Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors.

Yujia Cai,Martin R Jakobsen,Jacob Giehm Mikkelsen,Mads Valdemar Anderson,Shengting Li,Yonglun Luo,Anders Laustsen,Yan Zhou,Chenglong Sun,Niels Uldbjerg

doi:10.7554/elife.12213

Abstract

Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et al., 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34(+) hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell-based gene therapies.

Highlights

Uncontrolled genomic insertion of transgenes, leading to insertional mutagenesis, remains a primary challenge for safe genetic therapies
In a previous paper (Cai et al, 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR)
Building on these findings, we report a simple method for establishing targeted gene integration (TGI) by treating cells with integrase-defective lentiviral vectors (IDLVs) packaged with both the transgene-containing donor template and ZFN proteins fused to the N-terminus of lentiviral Gag and GagPol polypeptides

Summary

Introduction

Uncontrolled genomic insertion of transgenes, leading to insertional mutagenesis, remains a primary challenge for safe genetic therapies. We previously established genomic editing in cells treated with ‘all-in-one’ lentiviral particles loaded with locus-targeted ZFN proteins and a donor sequence flanked by homology arms (Cai et al, 2014a). Building on these findings, we report a simple method for establishing TGI by treating cells with integrase-defective lentiviral vectors (IDLVs) packaged with both the transgene-containing donor template and ZFN proteins fused to the N-terminus of lentiviral Gag and GagPol polypeptides. Locus-directed cleavage and TGI is evident in cell lines, CD34+ hematopoietic stem cells, and induced pluripotent stem cells (iPSCs) upon transduction with ZFN-loaded vectors

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Conclusion