Protein composition in the producer cellFigure 4. Generation, imaging, and evaluation of MHC-free LV. Western blot on protein extracts from LV batches created by B2M-positive (LV) or B2M-negative (MHC-free LV) cells. Representative photomicrographs of LV batches immunostained with anti-VSV.G or anti-MHC-I antibodies, as indicated and analyzed by electron microscopy. Scale bars: 100 nm. C Quantitative analysis (gold particles per virion) of LV (black squares) or MHC-free LV (orange squares) immunostained with anti-MHC-I antibodies (top rated panel, n = 57?3 virions per sample) or with anti-VSV.G antibodies (bottom panel, n = 35?8 virions per sample), or, as staining control, with no the major antibody (ctrl, black triangles), and analyzed by electron microscopy. D Flow cytometry analysis (contour plots with outliers) of LV packaging cell line untreated, CRISPR/Cas9 treated, B2M good or B2M adverse sorted as indicated, performed 1 month immediately after sorting. E, F Human Repair expression ( of standard) in the plasma (E) more than time and VCN (F) in liver DNA of hemophilia B mice treated with LV-FIX-Padua developed by transient transfection into B2M-positive (n = three, black squares or line) or B2M-negative (n = four, orange squares or line) 293T cells, or from B2M-negative producer cells (n = 4, orange circles or line). No significant variations. G, H Percentage of titer recovered, when compared with the no-serum manage (five independent assays performed in the indicated LV concentration) of transient transfection produced VSV.G- (G) or GP64- (H) pseudotyped LV (black lines) or MCH-free LV (orange lines) incubated with heat-inactivated (empty squares) or fresh complement-preserved (filled squares) human sera from allo-immunized men and women, hence containing antibodies against MHC-I (for VSV.G n = 1? per concentration, for GP64 n = two? per concentration). I, J Human peripheral blood-derived monocytes have been exposed to matched physical particle doses of VSV.G- (J, left panel) or GP64- (J, suitable panel) pseudotyped LV or MHC-free LV particles, co-cultured with T cells with the exact same blood donor, and activation of CD3-positive T cells was measured by interferon-c production (two independent assays with seven wholesome blood donors and two distinct packaging cell line-produced LV or MHC-free LV batches for VSV.G-pseudotyped LV; three independent assays with five wholesome blood donors for transient transfection made GP64-pseudotyped LV or MHC-free LV. Information facts: In (C, E ), information are presented as mean with SEM (for n 3), mean with variety (for n = two), and/or single values. Significance was assessed by Kruskal allis test with Dunn’s a number of comparison test in (C, F) or by two-way ANOVA for repeated measures (E) or by Wilcoxon matched pairs test (G, H, J). Source information are offered on line for this figure. A B?2017 The AuthorsEMBO Molecular Medicine Vol 9 No 11 EMBO Molecular MedicineAlloantigen-free lentiviral vectorsMichela Milani et alALV MHCfree LV MHC-I (43 kDa) p24 (24 kDa)BLV Anti-VSV.GLV Anti-MHC-IMHC-free LV Anti-MHC-IC50 Gold particles per virion 40 30 20 10LVAnti-MHC-Ip0.p=0.DUntreatedCRISPR/CasB2M+ sortedB2M- sortedMHCe -frV eLctrl80 Gold particles per DOTA-?NHS-?ester Antibody-drug Conjugate/ADC Related virionAnti-VSV.GB2M60 40 20LV MH fr CLVMHC-IEFeeGTiter recovery one hundred 75 50 20 ten 0VSV.G LVHTiter recoveryGP64 LV LVp=0.LVp=0.MHC-free LVMHC-free LV105 TU/mL105 TU/mLGP64 LVp=0.ILV or MHC-free LVJ500 IFN making CD3+ T cell per 106 400 300 200 100VSV.G LVp=0.1 2 3 four five 6monocytesautologous T cells+8 9 10 11T UN M L.
