Increased HbF levels in erythroblasts derived from β-thalassemic BM CD34+ cells transduced with the V5m3–400, BCL11A shRNA, or GG1-VP64 lentiviral vectors
| . | Donor 1 . | Donor 2 . | Donor 3 . | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| GFP . | GG1-VP64 . | V5m3–400 . | GFP . | GG1-VP64 . | Bcl11A shRNA . | V5m3–400 . | GFP . | GG1-VP64 . | Bcl11A shRNA . | V5m3–400 . | |
| Cell proliferation* | 265 | 77 | 186 | 172 | 75 | 87 | 150 | 193 | 80 | 80 | 187 |
| CD235+,† % | 93 | 91 | 94 | 95 | 83 | 86 | 89 | 78 | 77 | 70 | 80 |
| GFP+,‡ % | 68 | 46 | ND | 85 | 49 | 26 | ND | 76 | 35 | 29 | ND |
| CFU-C,§ % | ND | ND | 20 of 25 | ND | ND | ND | 22 of 24 | ND | ND | ND | 17 of 20 |
| Copy no. Southern‖ | 0.7 | 0.5 | 0.6 | 4.1 | 1.8 | 0.7 | 0.8 | 2 | 1.4 | 0.6 | 0.8 |
| HbF HPLC,¶ % | 38.3 | 63.1 | 61 | 26.7 | 56 | 45.3 | 61.9 | 24 | 46 | 33.4 | 46.2 |
| HbF densitometry,# % | 36.2 | 59.9 | 56.4 | 20.8 | 63 | 38 | 53.1 | 22.7 | 45.2 | 38.3 | 43.2 |
| . | Donor 1 . | Donor 2 . | Donor 3 . | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| GFP . | GG1-VP64 . | V5m3–400 . | GFP . | GG1-VP64 . | Bcl11A shRNA . | V5m3–400 . | GFP . | GG1-VP64 . | Bcl11A shRNA . | V5m3–400 . | |
| Cell proliferation* | 265 | 77 | 186 | 172 | 75 | 87 | 150 | 193 | 80 | 80 | 187 |
| CD235+,† % | 93 | 91 | 94 | 95 | 83 | 86 | 89 | 78 | 77 | 70 | 80 |
| GFP+,‡ % | 68 | 46 | ND | 85 | 49 | 26 | ND | 76 | 35 | 29 | ND |
| CFU-C,§ % | ND | ND | 20 of 25 | ND | ND | ND | 22 of 24 | ND | ND | ND | 17 of 20 |
| Copy no. Southern‖ | 0.7 | 0.5 | 0.6 | 4.1 | 1.8 | 0.7 | 0.8 | 2 | 1.4 | 0.6 | 0.8 |
| HbF HPLC,¶ % | 38.3 | 63.1 | 61 | 26.7 | 56 | 45.3 | 61.9 | 24 | 46 | 33.4 | 46.2 |
| HbF densitometry,# % | 36.2 | 59.9 | 56.4 | 20.8 | 63 | 38 | 53.1 | 22.7 | 45.2 | 38.3 | 43.2 |
ND indicates not done.
Fold increase in viable cell counts determined by trypan blue exclusion 11 days after lentiviral vector transduction.
Percentage CD235 (Glycophorin A)-positive cells in bulk population determined by flow cytometry on culture day 14.
Percentage of GFP+ fraction of bulk cell population determined by flow cytometry on culture day 13 or day 14.
PCR analysis for the presence of vector sequences in clonogenic progenitors grown in methylcellulose supplemented with cytokines.
Genomic DNA was isolated from the bulk cell population on day 8 of culture and copy number determined by quantitative PCR or Southern blot analysis.
Percentage of HbF production as determined by HPLC.
Percentage of HbF production determined by densitometry analysis of signal intensity for individual hemoglobin chains on cellulose acetate Hb electrophoresis gel images using the Un-Scan-IT software package v5.1 (Silk Scientific) followed by calculation using the formula HbF/(HbF + HbA).