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A. Dong et al.
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Figure 3. Representative reverse-phase high performance liquid chromatography profile of the separation of hemoglobins in IVS2-745/β0 heterozygous sample treated with 2'-MOE-SSO 91. (A) Presence of α-heme aggregates and low levels of adult hemoglobin (HbA) detected in untreated cells from P1. (B) After treatment with 2'-MOE-SSO 91 the production of HbA was increased 11-fold and α-heme aggregates were reduced 2-fold. Fetal and HbA2 levels were unaltered. (C) Percentage of α-heme aggregates detected by reverse-phase high performance liquid chromatography in treated samples. All statistics (ANOVA/Kruskal-Wallis) are compared to scramble control. *P<0.1; **P<0.01; ***P<0.001; ****P<0.0001.
correction on both heterozygous and homozygous speci- mens for the IVS2-745 mutation. We chose the 2'-MOE- SSO 91 at the 50 mM dose because of its higher perform- ance and the plateauing effect on HbA increase observed in previous experiments, based on Figures 5 and 6.
Treatment with 2'-MOE-SSO 91 in P4, derived from a compound heterozygous patient with an b-IVS2-745/b0 genotype, showed a 19.3-fold increase in WT HBB mRNA, when normalized by b-actin expression (Figure 7A), and a 11-fold increase in WT HBB mRNA, when normalized by the b-IVS2-745 mRNA expression (Online Supplementary Figure S6). The proportion of WT HBB increase was more dramatic in cells from a homozygous patient with an IVS2- 745/IVS2-745 genotype (Figure 7B). Treatment of specimen P5 with 2'-MOE-SSO 91 resulted in a >800-fold increase in WT HBB mRNA. This improvement in the efficiency of treatment with 2'-MOE-SSO 91 confirmed data previously obtained via Q-PCR and HPLC, and showed how the allele dose-dependency is much more evident when detected with a direct quantification method. Using ddPCR we con- firmed that untreated and scrambled-treated presented similar levels of WT HBB expression, indicating that the use of scramble 2'-MOE-SSO did not alter the splicing of the HBB gene in our specimens.
2'-MOE-SSO prevents sickling in samples with an IVS2-745/β-S genotype
Lastly, we investigated the effect of these 2'-MOE-SSO on cells with the IVS2-745 HBB mutation in combination
Figure 4. Comparison of the efficacy of adult hemoglobin production between 2'-MOE-SSO 91 and lentiviral transduction in IVS2-745/b0 heterozygous sam- ple. Percentage of adult hemoglobin (HbA) detected by reverse-phase high per- formance liquid chromatography in control and after treatment. Vector copy number determined via quatitative polymerase chain reaction. All statistical comparisons (ANOVA/Kruskal-Wallis) are tested against scramble control; n=3. ns=not significant, **P<0.01, ****P<0.0001.
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haematologica | 2021; 106(5)