Figure 1.

The LVβ gene therapy vector containing intronic miR30-shGFP reduces target gene expression in erythroid cells

(A) Schematic of the LVβ gene therapy vector, LentiGlobin BB305, modified to express the miR30-shRNA cassette. (B) Representative histograms of EGFP expression by MEL-βEGFP cells transduced with the BB305, LVβ-shSCR, and LVβ-shGFP vectors at equivalent MOIs. (C and D) Relative EGFP mRNA expression (C, n = 3) and β^A-T87Q-globin mRNA expression (D) in transduced MEL-βEGFP cells normalized to β-actin. Data are presented as mean ± SD of at least three separate experiments relative to untransduced (UT) cells following 10 days of DMSO induction. Significance was calculated by unpaired Student’s t test; ∗p ≤ 0.05, ∗∗∗p ≤ 0.001. (E) Flow cytometry analysis of individual MEL-βEGFP clones (n = 10) transduced with the BB305, LVβ-shSCR, and LVβ-shGFP vectors. Data represent median (lines), the 25^th and 75^th percentiles (boxes), and the 90^th and 10^th percentiles (error bars) (∗∗∗p ≤ 0.0001). (F–H) Correlation analysis of (F) shGFP expression to β-globin mRNA, (G) EGFP fluorescence to β-globin mRNA in LVβ-shGFP-transduced clones, and (H) EGFP MFI to β-globin mRNA in BB305-transduced (squares) and LVβ-shSCR-transduced (circles) clones following 10 days of DMSO induction. Correlation analysis was performed using the Spearman rank method. Scatterplot linear regression lines with 95% confidence intervals (dotted lines) are displayed for (F)–(H). A positive correlation between shGFP expression and β-globin mRNA expression in LVβ-shGFP clones was detected (n = 18, R = 0.864, p < 0.0001). A negative correlation between EGFP MFI and LV β-shGFP β-globin mRNA expression was detected (n = 18, R = −0.735, p < 0.0005). EGFP MFI and β-globin mRNA expression in BB305-transduced (squares) and LVβ-shSCR-transduced (circles) clones did not correlate (n = 16, R = 0.0751, p = 0.7811).