Volume 26, Issue 2, Pages 634-647 (February 2018) The Impact of the CD9 Tetraspanin on Lentivirus Infectivity and Exosome Secretion  Kai O. Böker, Nicolas Lemus-Diaz, Rafael Rinaldi Ferreira, Lara Schiller, Stefan Schneider, Jens Gruber  Molecular Therapy  Volume 26, Issue 2, Pages 634-647 (February 2018) DOI: 10.1016/j.ymthe.2017.11.008 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Overexpression of Exosomal Marker Proteins CD9, Alix, and TSG101 in HEK293 Cells Affects Vesicle Amounts and Sizes (A) qPCR analyses were performed and displayed a 4- to 22-fold higher expression of the respective transgenes. Nanoparticle tracking analysis of extracellular vesicles showed significantly enlarged particles for HEK293-TSG101 and HEK293-Alix compared to wild-type. (B) The overexpression of CD9 led to a decreased average size of secreted vesicles. (C) Total extracellular vesicle amount was decreased upon TSG101 and Alix overexpression, but the extracellular vesicle amount was significantly increased upon CD9 overexpression. (D) For vesicles within the size range of exosomes (30–100 nm), these trends were even more obvious. Error bars represent SD. Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 CD9 Locates to the Plasma Membrane and Cell-Cell Contact Surfaces in Stably CD9-GFP-Expressing Cell Lines Cell membrane localization of CD9-GFP was observed via fluorescence microscopy. Green fluorescence was detected after at least 2 weeks of Blasticidin selection (scale bar, 100 μm). Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Lentivirus Production and Comparison between Exosomes and LVs (A) HEK293FT or HEK293FT-CD9 cells were transfected with three different plasmids encoding for envelope glycoproteins (i.e., CD9 and/or VSVG), viral capsid proteins, and the gene of interest, here, an RFP. LVs are shed from the cell membrane, and LVs produced in HEK293FT-CD9 cells carry additional CD9-GFP and/or VSVG within their envelope. (B) The three different LVs used in this study, i.e., exposing VSVG, CD9, or both VSVG and CD9, are schematically shown. Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 Production Capacity and Transduction Efficiency of LV-VSVG and LV-VSVG-CD9 (A and B) Comparative analysis of three lentiviral productions revealed only minor differences in titer concentrations for LV-VSVG, LV-VSVG-CD9GFP, and LV-CD9GFP demonstrated by ELISA (A) and qPCR (B). Comparison of transduction efficiency was evaluated with LV-VSVG as a standard control and LV-VSVG-CD9GF on HEK293, HeLa, and SH-SY5Y cells. (C) Successful transduction was confirmed via fluorescence microscopy in regular intervals from 20 to 108 hr after transduction. (D) In fluorescence microscopy analysis, positive cells were quantified by manual counting and the transduction efficiency was calculated. (E) Comparing equal amounts of LV-VSVG and LV-VSVG-CD9 revealed an increased efficiency for LV-VSVG-CD9 over a broad MOI range (30–300 MOI). (F) Density plot representation of lentiviral efficiency at equal MOI (150) through FACS analysis. Scale bars, 200 μm. Error bars represent SD. Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 CD9 Mediates Fusion with Target Cell Membrane (A) HEK293 cells were transduced with CD9, VSVG, and VSVG-CD9 LVs. All tested LVs show expression after 72 hr. The highest efficiency was observed with LV-VSVG-CD9GFP, followed by LV-VSVG, but the virus without any viral glycoprotein (LV-CD9GFP) also successfully infected a minor proportion of cells. (B) Negative control without viral envelope proteins or CD9GFP shows no transduction. Scale bars, 100 μm (A) and 200 μm (B). Exposure time: 1,000 ms. Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 Density Gradient Centrifugation Separates Exosomes and Lentiviral Particles (A) Lentiviral supernatant was fractionated by centrifugation through a gradient of iodixanol to separate lentiviral particles and exosomes. (B) Western blot analysis revealed high concentrations of CD9-GFP and GFP in fraction 6+7 and 10+11, whereas Alix showed an equal distribution, indicating a successful separation of exosomes (fraction 6+7) and lentiviruses (fraction 10+11). (C) qPCR titer measurements clarified the presence of lentiviral particles in fractions 9–11. (D) Comparative analysis of LV-VSVG and LV-VSVG-CD9 with similar MOIs displayed no enhanced infectivity for fractions 8 and 11, whereas in fractions 9 and 11, increased transduction rates were observed for LV-VSVG-CD9 lentiviral constructs on HEK293 cells. Error bars represent SD. Molecular Therapy 2018 26, 634-647DOI: (10.1016/j.ymthe.2017.11.008) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions