Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.omtm.2018.11.006

Figure Lengend Snippet: H/F LV Production in CD46 Null HEK293T Cells Is Non-cytotoxic and Does Not Form Syncytia Observed in Wild-Type HEK293T Cells (A) 10× bright-field (left) and 488-nm (right) fluorescence microscopy of HEK293T cells transiently co-transfected with packaging plasmids on day 0 to generate H/F LV. Limited syncytia formation was observed in wild-type HEK293T cells after 1 day, with major syncytia apparent by 2 days and cytotoxicity observed after 3 days when supernatant was collected. (B) CD46 null HEK293T cells transfected for LV generation and imaged as above. Cells form no observable syncytia throughout the 3-day culture period and continue to grow and proliferate without observable cell cytotoxicity. Representative images from n = 7 LV productions.

Article Snippet: Puromycin selected CRISPR/Cas9-transfected cells were stained for CD46 expression (CD46-APC, E4.3; BD Biosciences), then sorted for CD46-negative cells using a FACSAria IIU (BD Bioscience).

Techniques: Fluorescence, Microscopy, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.omtm.2018.11.006

Figure Lengend Snippet: H/F LV Purity and Titer Increased When Generated in CD46 Null HEK293T Cells, Compared with Wild-Type Producer Cells (A) Wild-type or CD46 null HEK293T cells were co-transfected with H/F LV plasmids, and cell viability (dotted lines) and total p24 protein production per day (solid lines) in supernatant were determined each day for 1 week after transfection. Data are presented as line graphs (mean ± SD p24 ng/mL or % viable cells). (B) Wild-type (blue) or CD46 null HEK293T cells (red) were transfected on day 0 to generate H/F LV, and producer cell viability was evaluated by propidium iodide (PI) exclusion using flow cytometry 3 days post-transfection. Data are presented as a FACS plots from a representative experiment (n = 3). (C) Average functional (transducing units [TU]/mL) and physical (μg/mL) titer of vector preps (n = 7), measured by serial dilution flow cytometric assay of wild-type HEK293T cell transduction or by anti-p24 ELISA. (D) Comparison of average functional titer (TU/mL) of H/F LV produced in wild-type HEK293T cells, CD46 null HEK293T cells, and VSV-G LV, by serial transduction on indicated cell lines (n = 3). (E) Representative SDS-PAGE gel of H/F LV containing supernatant from wild-type and CD46 knockout HEK293T cells collected 24 (D1), 48 (D2), and 72 hr (D3) post-transfection. Collected supernatant was also concentrated by ultracentrifugation over 20% sucrose, before matching for total protein and visualization by Coomassie stain.

Article Snippet: Puromycin selected CRISPR/Cas9-transfected cells were stained for CD46 expression (CD46-APC, E4.3; BD Biosciences), then sorted for CD46-negative cells using a FACSAria IIU (BD Bioscience).

Techniques: Generated, Transfection, Flow Cytometry, Functional Assay, Plasmid Preparation, Serial Dilution, Transduction, Enzyme-linked Immunosorbent Assay, Produced, SDS Page, Knock-Out, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.omtm.2018.11.006

Figure Lengend Snippet: H/F LV Produced in CD46 Null Cells Has Increased Transduction Efficiency and Less Cytotoxicity Than LV Produced in Wild-Type Cells on CD34 + HSPCs (A) UCB-derived CD34 + cells were transduced with either H/F LV derived from WT (blue) or CD46 null HEK293T cells (red) or VSV-G LV (gray) at indicated MOI, and evaluated by flow cytometry for EGFP expression after 7 days. (B) Cell viability of mock or LV-treated UCB-derived CD34 + cells matched for MOI of VSV-G LV, or H/F LV derived from either WT or CD46 null HEK293T cells. CD34 + cell viability was calculated as percent of trypan blue excluding cells. (C) Vector copy number quantification of UCB-derived CD34 + HSPCS treated with specified vector at indicated MOI. (D) Comparison of EGFP expression 7 days after transduction in UCB-derived CD34 + HSPCs after treatment with VSV-G LV or CD46 null cell-produced H/F LV with vector matched for physical titer (p24 protein). All data presented in bar and line graphs show mean ± SD (n = 2, 2 donors, 2 vector lots). Statistical analysis using two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001. ns, not significant.

Article Snippet: Puromycin selected CRISPR/Cas9-transfected cells were stained for CD46 expression (CD46-APC, E4.3; BD Biosciences), then sorted for CD46-negative cells using a FACSAria IIU (BD Bioscience).

Techniques: Produced, Transduction, Derivative Assay, Flow Cytometry, Expressing, Plasmid Preparation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.omtm.2018.11.006

Figure Lengend Snippet: H/F LVs Demonstrate Distinct Cellular Trafficking Patterns and Improved Transduction Ability in Cytokine-Stimulated CD34 + HSPCs (A) Experimental workflow of transduction of UCB-derived CD34 + HSPCs by VSV-G or H/F LV. CD34 + cells were isolated and transduced as described in the . (B) Co-localization analysis of confocal images for EEA1 + and p24 + vesicles, at 15 min and 1 hr after vector addition. Data are presented as dot plots (mean ± SD). Pearson’s correlation coefficient was compared by Kruskal-Wallis test with Dunn’s multiple comparison correction; ***p < 0.0002; ****p < 0.0001. (C) UCB-derived CD34 + HSPCs were pre-treated for 4 hr with the indicated concentration of prostaglandin E 2 (PGE-2), EGA, or DMSO vehicle control, before 24-hr co-treatment with H/F (blue) or VSV-G LV (gray). Data are presented as bar graphs (mean ± SD). *p = 0.0332; ****p < 0.0001 by two-tailed Student’s t test. (D) Representative plots of UCB-derived CD34 + HSPCs (n = 3) freshly isolated (blue) or treated with stem cell transduction-promoting cytokines for 24 hr (red), then analyzed for CD150 or CD46 expression by flow cytometry. (E) Freshly isolated UCB-derived CD34 + HSPCs were cultured with stem-cell-promoting cytokines for 24 hr before transduction with VSV-G or H/F LV at the indicated MOI (n = 3 donors). 24 hr after LV addition, vector was removed and cells were cultured in cytokine-containing media, before analysis by flow cytometry 7 days later. (F) UCB-derived CD34 + HSPCs were freshly isolated, then immediately transduced with either VSV-G or H/F pseudotyped LV as above, without pre-stimulation with cytokines. 24 hr after LV addition, LV was removed and cells were cultured and evaluated as above (n = 2 donors). Data are presented as bar graphs (mean ± SD). Statistical analysis using unpaired Student’s t test; *p < 0.0332; **p < 0.0021.

Article Snippet: Puromycin selected CRISPR/Cas9-transfected cells were stained for CD46 expression (CD46-APC, E4.3; BD Biosciences), then sorted for CD46-negative cells using a FACSAria IIU (BD Bioscience).

Techniques: Transduction, Derivative Assay, Isolation, Plasmid Preparation, Concentration Assay, Two Tailed Test, Expressing, Flow Cytometry, Cell Culture

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells

doi: 10.1016/j.omtm.2018.11.006

Figure Lengend Snippet: H/F LV Generated in Wild-Type or CD46 Null HEK293T Shows Distinct Effects on the Early Endosome Compartment and Enters Cells with Improved Efficiency Compared with VSV-G LV (A) UCB-derived CD34 + HSPCs were treated for 15 min or 1 hr with VSV-G LV (MOI 15) or H/F LV generated in either WT or CD46 null HEK293T cells (MOI 5). 50 cells for each condition were imaged by confocal microscopy, quantifying (A) EEA1 + endosome number and (B) Gag p24 + intracellular vesicle number, (C) mean vesicle volume, and (D) mean vesicle staining intensity. Data are presented as dot plots (mean ± SD). Statistical analysis using Kruskal-Wallis test with Dunn’s multiple comparisons test; *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001. EEA1, early endosome antigen-1.

Article Snippet: Puromycin selected CRISPR/Cas9-transfected cells were stained for CD46 expression (CD46-APC, E4.3; BD Biosciences), then sorted for CD46-negative cells using a FACSAria IIU (BD Bioscience).

Techniques: Generated, Derivative Assay, Confocal Microscopy, Staining