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Letters to the Editor
(Figure 3G). We confirmed that the oligonucleotides (Online Supplementary Table S1D) designed to amplify the Silb-globin gene amplified the transgenic Silb-globin gene exclusively (Online Supplementary Figure S1F). Using a murine erythroleukemia cell line (MEL), we confirmed a linear relationship between VCN, chimeric HbA (mouse α-globin/human b-globin-T87Q tetramer; Online
A
Supplementary Figure S1G), protein levels (Online Supplementary Figure S1H), and human b-globin RNA (Online Supplementary Figure S1I) produced upon inte- gration of ALS10-T87Q, ALS10-T87Q-silent, ATM1.1 and ATM1.1-silent. Furthermore, in a pilot experiment on CD34+-derived SCD erythroblasts treated with ALS10-T87Q, ALS10-T87Q-silent, ATM1.1 and
BCD
E
F
Figure 2. Optimization of BCL11A miRNA design. (A) Illustration of changes introduced into BCL11A miRNA sequence of ATM1.1 vector. ATM1.2 contains 1 basepair (bp) mismatch between the guide and the passenger creating a ”bulge” in our design, based on evidence from Fellman’s group, which demonstrated that inclusion of such an element can boost the potency of the miRNA.11 ATM1.3 presents a 2 bp modification of the published miR-30 loop to improve perform- ance.11 The modifications tested in ATM1.1, -1.2 and -1.3 were combined in the final design of ATM1.4. (B) Adult hemoglobin (HbAT87Q) (C) fetal hemoglobin (HbF) and (D) HbAT87Q+HbF levels at low (0.6+/-0.1, top panels) or high (2.0+/-0.3, bottom panels) integrations per cell after transduction of M#9 with ALS10-T87Q, ATM1, and its variants ATM1.1, ATM1.2, ATM1.3, or ATM1.4. (E) Graphic representation of the proportion of curative HbAT87Q+HbF hemoglobin over a range vector copy number (VCN) after transduction of M#9 with ALS10-T87Q, ATM1, ATM1.1, ATM1.2, ATM1.3, or ATM1.4. Data are represented as mean ± standard devia- tion, n=3. (F) Representative western blot showing side by side the HBA, HBB, HBG and BCL11A expression in control and different vectors transduced M#9 cells at day 7 of differentiation.
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haematologica | 2021; 106(10)