Comparison of CD34+ cells isolated from frozen cord blood and fresh adult peripheral blood of sickle cell disease patients in gene correction of the sickle mutation at late‐stage erythroid differentiation

Abstract

Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to correct a point mutation on the b-globin gene (HBB). CD34 haematopoietic stem and progenitor cells (HSPCs) are a primary target for therapeutic strategies of gene editing. Mobilized peripheral blood has become a popular source of CD34 HSPCs rather than bone marrow due to more efficient collection. Another source of HSPCs, cord blood (CB), also has several advantages, including ease of preparation, off-the-shelf availability, tolerance of human leukocyte antigen mismatch, and a low incidence of severe graft-versus-host disease. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) geneediting system has recently been used to treat b-thalassaemia and SCD. This system creates a double-strand break using a guide RNA (gRNA) recognized by the DNA target site at a three-base protospacer adjacent motif to activate the Cas9 endonuclease, followed by DNA repair through either nonhomologous end joining (NHEJ) that produces genomic insertions and deletions (indels) or templated homology-directed repair (HDR) that allows replacement of specific DNA sequences. Although the previous studies recently reported the correction of the sickle mutation in SCD-derived CD34 cells using CRISPR/Cas9 technology, innovations in CRISPR/Cas9 clinical use in SCD remain an area of development. In addition, it is critical to define which source of HSPCs, fresh or frozen, is superior for the application of CRISPR/Cas9 to SCD. In this study, we compared the efficiency of CRISPR/Cas9 gene editing for correcting the sickle mutation between frozen CB-derived and fresh adult peripheral blood-derived CD34 cells at day 14 of erythroid differentiation. We initially evaluated CD34 cells isolated from frozen CB samples of SCD patients. After 24 h of pre-stimulation, we electroporated chemically modified single guide (sg-)RNA/ Cas9 ribonucleoprotein and a wild-type (WT) HBB allele-single-stranded oligonucleotide (ssODN) template into SCD-CB CD34 cells, followed by 14 days of erythroid differentiation. Treatment with genome editing reagents did not alter erythroid cell differentiation in SCD-CB-derived CD34 cells at day 14 (Fig 1A–D). The CD71 GPA (anti-glycophorin A) phenotype was not significantly different between mock-treated cells (11⋅9 0⋅7%) and HBB-edited SCD-CB CD34 cells (11⋅7 1⋅9%; P = 0⋅88). Next, we evaluated the frequency of indels and HDR rate at day 14 by tracking of indels by decomposition (TIDE) and amplicon deep sequencing. Mean indel frequencies for the HBB-targeted loci were 63⋅1 3⋅6% and 73⋅6 17⋅6% in HBB-edited SCD-CB CD34 cells by TIDE and amplicon deep sequencing, respectively (Fig 2A). In addition, we observed a 20⋅4 9⋅1% and 25⋅4 6⋅3% HDR rate in HBB-edited SCD-CB CD34 cells at day 14 by TIDE and amplicon deep sequencing, respectively. We next used amplicon deep sequencing at three different loci to investigate the sgRNA’s off-target activity. Two of