The Mechanism Of Beta-globin Gene Silencing In Embryonic-fetal Erythroid Cells: Application to Gene Therapy

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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. The CD71GPA+ (anti-glycophorin A) phenotype was not significantly different between mock-treated cells (119 07%) and HBB-edited SCD-CB CD34+ cells (117 19%; P = 088). Next, we evaluated the frequency of indels and HDR rate at day 14 by tracking indels by decomposition (TIDE) and amplicon deep sequencing. Mean indel frequencies for the HBB-targeted loci were 631 36% and 736 176% in HBB-edited SCD-CB CD34+ cells by TIDE and amplicon deep sequencing, respectively. In addition, we observed a 204 91% and 254 63% 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 sgRNAs off-target activity. Two of these off-target sites (at ITGA9 and chr2: 42851054-42851076) were predicted by COSMID software (http://crispr.bme.gatech.edu); we further included one for HBD because it has high homology to the HBB gene (Table SI). We found that the off-target activity at ITGA9, chr2: 42851054-42851076, and HBD was 351 19%, 00 003%, and 005 006% indels in HBB-edited SCD-CB CD34+ cells at day 14, respectively (Fig 2B). As expected for CB- and in vitro-derived erythroblasts, hemoglobin typing demonstrated that background fetal hemoglobin (HbF) protein levels were high at day 14 (817 52%) in mock-treated cells and were slightly increased in HBB-edited SCD-CB CD34+ cells (901 44%) compared with mock-treated cells (P = 010) (Fig 2C, D). In addition, a WT adult hemoglobin (HbA) high-performance liquid chromatography (HPLC) peak occurred only in HBB-edited SCD-CB CD34+ cells and averaged 67 26%, with correction levels up to 83% observed (P = 001). Correspondingly, sickle hemoglobin (HbS) levels were significantly decreased in HBB-edited SCD-CB CD34+ cells (32 18%) compared with mock-treated cells (183 52%: P = 0009). Next, we assessed the efficiency of CRISPR/Cas9 editing of HBB at the sickle mutation in CD34+ cells isolated from fresh peripheral blood collected from adult SCD patients. Like SCD-CB CD34+ cells, we found no differences in erythroid cell differentiation in HBB-edited SCD-adult CD34+ cells at day 14 compared with mock-treated cells (Fig 1AD). No significant change was detected in the CD71GPA+ phenotype in HBB-edited SCD-adult CD34+ cells (125 33%) compared with mock-treated cells (122 24%) (P = 089). Indel frequency and HDR rate were 519 131% and 198 63%, respectively, in HBB-edited SCD-adult CD34+ cells by TIDE analysis; consistently, amplicon deep sequencing showed 509 104% indels and 150 87% HDR (Fig 2A). We observed near-background off-target activity of the sgRNA at ITGA9 (428 31% indels), chr2: 42851054-42851076 (001 004% indels), and HBD (0003 002% indels) in HBB-edited SCD-adult CD34+ cells (Fig 2B). Haemoglobin typing of HBB-edited SCD-adult CD34+ cell lysates showed significantly substantial levels of HbA (128 65%) and a concomitant decrease in HbS (241 46%) compared with mock-treated cells (00 00% HbA, P = 003; 778 23% HbS, P = 000006; Fig 2C, D). HbF levels were also significantly increased in HBB-edited SCD-adult CD34+ cells (541 33%) compared with mock-treated cells (160 30%) (P = 00001), while HbA2 levels were not significant difference between mock-treated cells (62 14%) and HBB-edited SCD-adult CD34+ cells (89 09%) (P = 006). These results demonstrate efficient in vitro editing of SCD-CB and SCD-adult CD34+ cells using the CRISPR/Cas9 technology. Corrected SCD CD34+ cells from both blood sources showed a significant increase in HbA, indicating that the gene correction led to the functional conversion of the S allele to A allele and a corresponding decrease in HbS levels. Indels within HBB may cause the HbF enhancement observed in HBB-edited CD34+ cells. These indels may be involved in the selective expansion of HbF-expressing cells during differentiation or stimulate upregulation of HbF within cells. Therefore, the molecular basis by which alterations at the -globin locus cause changes in HbF abundance requires further investigation. In conclusion, our studies establish frozen CB as an alternative source of SCD CD34+ HSPCs for CRISPR/Cas9 correction of the sickle mutation.