Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps

doi: 10.1016/j.omtm.2022.01.008

Figure Lengend Snippet: P5-associated rAAV contaminants can be translated and immunogenic after infection (A) Bar graph depicting transcriptional activity of forward and reverse direction of AAV2 P5 promoter and outside of AAV2ITRs after plasmid transfection of 293T cells. GFP cassette positioned relative to P5: upstream (blue), downstream (orange), upstream but reverse orientation (green), no GFP cassette (red). GFP upstream of AAV2 ITR: wild-type (gray), self-complementary (purple). Error bars indicate SEM. (B) Bar chart showing quantification of GFP-expressing 293T cells by FACS 72 h after transduction with AAV8 GFP_P5 F8 (red), AAV8 RevGFP_P5 F8 (orange), or AAV8 Empty_P5 F8 (blue) (n = 3). Error bars indicate SEM. (C) Time course graph depicting detectable GFP-positive cells in post-infection samples (5 × 10 6 MOI) with AAV8 GFP_P5 F8 (red), AAV8 RevGFP_P5 F8 (orange), or AAV8 Empty_P5 F8 (blue). Error bars indicate SEM. (D) Immunohistochemistry (IHC) images of C57BL/6J mouse liver sections stained for GFP protein 1 week after 9.4 × 10 11 vg infection with AAV8 GFP_P5 F8 (top left), AAV8 RevGFP_P5 F8 (top right), AAV8 Empty_P5 F8 (bottom left), or 2 × 10 10 vg AAV8-GFP (bottom right) (n = 5). (E) Representative FACS plots and bar chart depicting GFP reactive tetramer positive splenic CD8 + T cells from BALB/cJ mice 9 days after 1 × 10 12 vg infection with AAV8 GFP_P5 F8 or AAV8 Empty_P5 F8 (nonparametric t test n = 5, p = 0.0079). Error bars indicate SEM.

Article Snippet: Before transfection, adherent human embryonic kidney 293T cells (ATCC CRL-3216) were cultured in DMEM (Lonza Bio Whittaker Catalog #12-733Q) supplemented with 10% fetal bovine serum (Fisher Scientific Catalog #SH3007103) and 2 mM L-glutamine (Corning Catalog #MT25005CI) at 37°C 10% CO 2 .

Techniques: Infection, Activity Assay, Plasmid Preparation, Transfection, Expressing, Transduction, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

doi: 10.1101/2024.06.14.599118

Figure Lengend Snippet: ( A ) Live cell count, ( B ) cell viability and ( C ) AAV9 capsid titer was measured for 10 days after triple transfection in 293F and AC1P. n = 3 replicates per sample. Data are shown as mean ± SD. ( C ) Representative single-cell derived clonal colony in a 96-well plate. Dashed lines show colony boundary at 3 (green), 5 (purple), 7 (pink), and 10 (blue) days after single-cell seeding. Scale bar, 1000 µm. ( D ) Cell viability and ( F ) live cell count of top 24 clones was measured during the suspension adaptation phase. After 9 days of suspension adaptation, the top 12 clones (based on viability and cell count) were selected and progressed to 125mL shake flasks.

Article Snippet: Adherent polyclonal HEK293 cells (“AC1P” for short) (Cytion) were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 37°C, 5% CO 2 incubator.

Techniques: Cell Counting, Transfection, Derivative Assay, Clone Assay, Suspension

Journal: bioRxiv

Article Title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

doi: 10.1101/2024.06.14.599118

Figure Lengend Snippet: ( A ) Schematic of workflow for single-cell cloning and isolation of HEK293 clones with improved AAV production capacity. The Solentim VIPS™ single cell seeder was used to isolate clonal HEK293 cells using a two-step clonality verification process. ( B ) Subclone selection strategy for identification of top AAV9 producer clonal cells. ( C ) Approximately 300 single-cell derived HEK293 clones were identified based on growth, and top 24 performing colonies were selected based on transfection efficiency and AAV9 production. ( D ) After suspension adaptation, AAV productivity of the best suspension adapted clones was measured relative to AC1P (parental polyclonal cell line). n = 1–2 replicates per cell line. Data are shown as mean ± SD fold-change from AC1P (control).

Article Snippet: Adherent polyclonal HEK293 cells (“AC1P” for short) (Cytion) were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 37°C, 5% CO 2 incubator.

Techniques: Clone Assay, Isolation, Selection, Derivative Assay, Transfection, Suspension, Control

Journal: bioRxiv

Article Title: Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

doi: 10.1101/2024.06.14.599118

Figure Lengend Snippet: ( A ) Workflow and experimental design for RNA-sequencing analysis during AAV production. 293F, AC1P (polyclonal cell line) and 3 clonally derived cell lines from AC1P were used for the analysis. Samples were collected before transfection (0 hr) and at 24 and 72 hr after triple-plasmid transfection. 3 small-scale shake flasks were used per condition. ( B ) AAV9 vector genome titer was measured at 72 hr from the 5 cell lines. 293F and AC1P cells were designated as “base producers” with an average titer of ∼2.4E9 vg/mL. AC112 and AC230 were designated as high producers with an average titer of ∼1.1E10 vg/mL. n = 3 replicates per cell line. Data are shown as mean ± SD. ( C ) Uniform manifold approximation and projection (UMAP) and ( D ) gene correlation matrix of the 5 HEK293 cell lines show high reproducibility among replicates. RNA expression differences between cell lines are greater drivers of variability than differences between timepoints after transfection. ( E ) Heatmap of differentially enriched genes and functional categories in higher AAV producer cells (AC230 and AC122) compared to baseline producer cells (AC1P and 293F). Differentially enriched transcripts were manually categorized into cell cycle modulators, cell signaling, Extracellular matrix / adhesion, Ion transport, mitochondrial, heat shock protein, and cell fate and differentiation.

Article Snippet: Adherent polyclonal HEK293 cells (“AC1P” for short) (Cytion) were expanded in DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 37°C, 5% CO 2 incubator.

Techniques: RNA Sequencing Assay, Derivative Assay, Transfection, Plasmid Preparation, RNA Expression, Functional Assay

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: TEM8 co-localizes with endosome markers and associates to transferrin receptor-containing protein complexes. A Immunofluorescence staining for TEM8 (HA) demonstrating co-localization with Alexa 488 labeled human transferrin (TF), with TFR- and Rab11a- but not Rab4-positive intracellular compartments. To label intracellular compartments with transferrin, serum deprived Hek298 cells were incubated with 50 μg/ml Alexa 488-labeled human transferrin for 1h prior to fixation. Size bar is 10μm. B Quantification of co-localization of the endosomal markers TF, TFR, Rab11 and Rab4 with TEM8. The number of analyzed cells is indicated at the bottom of the bar. P value was determined with a t-test C Detection of TEM8-associated to multiprotein complexes containing transferrin receptor after chemical cross-linking using DSP in TEM8 expressing Hek293 cells. Cells were treated in the absence (odd lanes) or presence of DSP (even lanes). Detergent extracts were immunoprecipitated with magnetic beads alone (lanes 1-2) or beads decorated with TFR antibodies (lanes 3-4). Immune complexes were analyzed by immunoblot to detect TEM8, TFR and actin. Inputs represent 5%. 1 of 8 independent experiments is shown. D Co-immunoprecipitation of TEM8 with transferrin receptor after chemical cross-linking in Hek293 cells treated with vehicle or with 2μM monensin for the indicated time. The experimental design is similar to C, except that cells were treated in the absence or presence of monensin (2μM) at 37°C before incubation with DMSO vehicle control (DSP- lanes) or DSP (DSP + lanes). 1 of 3 experiments is shown.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Immunofluorescence, Staining, Labeling, Incubation, Expressing, Immunoprecipitation, Magnetic Beads, Western Blot

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: PA addition does not affect TEM8 recycling and induces receptor degradation. A TEM8-expressing Hek293 cells were surface biotinylated at 4°C with 0.5 mM EZ-Link Sulfo-NHS-Biotin. Unbound biotin was washed and cells were shifted for the indicated times to 37°C in the absence (PA-) or presence (PA+) of 1μg/ml PA. Cells were incubated with GSH to remove remaining surface biotin, washed and lysed at 4°C. Biotinylated proteins were isolated with streptavidin conjugated agarose beads and bound complexes resolved by SDS-PAGE and analyzed by Western Blot with antibodies against TEM8 (1 of 2 experiments). B Graphic representation of the quantification of the experiment shown in A. C TEM8 expressing Hek293 cells were treated or not with 1μg/ml PA for 30min. Cell were washed and treated at 4°C with DMSO vehicle control (DSP- lanes) or with DSP (DSP+ lanes). Detergent extracts were immunoprecipitated with magnetic beads decorated with LAMP1 antibodies or beads decorated with TFR antibodies. Immune complexes were analyzed by immunoblot to detect TEM8, TFR and LAMP1. Inputs represent 5%. 1 of 3 independent experiments is shown. D TEM8 expressing Hek293 cells were labeled overnight with 100μCi/ml [S35]-methionine and chased in media containing cold methionine either in the absence or presence of 1μg/ml PA for the indicated times. Chase was ended by cell lysis and TEM8 and TFR were immunoprecipitated. Radiolabeled immunocomplexes were resolved by SDS-PAGE and radioactive bands detected by fluorography.1 of 3 independent experiments is shown.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Expressing, Incubation, Isolation, SDS Page, Western Blot, Immunoprecipitation, Magnetic Beads, Labeling, Lysis

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: Recycling of cell surface labeled TEM8. Cell surface proteins in TEM8 expressing Hek293 cells were surface labeled by biotinylation at 4°C with 0.5 mM EZ-Link Sulfo-NHS-Biotin. Protein trafficking was allowed by incubating the cells at 16°C or 37°C for the indicated times. Cells were returned to 4°C to remove cell surface biotin by two GSH washes (GSH+ lanes) or were maintained in buffer in the absence of GSH (GSH- lanes). Proteins remaining biotinylated were recovered by streaptavidin-agarose beads precipitation from cell detergent lysates. Proteins associated to streptavidin beads were analyzed by western blot and probed for HA and epidermal growth factor receptor (EGFR).

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Labeling, Expressing, Western Blot

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: TEM8 and transferrin receptor distribution is altered by agents that perturb recycling endosomes. TEM8 expressing Hek293 cells were transfected with wild type EGFP-Rab11a, dominant negative EGFP-Rab11S25N, or a dominant negative EGFP-Myosin Vb tail construct. Fixed cells were double stained with antibodies against GFP and either HA to detect TEM8 or transferrin receptor. Size bar is 10μm.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Expressing, Transfection, Dominant Negative Mutation, Construct, Staining

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: Receptor recycling leads to increased PA accumulation, but not to ligand targeting to recycling endosomes. A Specific binding of FITC labeled PA63 to non-transfected or TEM8 expressing Hek293 cells at 4°C or 22°C for 60 min. B FITC-PA63 binding to TEM8 expressing Hek293 cells untreated or pre-incubated for 15 min with 2μM monensin or for 60 min with 10μg/ml with cycloheximide respectively (1 of 4 experiments). C Immunoflurescence staining with anti FITC and Alexa 555 secondary antibody in TEM8-Hek293 cells transfected with the indicated EGFP constructs. Cell were fixed after a 1h incubation with 1μg/ml FITC-PA63 at 37°C. One of two experiments is shown. Size bar is 10μm.

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Binding Assay, Labeling, Transfection, Expressing, Incubation, Staining, Construct

Journal:

Article Title: Endosomal Recycling Regulates Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8-Dependent Cell Spreading

doi: 10.1016/j.yexcr.2010.03.026

Figure Lengend Snippet: Myosin Vb tail does not affect lethal toxin delivery to the cytosol, but interferes with TEM8-mediated cell spreading on PA coated surfaces. A Control or TEM8 expressing Hek293 either transfected with GFP or EGFP-Myosin Vb tail were incubated in the absence or presence of Lethal Toxin (1ug/ml PA, 0.2ug/ml Lethal Factor) for the indicated time. Cell intoxication was assessed by Western Blot analysis for N-terminus MEK-1 cleavage. B EGFP-Myosin Vb tail transfected TEM8-Hek293 cells were plated on PA or Matrigel (MA) coated glass coverslips and stained with Alexa labeled phalloidin to score for spreading area and GFP expression in the same cell. Size bar is 10μm. C Box plot graph shows the spreading area distribution of 30 cells from each indicated condition. The middle line in each condition represents the mean, the boxes represent the distribution of 50% of the cells, the lines represent the maximum and minimum values and the white dots represent outliers as determined by the graphing software. p value was determined using a t-test. D TEM8 expressing Hek293 cells either transfected with GFP or EGFP-Myosin Vb tail were plated on 0.1%BSA or 10μg/ml PA coated dishes in media containing 0.5 mM EZ-Link Sulfo-NHS-Biotin for 60 minutes at 37°C. Cells were washed and detergent soluble extracts were incubated with streptavidin conjugated agarose beads and bound complexes resolved by SDS-PAGE. Biotinylated proteins were analyzed by Western Blot using antibodies against TEM8 and EGF receptor (EGFR).

Article Snippet: Cell lines & Transfections Hek293 cells (adherent cell strain) were obtained from QBiogene and cultivated in 10%FBS DME, 100U/ml penicillin and 100μg/ml streptomycin in 10%CO 2 .

Techniques: Expressing, Transfection, Incubation, Western Blot, Staining, Labeling, Software, SDS Page