55. AAVHSC Vectors Encoding Zinc-Finger Nucleases Mediate Efficient Targeted Integration at the Human AAVS1 Locus in CD34+ Human Hematopoietic Stem Cells

Abstract

Zinc-finger nucleases (ZFN) are powerful genome editing tools that may be used to prevent expression of specified genes, correct mutations, or insert transgenes at safe harbor loci. ZFNs create double-stranded breaks at specified locations in the genome which may subsequently be repaired via the non-homologous end joining pathway to interrupt expression of a gene. Targeted integration of genes at pre-determined sites in the genome can be achieved through homology-directed repair of ZFN-mediated targeted double-strand breaks using a co-delivered donor construct. We recently isolated and characterized a panel of novel AAVs (AAVHSC) derived from CD34+ human hematopoietic stem cells (HSC) from healthy donors. We have previously shown that vectors derived from AAVHSCs have the ability to efficiently transduce CD34+ cells. Here we tested the hypothesis that delivery of ZFNs and donor DNA by AAVHSC vectors to CD34+ human cells results in the creation of double stranded site-specific breaks and subsequent efficient targeted transgene integration. A promoterless GFP gene was targeted to Intron 1 of the PPP1R12C gene in the AAVS1 locus such that expression would be driven by the endogenous PPP1R12C promoter following site specific transgene integration. ZFNs specific for PPP1R12C Intron 1 and donor DNA were packaged in AAVHSCs and used to transduce either K562 cells or primary human cytokine-primed peripheral blood CD34+ cells (PBSC). Flow-cytometric analysis at different time points post-transduction revealed specific GFP expression in ZFN + Donor-transduced cells but not in cells transduced with ZFN alone. The multiplicities of infection, ratio of ZFN:donor, and timing of transduction were optimized in K562 cells. Simultaneous transduction of AAVHSC encoding both ZFN and donor DNA at multiplicities of infection of 50,000 and 150,000 respectively was found to be optimal and resulted in GFP expression in up to 50% of transduced cells. Optimized conditions were confirmed in primary CD34+ PBSC from different donors. Targeted integration into AAVS1 was consistently found to be more efficient in CD34+ PBSC as compared with K562 cells, with GFP expression ranging from 10 to 65% of cells. Integrated GFP was expressed long term, with approximately 20% GFP positive cells persisting at 35 days post-transduction in vitro in the absence of selection. Targeted integration into the AAVS1 locus was independently confirmed by amplification using transgene-specific and chromosome-specific primers. Sequencing of the amplified vector chromosome junctions confirmed site-specific integration into AAVS1. No unexpected modifications were found at the junction of the AAVS1 chromosomal sequence and donor homology arms. These results indicate that AAVHSCs function as efficient delivery tools for genome editing in therapeutically relevant cells such as primary PBSC.