Rapamycin increases in vitro LV transduction efficiency in human and mouse hematopoietic progenitors. (A) Scheme of human CD34⁺ and mouse Lin⁻ cell transduction and assessment. (B) Human cord blood CD34⁺ cells were transduced with CG-UbiC-EGFP LV in the presence of rapamycin or DMSO only as a diluent control. Black triangles, DMSO only; red circles, 10 μg/mL rapamycin. Cells were analyzed 10 to 14 days posttransduction. (C) Human cord blood CD34⁺ cells were transduced with CG-UbiC-EGFP, MOI = 25, in the presence of various concentrations of rapamycin or (D) Torin 1, an active site mTOR inhibitor. In most cases, data are pooled from 4 to 5 independent experiments, each with different cord blood donors and done in duplicate. At lower MOI or rapamycin concentrations, 2 different donors in duplicated experiments were assessed. (E) MFI of EGFP from a representative titration series in panel C. (F) Fold change over DMSO-treated controls in integrated LV copy number per cell, determined by qPCR, in CD34⁺ cells transduced in the presence of various concentrations of rapamycin and analyzed 10 to 14 days posttransduction. Data are pooled from 2 to 5 independent experiments, each with different cord blood donors. (G) EGFP cell marking and (H) fold change in integrated LV copy number in mouse bone marrow Lin⁻ cells transduced with RRL-MND-GFP at various MOIs in the absence (DMSO only) or presence of 1 or 5 μg/mL of rapamycin and analyzed 10 to 12 days posttransduction. Data shown are derived from 4 independent experiments comprising 48 donor animals. LV copy numbers are shown as fold change over the average LV copy number of the DMSO-treated controls. For all panel, lines represent group mean and error bars represent standard deviation. *P < .05, **P < .01, ***P < .001, and ****P < .0001 from a parametric 2-tailed unpaired Student t test. BM, bone marrow; hu, human; mu, murine; Rapa, rapamycin.