Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors

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 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 CD34+CD133+CD90+ 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.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells

“…However, recently, high rates of HDR were achieved in primitive HSCs using ZFNs (delivered by electroporation of in vitro transcribed mRNA) and a donor template delivered by adeno-associated virus serotype 6 (AAV6). 33 The use of AAV6 as a donor template could be applied for the correction of the SCD mutation described here as well. By delivering both the gRNA and the homologous donor template in a single AAV6, high rates of HDR in primitive HSCs may be possible and is currently under investigation.…”

Engineering HIV-Resistant, Anti-HIV Chimeric Antigen Receptor T Cells

“…11,46 Using new gene-editing techniques, it has recently become possible to achieve high rates of homology-directed recombination (HDR) of therapeutic cassettes into targeted loci, including CCR5 in primary T cells. [47][48][49][50] We have previously shown introduction of cDNA expression cassettes at the CCR5 locus in primary human T cells using an mRNA-delivered megaTAL nuclease and a homologous AAV donor template at rates of up to 60%. 48 HDR has the potential advantage of simultaneous introduction of a CAR and disruption of CCR5 to protect engineered cells from HIV.…”

Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

“…This is inline with recent reports that have described efficient CD34 1 HPC targeted corrections or insertions. [19][20][21][22] We demonstrated our gene-targeting correction in iPSCs from 3 subjects and in HPCs from a fourth subject with p47-CGD. Three of these patients had the common homozygous DGT mutation at the NCF1 locus while one patient (subject 5; the source of iPSC of the P47-05 correction series) had normal GTGT at the start of exon 2 of NCF1 and instead had a homozygous D734-748 mutation in exon 8 of NCF1.…”