Table 1.
High levels of anti-sickling hemoglobin in erythroblasts derived from BM CD34+ cells from SCD patients transduced with V5m3-400 or βAS3-FB lentiviral vectors.
| Condition |
aCell Growth |
b(%) CD235+/Draq5− |
cVector Copy No. |
dmRNA Levels |
e(%) HbA/AS3 |
e(%) HbF |
f(%) Correction |
|---|---|---|---|---|---|---|---|
| HD CD34 | 82 ± 18 | 85 ± 4 | ND | ND | 83.6 ± 1.8 | 5.8 ± 0.1 | ND |
| SCD mock | 76 ± 21 | 86 ± 5 | ND | 0 | 4.9 ± 1.2 | 6.9 ± 0.4 | 0 |
| βAS3-FB | 83 ± 19 | 86 ± 5 | 1.1 ± 0.1 | 7.6 ± 1.9 | 18.9 ± 3.1 | 8.1 ± 1.3 | 21.5 ± 5.0 |
| V5m3-400 | 70 ± 13 | 88 ± 2 | 0.9 ± 0.2 | 7.3 ± 2.2 | 7.0 ± 2.2 | 20.8 ± 1.6 | 22.1 ± 7.7 |
Mean ± s.e.m (n=3 independent donors with matched controls) are reported for each characteristic except for % Corrected (mean ± s.d., n=2 independent donors with matched controls)
Fold-increase in viable cells from day of transduction to culture day 11 or 12
Erythroid differentiation was determined by flow cytometry analysis of bulk cell populations for enucleated RBC (CD235+/ Draq5−) on culture day 21
Genomic DNA was isolated from the bulk cell population on day 8 of culture and copy number determined by quantitative PCR and/or Southern blot analysis
Relative levels of βAS3- and γ-globin transcripts determined by droplet digital quantitative RT-PCR with normalization to α-globin before subtraction of results for SCD mock
% of adult hemoglobin (HbA) or HbAS3 and fetal hemoglobin (HbF) quantified by densitometry analysis of cellulose acetate Hb electrophoresis gel results or HPLC
% red blood cells that did not sickle when deoxygenated with sodium metabisulfite and normalized per vector copy using the formula: % correction = (% sickle RBC in SCD mock - % sickle RBC in vector treated sample)/vector copy number.
Abbreviaions: HD, healthy donor; SCD, sickle cell disease; ND, not determined.