Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy

doi: 10.1016/j.omtm.2019.11.005

Figure Lengend Snippet: Experimental Design in which Samples from Two Doses of rAAV2-REP1 Were Collected at Baseline and Over Time Using Three Replicate Loading Syringes (19G Needle) and Three Replicate Dosing Syringes (23G with 41G Teflon Tip) Kept at 4°C and 23°C, Respectively

Article Snippet: Positive controls were prepared using untransduced cell lysate spiked with a recombinant REP1 protein (fish His-REP1; Jena Biosciences, Jena, Germany).

Techniques: Injection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy

doi: 10.1016/j.omtm.2019.11.005

Figure Lengend Snippet: Titer of rAAV2-REP1 Vector Samples following Dilution and Passage through Surgical Devices at Several Time Points and Temperatures (A) Sample titers were determined by qPCR and plotted as individual values (in DRP/mL). Dotted lines mark the nominal titers for both high (1E+12 DRP/mL) and low doses (1E+11 DRP/mL). (B) Plot of the difference of the mean titer to baseline at each time point for all samples collected. Symbols represent mean of three replicates ± SD, except for 1E+12 DRP/mL, where only two replicates were considered. # Only two replicates were analyzed for high dose (1E+12 DRP/mL). A two-way ANOVA found BSS-diluted samples to have a significant low titer compared with both high-dose samples and low-dose samples prepared in formulation buffer (*0.01 < p < 0.0001; dilution and time points as factors; Dunnett’s multiple comparison test for the effect of the dilution within each time point; dosing syringe at 90+90 min time point excluded from analysis because of high CV).

Article Snippet: Positive controls were prepared using untransduced cell lysate spiked with a recombinant REP1 protein (fish His-REP1; Jena Biosciences, Jena, Germany).

Techniques: Plasmid Preparation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy

doi: 10.1016/j.omtm.2019.11.005

Figure Lengend Snippet: Biological Activity of rAAV2-REP1 Samples Containing PF68 following Passage through the Surgical Device (A) 293 cells were transduced with rAAV2-REP1 samples prepared in PF68 0.001% formulation buffer (high and low doses) at MOI 10,000 DRP/cell. Sample replicates for each condition (baseline, loading and dosing 90+90 min, syringe and injected) were run in parallel for both high dose (top panel) and low dose (bottom panel). Protein expression (human REP1 and β-actin) and biotin incorporation in RAB6A were detected in prenylation reaction products following SDS-PAGE and immunoblot analysis. Positive (+ve) control: untransduced cell lysate spiked with recombinant fish REP1 (25 nM). (B and C) Semi-quantification data (band density values normalized to actin as loading control) are plotted as percentage of difference to baseline for each dose; REP1 expression (B) and biotinylated-RAB6A (C). Symbols are mean of three replicates ± SD. A two-way ANOVA confirmed the levels of biotinylated Rab substrate did not vary significantly from baseline in either high- or low-dose samples (p > 0.5; dilution and time points as factors; Bonferroni’s multiple comparison test for the effect of the time points within each dilution).

Article Snippet: Positive controls were prepared using untransduced cell lysate spiked with a recombinant REP1 protein (fish His-REP1; Jena Biosciences, Jena, Germany).

Techniques: Activity Assay, Transduction, Injection, Expressing, SDS Page, Western Blot, Recombinant

Journal: eLife

Article Title: Recognition of galactose by a scaffold protein recruits a transcriptional activator for the GAL regulon induction in Candida albicans

doi: 10.7554/eLife.84155

Figure Lengend Snippet: ( A ) Cells of wild type, rep1 mutant or rep1 mutant carrying a wild-type copy of REP1 were diluted 10-fold and spotted onto YNB solid medium containing 25 mM of the indicated sugar. Photographs were taken after 40 hr of growth at 30 °C. ( B ) qRT-PCR analysis of galactose catabolic genes GAL1 and GAL10 upon galactose induction in the same strains shown in ( A ). Cells were grown in liquid SC medium with 2% galactose, glucose or glycerol for 2 hr at 30 °C for RNA extraction. ( C ) Domain structure of Rep1. The transcriptional activation domain (AD) is colored in blue, the DNA binding domain (BD) is colored in orange and the domain located in C-terminus of Rep1 is colored in green. rep1 mutant cells carrying a wild type copy of Rep1, Rep1truncationsAD, BD, C, as well as Rep1 ΔC ,Rep1 ΔAD or vector alone were serially diluted 10-fold and spotted onto YNB solid medium containing 2.5 mM galactose, glucose or glycerol. Graphs were taken after 40 hr of growth at 30 °C. aa: amino acids. ( D ) The AD domain is unnecessary for Rep1-mediated induction of GAL genes. qRT-PCR analysis of GAL1 and GAL10 upon galactose induction in the same strains shown in ( C ). ( A and C ) Representative images of three independent experiments are shown. ( B and D ) Data shown as means ± SD of three independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. Figure 1—source data 1. Raw xlsx files used for analysis of the dataset.

Article Snippet: Recombinant Rep1 ∆AD -His and its variants were incubated with 50 μl D–Galactose agarose beads (Thermo Scientific) overnight at 4 °C.

Techniques: Mutagenesis, Quantitative RT-PCR, RNA Extraction, Activation Assay, Binding Assay, Plasmid Preparation, Two Tailed Test

Journal: eLife

Article Title: Recognition of galactose by a scaffold protein recruits a transcriptional activator for the GAL regulon induction in Candida albicans

doi: 10.7554/eLife.84155

Figure Lengend Snippet: ( A ) Rep1 is constitutively presented at the GAL1-GAL10 promotor. Overnight culture of wild type cells carrying Rep1-Myc and untagged control was inoculated into SC medium containing 2% indicated sugar at 30 °C for 4 hr for the ChIP experiment. The enrichment is presented as a ratio of GAL1-GAL10 promotor IP (bound/input) over the control region ACT1 IP (bound/input) and is then normalized to the untagged strain. The ChIP data showed the average of three independent experiments with error bars representing the SD. ( B ) Structure model for Rep1 predicted by AlphaFold2. A galectin-like fold with the characteristic two twisted β-sheets separated by a cleft is shown in Rep1. ( C ) Rep1 ΔAD directly binds to galactose.Recombinant Rep1 ΔAD and its mutation in Y526 were incubated with Galactose-agarose or control beads at 4 °C overnight in the absence or presence of 100 mM galactose.The input and bound fractions were analysed on Western blots probed with His antibody. Representative blots of three independent experiments are shown. ( D ) Molecular docking of galactose onto Rep1 ΔAD . The highest-ranked docked galactose is shown in the ball-and-stick model, and the protein is shown with a transparent surface. The inset shows how galactose interacts with Rep1 ΔAD residues. The galactose and the Rep1 ΔAD residues are shown in ball-and-stick and stick models, respectively. Hydrogen bond interactions are shown in dashed lines. ( E ) ITC binding curves using recombinant Rep1 ΔAD orits Y526A mutation and galactose (n=3; mean ± SD). ( F ) Disrupting the binding of Rep1 to galactose leads to a defect in galactose utilization in C. albicans. rep1 mutant cells carrying vector alone , Rep1 ΔAD or its Y526 mutationwere serially diluted 10-fold and spotted onto YNB solid medium containing 25 mM galactose or glucose. Graphs were taken after 40 hr of growth at 30 °C. Representative images of three independent experiments are shown. ( G ) qRT-PCR analysis of GAL1 and GAL10 upon galactose induction in the same strains shown in ( F ). A & G, Data shown as means ± SD of three independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. Figure 2—source data 1. Raw xlsx files used for analysis of the dataset. Figure 2—source data 2. Raw Western blot images with labeled band of interest.

Article Snippet: Recombinant Rep1 ∆AD -His and its variants were incubated with 50 μl D–Galactose agarose beads (Thermo Scientific) overnight at 4 °C.

Techniques: Recombinant, Mutagenesis, Incubation, Western Blot, Binding Assay, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test, Labeling

Journal: eLife

Article Title: Recognition of galactose by a scaffold protein recruits a transcriptional activator for the GAL regulon induction in Candida albicans

doi: 10.7554/eLife.84155

Figure Lengend Snippet: ( A ) Rep1 does not possess the ability to activate transcription, but Cga1 has it. Liquid β-galactosidase assays (in Miller units) of the indicated fusions. ( B ) A putative model for Rep1-mediated induction of the GAL genes is shown. Protein X represents a transcriptional activator. ( C ) Putative Rep1 interaction partners specifically in the presence of galactose identified by Mass Spectrometry. Data were obtained from three independent experiments, and the number of unique peptides from each experiment shown together with an average ion score. ( D ) Cells of indicated strains were diluted 10-fold and spotted onto YNB solid medium containing 25 mM of galactose, glucose or glycerol. Photographs were taken after 40 hr of growth at 30 °C. ( E ) Cga1 is essential for galactose-inducible expression of GAL genes. Data shown as means ± SD of three independent experiments.( F ) Rep1 interacts with Cga1 specifically in galactose in vivo. Overnight culture of wild type cells carrying Rep1-FLAG and Cga1-Myc or Rep1-FLAG alone was diluted 1:50 into SC medium with 2% glucose, galactose or glycerol at 30 °C for 4 hr. Protein lysates were subjected to immunoprecipitation with anti-Myc antibody, and theprecipitated proteins were probed with anti-FLAG antibody. As an inputcontrol, cell lysates were analysed by Western blotting with the anti-FLAGantibody. ( G ) Rep1 recruits Cga1 to the GAL gene promoter in a galactose-dependent manner. Cells of wild type or rep1 mutant strain carrying Cga1-Myc were diluted at 1:50 into SC medium containing indicated sugars at 30 °C for 4 hr. The enrichment is presented as a ratio of GAL1-GAL10 promotor IP (bound/input) over the control region ACT1 IP (bound/input) and is then normalized to the untagged strain.Data shown as means ± SD of three independent experiments. ( H ) Rep1 directly interacts with Cga1 in the presence of galactose. Recombinant GST-Cga1 and Rep1 ∆AD -His were incubated with Glutathione-agarose in the presence of glucose, galactose or glycerol at 4 °C for 2 hr. Samples were assayed by immunoblot with the anti-His antibodies. ( A, E and G ) Data shown as means ± SD of three independent experiments. Statistical analysis was performed using an unpaired two-tailed Student’s t -test. ( F and H ) Representative blots or images of three independent experiments are shown. Figure 3—source data 1. Raw xlsx files used for analysis of the dataset. Figure 3—source data 2. Raw Western blot images with the labeled band of interest. Figure 3—source data 3. The source file for the table.

Article Snippet: Recombinant Rep1 ∆AD -His and its variants were incubated with 50 μl D–Galactose agarose beads (Thermo Scientific) overnight at 4 °C.

Techniques: Mass Spectrometry, Expressing, In Vivo, Immunoprecipitation, Western Blot, Mutagenesis, Recombinant, Incubation, Two Tailed Test, Labeling

Journal: eLife

Article Title: Recognition of galactose by a scaffold protein recruits a transcriptional activator for the GAL regulon induction in Candida albicans

doi: 10.7554/eLife.84155

Figure Lengend Snippet: ( A ) The AD domain of Rep1 is essential for GlcNAc signaling. Cells of the rep1 mutant carrying Rep1,Rep1 ΔAD or vector alone were spotted onto YNB solid medium containing 2.5 mM glucose, glycerol or GlcNAc. Photographs were taken after 40 hr of growth at 30 °C. Representative images of three independent experiments are shown. ( B ) Both the AD domain of Rep1 and Ngs1 are required for the induction of GAL1/GAL10 in GlcNAc. Data shown as means ± SD of three independent experiments.Statistical analysis was performed using an unpaired two-tailed Student’s t -test.( C ) The AD domain of Rep1 is required for the recruitment of Ngs1. Protein lysates from wild type cells carrying Rep1-Myc, FLAG-Ngs1 & Rep1-Myc, Rep1 ΔAD -Myc, or FLAG-Ngs1 &Rep1 ΔAD -Myc were subjected to immunoprecipitation with anti-FLAG antibody, and the precipitated proteins were probed with anti-Myc antibody. As an input control, cell lysates were analysed by Western blotting with the anti-Myc antibody. Representative blots of three independent experiments are shown. ( D ) The cis -regulatory motif most highly enriched at locations of Rep1 in C. albicans . Motifs were generated using MEME. ( E ) The major terms after performing GO enrichment analysis. Bar graphs represent the corrected p-value. Representative genes for each GO term are listed below and the number of genes is shown on the right. ( F ) Presence–absence of Leloir genes, key regulators (Rep1, Cga1) and growth on galactose media. Y, present; N, absent; NA, not available. Growth data were obtained from the review article . ( G ) Model of Rep1-mediated transcriptional activation in response to galactose (top) or GlcNAc (bottom). Rep1 recognizes galactose via directly physical interaction, enabling the recruitment of Cga1 to activate transcription. While Rep1 constitutively interacts with GlcNAc sensor Ngs1, the transcriptional activation upon GlcNAc induction is achieved by promoter histone acetylation via GlcNAc binding to Ngs1. Figure 4—source data 1. Raw xlsx files used for analysis of the dataset. Figure 4—source data 2. Raw Western blot images with the labeled band of interest. Figure 4—source data 3. The source file for the table.

Article Snippet: Recombinant Rep1 ∆AD -His and its variants were incubated with 50 μl D–Galactose agarose beads (Thermo Scientific) overnight at 4 °C.

Techniques: Mutagenesis, Plasmid Preparation, Two Tailed Test, Immunoprecipitation, Western Blot, Generated, Activation Assay, Binding Assay, Labeling

Journal: eLife

Article Title: Recognition of galactose by a scaffold protein recruits a transcriptional activator for the GAL regulon induction in Candida albicans

doi: 10.7554/eLife.84155

Figure Lengend Snippet:

Article Snippet: Recombinant Rep1 ∆AD -His and its variants were incubated with 50 μl D–Galactose agarose beads (Thermo Scientific) overnight at 4 °C.

Techniques: Sequencing, Recombinant, Purification, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

doi: 10.1007/s00109-013-1006-4

Figure Lengend Snippet: Expression of CHM / REP1 cDNA and EGFP transgenes in dog D17 cell line. a Plasmids pAAV2-EFS-REP1 and pAAV2-CBA-REP1 carry the CHM / REP1 cDNA transgene with Kozak sequence at the 5′-end. Plasmids pAAV2-EFS-GFP and pAAV2-CBA-GFP were used to generate control viral vectors. WPRE* is a WPRE that has been modified by deleting the We2 promoter/enhancer and mutating the We1 promoter (for other details, see “ ”). b – d D17 cells were transduced with AAV2/2-EFS-GFP, AAV2/5-EFS-GFP and AAV2/2-CBA-GFP. Expression of GFP was analysed 2 days post-transduction (P0). Cells were replated, cultured for additional 6 days and analysed (P1), then replated and analysed after 4 days in culture (P2). b FACS analysis shows prevalence of transduced cells (% of total cell number). c Immunoblot analysis of the total protein lysate using GFP antibody and α-tubulin antibody as a loading control. d Quantification of the western blot shown in c using ImageJ software. Data are presented as relative density of GFP signal in relation to α-tubulin signal. e Immunoblot analysis of the D17 cells transduced with AAV2/2-EFS-REP1, AAV2/5-EFS-REP1 and AAV2/2-CBA-REP1 using 2F1 antibody specific for human REP1 and α-tubulin antibody as a loading control. Cells were analysed 48 h post-transduction. f Quantification of the western blot shown in e using ImageJ software. Data are presented as relative density of GFP signal in relation to α-tubulin signal

Article Snippet: Antibodies used in the current study were: mouse monoclonal 2F1 (specific for human REP1, dilution 1:1,000); rabbit serum J905 (pan-REP, specific for mouse, rat and human isoforms of REP1 and REP2); mouse monoclonal α-tubulin (Sigma, dilution 1:5,000) and mouse monoclonal anti-GFP (Zymed, dilution 1:2,000).

Techniques: Expressing, Sequencing, Modification, Transduction, Cell Culture, Western Blot, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

doi: 10.1007/s00109-013-1006-4

Figure Lengend Snippet: a Immunoblot analysis of choroideremia patient fibroblasts ( CHM ) transduced with AAV2/2-EFS-GFP, AAV2/5-EFS-GFP and AAV2/2-CBA-GFP using GFP antibody and α-tubulin antibody as a loading control. Expression of GFP was analysed 7 days post-transduction. b Immunoblot analysis of CHM transduced with AAV2/2-EFS-REP1, AAV2/5-EFS-REP1 and AAV2/2-CBA-REP1 using 2F1 antibody and α-tubulin antibody as a loading control. Expression of REP1 was analysed 7 days post-transduction. c Immunoblot analysis of choroideremia patient fibroblasts that were untransduced (CHM) and transduced with AAV2/2-CBA-REP1 and control (WT) fibroblasts using 2F1 antibody and α-tubulin antibody as a loading control. Expression of REP1 was analysed 10 days post-transduction. Amount of loaded cell lysate (micrograms) is indicated above each lane . Recombinant human protein (hREP1) was used as a positive control. d Quantification of the GFP signal intensity from the western blot shown in c using ImageJ software. e In vitro prenylation analysis was performed using 5 and 20 μg of cytosolic fractions of the cell lysates isolated from untransduced ( white diamond ) and transduced with AAV2/2-CBA-GFP ( black square ) and AAV2/2-CBA-REP1 ( white triangle ) CHM fibroblasts. f In vitro prenylation analysis was performed using 2, 4, 8 and 16 μg of cytosolic fractions of the cell lysates isolated from untransduced D17 cell ( white diamond ) and D17 transduced with AAV2/2-CBA-GFP ( black square ) and AAV2/2-CBA-REP1 ( white triangle )

Article Snippet: Antibodies used in the current study were: mouse monoclonal 2F1 (specific for human REP1, dilution 1:1,000); rabbit serum J905 (pan-REP, specific for mouse, rat and human isoforms of REP1 and REP2); mouse monoclonal α-tubulin (Sigma, dilution 1:5,000) and mouse monoclonal anti-GFP (Zymed, dilution 1:2,000).

Techniques: Western Blot, Transduction, Expressing, Recombinant, Positive Control, Software, In Vitro, Isolation

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

doi: 10.1007/s00109-013-1006-4

Figure Lengend Snippet: a , b Illustration of delivery route for AAV viral vectors by subretinal injection. The subretinal injection was performed through posterior retina using a 10-μl Hamilton syringe and a 30° bevelled 34G needle. c Immunoblot analysis of RPE collected from the eyes of choroideremia mice ( Chm null / WT ) injected with AAV2/2-EFS-REP1, AAV2/5-EFS-REP1 and AAV2/2-CBA-REP1 using 2F1 antibody and α-tubulin antibody as a loading control. Expression of REP1 was analysed 5 weeks post-injection. d – f Histological analysis of the AAV2/2-CBA-GFP-injected mouse eyes 5 weeks post-injection. e Phase contrast image corresponding to d . f Enlargement image of the area boxed in d . Arrows indicate nuclei of rod photoreceptors that express GFP confirming successful transduction. RPE retinal pigment epithelium, ONL outer nuclear layer, IS inner segments, OS outer segments. Bar is 50 μm

Article Snippet: Antibodies used in the current study were: mouse monoclonal 2F1 (specific for human REP1, dilution 1:1,000); rabbit serum J905 (pan-REP, specific for mouse, rat and human isoforms of REP1 and REP2); mouse monoclonal α-tubulin (Sigma, dilution 1:5,000) and mouse monoclonal anti-GFP (Zymed, dilution 1:2,000).

Techniques: Injection, Western Blot, Expressing, Transduction

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

doi: 10.1007/s00109-013-1006-4

Figure Lengend Snippet: ERG analysis of wild-type mice treated with AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP. a Representative averaged ERG traces from the eyes injected with high-dose (1 × 10 9 gp) AAV2/2-CBA-REP1 (shown in black ) and AAV2/2-CBA-GFP (shown in grey ). b , c Quantification of the amplitude of a- and b-waves ( b ) and implicit time data ( c ) recorded across a range of stimulus intensities in high-dose (1 × 10 9 gp) AAV2/2-CBA-REP1-injected ( filled black circles and solid black lines ) and AAV2/2-CBA-GFP-injected ( open grey circles and dashed grey lines ) eyes. Plotted symbols show mean ± SEM, n = 5. d , e Quantification of the amplitude of a- and b-waves ( d ) and implicit time data ( e ) recorded across a range of stimulus intensities in low-dose (1 × 10 8 gp) AAV2/2-CBA-REP1-injected ( filled black circles and solid black lines ) and AAV2/2-CBA-GFP-injected ( open grey circles and dashed lines ) eyes. Plotted symbols show mean ± SEM, n = 4

Article Snippet: Antibodies used in the current study were: mouse monoclonal 2F1 (specific for human REP1, dilution 1:1,000); rabbit serum J905 (pan-REP, specific for mouse, rat and human isoforms of REP1 and REP2); mouse monoclonal α-tubulin (Sigma, dilution 1:5,000) and mouse monoclonal anti-GFP (Zymed, dilution 1:2,000).

Techniques: Injection