Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration

Abstract

Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated sitespecific integration utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in HSPCs, where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Recently, a novel NHEJ-based approach to CRISPR-Cas9-mediated transgene knockin, known as homology-independent targeted integration (HITI), has demonstrated improved site-specific integration frequencies in non-dividing cells. Here we utilize a HITI-based approach to achieve robust site-specific transgene integration in human mobilized peripheral blood CD34+ HSPCs. As proof of concept, a reporter gene was targeted to a clinically relevant genetic locus using a recombinant adeno-associated virus serotype 6 vector and single guide RNA/Cas9 ribonucleoprotein complexes. We demonstrate high levels of stable HITI-mediated genome editing ($21%) in repopulating HSPCs after transplantation into immunodeficient mice. Our study demonstrates that HITImediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs.

Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy

“…Recently, HITI-mediated reporter gene integration was reported in primary human CD34+ hematopoietic progenitor cells with long-term transgene expression following transplantation into immunodeficient mice. 15 These proof-of-principle studies demonstrate the exciting potential of HITI-based gene therapy approaches to correct genetic disease by targeted insertion strategies.…”

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

“…Using a Cas9 RNP and AAV6 with a GFP-vector targeting exon 1 of the ITGB2 locus, mutated in LAD-1, we could successfully achieve an average of 11% GFP positivity in HSPCs, persisting in culture up to four weeks post editing. Colony forming unit assays reveal that HITI does not result in lineage skewing, and HITI-edited HSPCs transplanted into immunodeficient NSG mice comprised an average of 21% of engrafted human CD45+ cells within the bone marrow 18 weeks post-transplantation [120]. These promising results indicate that HITI has the potential to complement HDR strategies currently used for HSPC gene editing.…”

High-affinity CD16 integration into a CRISPR/Cas9-edited CD38 locus augments CD38-directed antitumor activity of primary human natural killer cells

“…Based on a previously reported strategy, 28 the human genome was queried for potential Cas9 off-target sites using the Cas-OFFinder search tool 29 ( http://www.rgenome.net/cas-offinder/ ) with the CD38-targeting gRNA from this study as a query sequence. Up to three mismatched base pairs between the gRNA and potential off-target sites were allowed.…”