Homology-driven Genome Editing in Hematopoietic Stem and Progenitor Cells Using ZFN MRNA and AAV6 Donors

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2015 Nov 10

PMID 26551060

Citations 167

Authors

Jianbin Wang

Colin M Exline

Joshua J DeClercq

G Nicholas Llewellyn

Samuel B Hayward

Patrick Wai-Lun Li

David A Shivak

Richard T Surosky

Philip D Gregory

Michael C Holmes

Paula M Cannon

Affiliations

Soon will be listed here.

Abstract

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34 HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34CD133CD90 cell population, a minor component of CD34 cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.

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