Vector stability and the effects of sh1005/sh516 co-expression on cell viability and HSPC differentiation potential.

<p>IL-2/PHA-stimulated PBMCs (4×10⁵) were transduced with lentiviral vectors at MOI 0.6–1.0. <b>A.</b> Representative data showing EGFP expression in vector-transduced PBMCs over time. <b>B.</b> Vector-transduced cells were subjected to CytoTox-Glo^TM Cytotoxicity Assay (Promega) four and six days post-transduction. Relative cytotoxicities were calculated by dividing the dead cell count by the total cell count and normalizing to that of mock-transduced cells. 1 μM Staurosporine (STS) and U6 promoter-driven sh1005 expression (U6-sh1005) served as positive controls. Error bars: mean + SD. <b>C.</b> OAS1 mRNA expression relative to β-actin was assessed by qRT-PCR analysis of total RNA isolated from vector-transduced PBMCs. PBMCs harvested two days post-electroporation with 500 pg/μL poly(I:C) served as positive control. Values were normalized to those of mock-transduced cells. Error bars: mean + SD. <b>D.</b> Cytokine-pre-stimulated mPB-CD34⁺ cells were transduced with lentiviral vectors at MOI 10. HSPC differentiation potential was assessed by counting colony forming units produced from mock- and vector- transduced mPB-CD34⁺ cells plated on semi-solid methylcellulose plates one day after transduction. CFU-GEMM: granulocyte/erythrocyte/macrophage/megakaryocyte colony forming units. C/BFU-E: erythroid colony/burst forming units. CFU-GM: granulocyte/monocyte colony forming units. Error bars: range of values between duplicate samples. Representative data of three independent experiments.