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A. Dong et al.
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Figure 1. Cell viability and differentiation rate in patients’ erythroblasts. (A) Percentage of live/dead cells counted by trypan blue assay. (B) Number of hemoglo- binized cells (benzidine-positive count).
previously reported in other studies based on the use of 2'- MOE-SSO.28 We observed no statistical difference in via- bility or cell differentiation in the majority of treatments at the aforementioned treatment dosages (Figure 1).
MOE-SSO 91 (Figure 3C), in specimen P1, with up to 54% decrease. In specimen P2, a significant reduction in aggre- gates was seen starting at a 25 mM dose. Although baseline levels of α-heme aggregates in P3 and P4 were lower, 2'- MOE-SSO 91 and 93 still elicited a significant α-heme aggregate reduction at a 50 mM dose. Consistent with our previous observations, 2'-MOE-SSO 91 was the most effective, with as high as an 87% reduction in α-heme aggregates (specimen P3).
We further compared the potency of 2'-MOE-SSO treat- ment to the effect of a lentiviral vector, AnkCT9W, carry- ing a WT copy of the HBB gene in IVS2-745/b0 heterozy- gous specimens (P1). At an average VCN of 1.13, erythrob- lasts transduced with this vector induced an increase of HbA to 50% (Figure 4), which was comparable to the effect obtained using 2'-MOE-SSO 91 at the concentration of 50 mM. This indicates that the 2'-MOE-SSO dose used for our assessment can generate an effect that is compara- ble to that of a safe single copy gene addition approach.
2'-MOE-SSO show the most robust effects on cells from homozygous patients with an IVS2-745/IVS2-745 genotype
After demonstrating a dose-dependency in response to 2’-MOE-SSO treatment, we next sought to determine if there was an “allele dose-dependency”, e.g., we investigat- ed whether treatment with 2'-MOE-SSO had a higher impact on a homozygous sample with two 745 target alle-
Despite the presence of detectable levels of WT HBB mRNA, the amount of HbA measured by HPLC was rela- tively low in untreated specimens (Figure 2A). Upon 2'- MOE-SSO treatment, HbA levels were increased and a significant reduction of α-heme aggregates was observed. Once again, treatment with 2'-MOE-SSO 91 induced the most robust outcome. At the 5 mM dose, 2'-MOE-SSO 91 induced HbA increase from baseline levels of 2.60±0.17% and 4.65±0.46% to 29.21±2.16% and 27.94±1.09%, in specimens P1 and P2, respectively. At the 50 mM dose, 2'- MOE-SSO 91 induced an HbA increase from baseline lev- els of 4.20±0.75% and 2.97±0.23% HbA to 59.14±1.34% and 60.21±2.61%, in specimens P3 and P4, respectively, indicating a corresponding 15- and 20-fold increase in HbA. Q-PCR analyses of the mRNA obtained from the same specimens show a similar trend (Figure 2B). In these, the amount of correctly spliced WT HBB mRNA increased up to 100-fold, relative to baseline levels, at the 50 mM dose of 2'-MOE-SSO 91.
The baseline α-heme aggregate26 level in heterozygous cells was variable across samples, with means ranging from 4.28±1.12%, in P4, to 22.00±0.76%, in P1 (Figures 3A and B). We detected a significant reduction in aggregates across all 2'-MOE-SSO treatments, more pronounced for 2'-
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haematologica | 2021; 106(5)