High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

Joachim Schwäble,Roy Ehling,Harald von Melchner,Nina Kurrle,Duran Sürün,Frank Schnütgen,Stefan Stein,Ana Tomasovic

doi:10.1016/j.omtn.2017.11.001

Joachim Schwäble, Roy Ehling

Open Access

https://doi.org/10.1016/j.omtn.2017.11.001

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Abstract

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Highlights

The vast majority of inherited monogenic disorders are caused by patient-specific mutations dispersed over the entire locus of the affected gene.[1]
Because double-strand breaks (DSBs) repair by non-homologous end joining (NHEJ) in mammalian cells significantly exceeds homology directed repair (HDR) and, more importantly, is the dominant DSB-repair pathway in hematopoietic stem and progenitor cells (HSPCs),[13,14] we exploited NHEJ for gene repair because, in theory, approximately one-third of indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation
We show that gene-inactivating point mutations introduced into EGFP transgenes expressed in PLB cells (PLBs)-985 myeloid leukemia cells are effectively repaired by donor template-free RNA-guided CRISPR/ Cas[9] endonucleases (RGNs) delivered by integrase-defective lentiviruses (IDLVs)

Summary

INTRODUCTION

The vast majority of inherited monogenic disorders are caused by patient-specific mutations dispersed over the entire locus of the affected gene.[1]. HDR offers precision, efficiency is low and most editing protocols rely on positive selection to enrich for gene-corrected cells.[5,6,7,8,9,10,11,12] Because DSB repair by NHEJ in mammalian cells significantly exceeds HDR and, more importantly, is the dominant DSB-repair pathway in hematopoietic stem and progenitor cells (HSPCs),[13,14] we exploited NHEJ for gene repair because, in theory, approximately one-third of indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation This could lead to many ORF reconstitutions, of which some, depending on the position and type of the original mutation, should completely or partially recover protein function, as has been shown recently for the dystrophin gene in patients with Duchenne’s muscular dystrophy (DMD).[15].

Molecular Therapy

RESULTS AND DISCUSSION

MATERIALS AND METHODS