare³, Hilda A Pickett², Markus Grompe⁴, Kimberley Dilworth⁵, Leszek Lisowski^{5

6}, Ian E Alexander^{1

7}

Affiliations

¹ Gene Therapy Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney and Sydney Children's Hospitals Network, Westmead, Australia.
² Telomere Length Regulation Group, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia.
³ Genome Integrity Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia.
⁴ School of Medicine, Oregon Health & Science University, Portland, Oregon.
⁵ Translational Vectorology Group and Vector & Genome Engineering Facility, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia.
⁶ Military Institute of Hygiene and Epidemiology, Pulway, Poland.
⁷ Discipline of Child and Adolescent Health, Sydney Medical School, Faculty of Medicine and Health, The University of Sydney, Westmead, Australia.

Abstract

Background & aims: Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. In this study, we aimed to repair a disease-causing point mutation in the ornithine transcarbamylase (OTC) locus in patient-derived primary human hepatocytes in vivo at therapeutically relevant levels.

Methods: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human OTC locus were first evaluated against an OTC minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native OTC locus in patient-derived primary human hepatocytes xenografted into the FRG (Fah ^-/- Rag2 ^-/- Il2rg ^-/-) mouse liver. A highly human hepatotropic capsid (NP59) was used for adeno-associated virus (AAV)-mediated gene transfer. Editing events were characterised using next-generation sequencing and restoration of OTC expression was evaluated using immunofluorescence.

Results: Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes in vivo, OTC locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of OTC alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 in silico-predicted sites in the genome.

Conclusions: We report efficient single-nucleotide correction of a disease-causing mutation in the OTC locus in patient-derived primary human hepatocytes in vivo at levels that, if recapitulated in the clinic, would provide benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on- and off-target editing events.

Lay summary: The ability to efficiently and safely correct disease-causing mutations remains the holy grail of gene therapy. Herein, we demonstrate, for the first time, efficient in vivo correction of a patient-specific disease-causing mutation in the OTC gene in primary human hepatocytes, using therapeutically relevant vector doses. We also highlight the challenges that need to be overcome for this technology to be translated into clinical practice.

Keywords: 7 NGS, next-generation sequencing; AAV, adeno-associated virus; BrdU, bromodeoxyuridine; CRISPR-Cas9; FRG, Fah-/-Rag2-/-Il2rg-/-; HDR, homology-directed repair; ITR, inverted terminal repeats; InDels, insertions and deletions; LSP1, liver-specific promoter; NHEJ, non-homologous end joining; NP59 capsid; OTC deficiency; PAM, protospacer adjacent motif; PRE, mutant form of the Woodchuck hepatitis virus posttranscriptional regulatory element; RTA, Real Time Analysis; SV40 pA, SV40 polyadenylation signal sequence; SaCas9, Staphylococcus aureus Cas9 nuclease; TBG, human thyroxine binding globulin promoter; U6, RNA polymerase III promoter for human U6 snRNA; WT, wild-type; genome editing; homology-directed repair; humanised FRG mice; pA, bovine growth hormone polyadenylation signal sequence; primary human hepatocytes; rAAV, recombinant adeno-associated virus; recombinant AAV; sgRNA, single guide RNA; synthetic capsid.