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. 2017 Aug 23;9(11):1558–1573. doi: 10.15252/emmm.201708148

Figure 2. Evaluation of LV produced by the producer cell lines.

Figure 2

  • A, B
    LV infectious titer (TU/ml, black bars or line, plotted on left y‐axis), physical particles (ng p24/ml, dashed bars or line, plotted on right y‐axis), and specific infectivity (TU/ng p24, gray bars or line, plotted on left y‐axis) in conditioned medium of (A) bulk GFP‐positive (+) sorted populations and single‐cell clones obtained from three independent T.I. experiments performed with the indicated donor DNA, 3 days after dox induction, or (B) at the indicated time (months) of continuous culture in the absence of antibiotic selective pressure. Axis interruption indicates a freeze/thaw cycle. The n of independent inductions of LV production from bulk‐sorted populations (+) or single‐cell clones is shown on top of the bars in panel (A), when not 1.
  • C–F
    Percentage of GFP‐positive cells (C, D) and VCN (E, F) in the CD34‐positive cells culture (C, E) or pooled colonies (D, F) from CFC assays (MOI 10 and 100, n = 4 transductions in 2 independent experiments using three different LV batches per production method; MOI 300, n = 2 transductions). HSPC (n = 4 healthy cord blood donors) were transduced with LV produced by transient transfection (“transfection”, white squares) or by LV‐GFP producer cell line (“cell line”, black circles) at the indicated MOI and analyzed at 7 (in C) or 14 (in D–F) days after transduction.
  • G, H
    Percentage of GFP‐positive cells (G) and VCN (H) in T lymphocytes transduced and analyzed as in (A–D) (“cell line”, black circles, n = 4 transductions using two different LV batches; “transfection”, white squares, n = 2 transductions).
  • I, J
    Human FIX expression (% of normal) in the plasma over time (I) and VCN (J) in liver DNA of hemophilia B mice treated with LV‐FIX produced by transient transfection (n = 4, white squares) or from producer cell line (n = 10 using two different LV batches, black circles). No significant differences.
Data information: In (A–J), data are presented as mean with SEM (for n ≥ 3), mean with range (for n = 2), and/or single values. Significance was assessed by Mann–Whitney test in (C–F) and (J) or by two‐way ANOVA for repeated measures in (I).