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USC, Sangamo Scientists Show Improved ZFN Activity in Hematopoietic Stem Cell Genome Editing

NEW YORK (GenomeWeb) – Scientists have shown an improved ability to edit genomes in human hematopoietic stem and progenitor cells (HSPCs) using zinc finger nucleases by introducing the enzymes into the cell as mRNA.

Scientists led by Paula Cannon of the University of Southern California and Michael Holmes of Richmond, California-based Sangamo Biosciences were able to introduce a reporter gene into HSPCs — including the most primitive kinds such as CD34+ cells, which had long been a challenge to achieve genome editing in — by electroporating mRNA and supplying the donor template for homology directed repair via adeno-associated virus (AAV).

They successfully inserted a green fluorescent protein cassette at the CCR5 gene locus at an average rate of 17 percent in CD34+ and 19 percent in fetal liver HSPCs; they successfully inserted the GFP gene at the AAVS1 locus at an average rate of 26 percent and 43 percent in the respective cell types.

They published the results of their study yesterday in Nature Biotechnology.

"We successfully modified a class of the most pluripotent stem cells, leading to long-term production of ZFN-modified cells across all blood cell lineages following transplantation into immune-deficient mice," Holmes said in a statement. "Our results in these two studies provide a robust strategy for additional therapeutic applications of ZFN-mediated genome editing in HSPCs and T-cells."

CCR5 is an important immune receptor used by HIV to enter human cells.

Sangamo is developing ZFNs for use in gene editing therapies for HIV.

The authors noted that cytotoxicity was a problem for genome editing in HPSCs and that delivering the ZFNs as mRNAs was not as toxic as delivering them via plasmid DNA, meshing with previous results from other genome editing studies. In June, scientists from Stanford University and Agilent Technologies found that delivering the CRISPR/Cas9 genome editing system as RNA, instead of DNA, greatly increased its effectiveness in HPSCs.