Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease [CHANGE-seq]

To define the genome-wide activity of Cas9-sgRNA directed at the gamma-globin promoter genes, we performed CHANGE-seq and identified 678 off-target sites from plerixafor-mobilized normal donors of African American ancestry (pre-GMP engineering runs, n = 3). Most of these off-targets are in intergenic and intronic regions of the human genome, as expected based on their relative genomic proportions. We selected the 277 CHANGE-seq candidate sites reproducibly identified in 2 or more biological donors and in silico predicted sites for multiplexed targeted amplicon sequencing (rhAmp-seq, IDT). We applied CHANGE-seq, a highly-sensitive in vitro based biochemical assay to detect potential off-target sites from plerixafor-mobilized normal donors of African American ancestry (pre-GMP engineering runs, n = 3).