118. Characterization of Chromosomal Alterations Using a Zinc-Finger Nuclease Targeting the Beta-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

The use of engineered nucleases for targeted gene correction of the sickle cell disease (SCD) mutation in hematopoietic stem and progenitor cells (HSPCs) combined with a homologous DNA donor template can result in successful targeted gene correction. However, due to a high sequence homology existing between the beta- and delta-globin genes, off-target endonuclease cleavage events can occur when using endonucleases targeted to beta-globin. Moreover, the introduction of multiple double stranded breaks by site-specific endonucleases has the potential to induce chromosomal alterations such as translocations, deletions, and inversions. This work focused on the use of a novel Droplet Digital PCR (ddPCR) assay to characterize the frequency of deletions, inversions and translocations between the beta- and delta-globin paralogs when delivering nucleases that can cleave at beta- and show off-target activity at the delta-globin locus in HSPCs. The impact of co-delivering these nucleases with or without a homology-directed repair (HDR) donor template (as an integrase defective lentiviral vector [IDLV] or as a single stranded oligonucleotide) was also assessed. Bone marrow (BM) CD34+ cells from a SCD patient were treated with ZFNs with high levels of site-specific cleavage activity at both the beta- and delta-globin loci (32-44% indels at beta-globin and 2-12% indels at delta-globin by CEL1 assay). Samples treated with nuclease-only, nuclease+Oligo and nuclease+IDLV respectively, showed that the different events were present at the following frequencies: 41.3%, 45.3% and 50.2% for the deletion; 9.7%, 6.5% and 8.8% for the inversion; and 0.6%, 0.5% and 0.2% for the translocations involving beta- and delta-globin. The same analysis performed in colony forming units (CFUs), derived from the SCD BM CD34+ cells treated with nuclease or nuclease with HDR Donor, showed that the deletion was the most frequent event, followed by the inversion, with the translocation least frequent, mirroring what was observed in the CD34+ pooled populations. Thus, for each event, the frequency was independent of the presence of oligo or IDLV donor template, in both the pooled and the clonal populations. Additionally, the ddPCR analysis of CFUs gave insight into the mono-allelic vs. bi-allelic nature of each rearrangement event, and thus the mono-allelic vs. bi-allelic modification frequency of the nuclease-driven editing. Analysis of the nuclease only treated CFUs showed that 30% of deletion events were bi-allelic; while the majority of inversions and translocations were mono-allelic. These results confirmed that due to the high sequence homology existing between the target locus in beta-globin and the off-target locus in delta-globin, there was a significant frequency of chromosomal alterations induced by cleaving both paralogs in the same human HSPCs. This may partly reflect the unique chromosomal conformation of the globin genes due to their co-regulation in hematopoiesis, and looping to the same cis-regulatory element. More generally, these findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted chromosomal alterations.