cx3cl1 targeting shrna  (Santa Cruz Biotechnology)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: A workflow of APEX2-mediated proximity labeling and generation of inducible HEK293 cell lines expressing MAAP2-APEX2 and APEX2 (A) Diagram of workflow for APEX2-mediated proximity labeling. 293 iMAAP2−APEX2 or 293 iAPEX2 cells were infected with AAV2 ΔMAAP and transfected with pHelper. At 16 hpi, doxycycline (Dox) was added to induce expression of MAAP2-APEX2 or APEX2, and infected with AAV2 ΔMAAP . Upon addition of biotin-phenol (BP) and H 2 O 2 , APEX2 catalyzes the oxidation of BP to biotin-phenoxyl radicals within ∼20 nm in diameter, thereby resulting in the biotinylation of MAAP2-associated proteins (APs) in 1 min. The reaction is then quenched and followed by a pulldown of MAAP2-APs using streptavidin magnetic beads. MAAP2-APs are identified by quantitative mass spectrometry (qMS). (B) MAAP2-APEX2 and APEX2 only expressing lentiviruses. TripZ-APEX2 and TripZ-MAAP2-APEX2 lentiviruses express APEX2 only (serves as negative control) and MAAP2-APEX2, respectively. APEX2 and MAAP2-APEX2 were expressed with a V5-tag at the N terminus of APEX2 as indicated. (C) Dox-inducible cell lines. 293 iAPEX2 and 293 iMAAP2−APEX2 cells were seeded on wells of a six-well plate, Dox was added to induce expression of APEX2 (∼29 kDa with the linker and V5 tag) or MAAP2-APEX2 (∼43 kDa with the linker and V5 tag). Cell lysates of mock (−) and Dox-added (+) 293 iAPEX2 or 293 iMAAP2−APEX2 cells were immunoblotted for V5 tag. β-actin is shown as a loading control.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Labeling, Expressing, Infection, Transfection, Magnetic Beads, Mass Spectrometry, Negative Control, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: Identification of MAAP2-associating host proteins by APEX2-mediated proximity labeling (A) Optimized biotinylation for sampling of quantitative mass spectrometry (qMS). 293 iMAAP2−APEX2 and 293 iAPEX2 cells in a T25 flask were infected with AAV2 ΔMAAP at an MOI of 5,000 vgc/cell. At 16 hpi, Dox was added. At 2 dpi, APEX2-mediated biotinylation was carried out as described in . Cells were incubated with biotin-phenol for 30 min at 37°C. H 2 O 2 was added and incubated for 1 min at room temperature to biotinylate proximal proteins. The reaction was then quenched. The cells were harvested and lysed in RIPA buffer. The supernatant of the lysates was then incubated with streptavidin beads. Ten percent of the washed beads were separated on SDS-PAGE for Coomassie staining and western blotting (WB), respectively. The western blot was probed with Alexa Fluor 680-conjugated streptavidin (Thermo Fisher). (B) Analysis of the identified proteins by qMS. The remaining 90% of the washed beads were subjected to on-bead digestion and liquid chromatography-tandem mass spectrometry analysis. Three repeats were carried out. The qMS data listed in Table S1 were analyzed. The volcano plot shows statistical significance (−log 10 , p value) vs. the magnitude of change (log 2 , fold-change) for differentially interacted proteins between MAAP2-APEX2-and APEX2-expressing cells. Pink dots indicate enriched proteins by >2-fold and green dots indicate depleted proteins by >2-fold. The t test (unpaired, two-tailed) was employed for determination of statistical significance ( p value). The horizontal dash line indicates p < 0.05. (C) Gene Ontology (GO) annotation of top enriched proteins. In the volcano plot (B), proteins clustered in the upper/right corner as divided by the blue dash lines, which were enriched by >16 times and had significance ( p value) of >0.01 in the MAAP2-APEX2-expressing cells, were analyzed by GO. Proteins are shown in circles with colors indicating their functions. The size of the circles represents the enrichment score (fold-change in log 2 ).

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Labeling, Sampling, Mass Spectrometry, Infection, Incubation, SDS Page, Staining, Western Blot, Liquid Chromatography, Expressing, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: STX7 or SNAP23 colocalizes with MAAP2 in both wtAAV2-infected cells and rAAV2-producing cells (A and D) wtAAV2 infection. HEK293 cells were mock-infected or infected with wtAAV2 followed by pHelper transfection. At 2 dpi, the cells were co-immunostained for MAAP2 and SNAP23 (A) or MAAP2 and STX7 (C). (B and E) rAAV2 production. HEK293 cells were mock or transfected with pR2C2, pHelper, and prAAV2. At 2 dpt, the transfected cells were co-immunostained for MAAP2 and SNAP23 (B) or MAAP2 and STX7 (D). SNAP23 (B) or STX7 (D) cells were co-immunostained with a secondary antibody conjugated with a far-red dye. Co-immunostained cells were observed under a Leica STED microscope with a 100× objective lens. Images captured in the far-red wavelength were pseudo-colored in red. The colors of confocal images correspond to blue for DAPI, green for MAAP, and red for SNAP23 or STX7 as indicated. Scale bar, 5 μm. Representative confocal images are shown. (C and F) Quantification of colocalization. Pearson’s correlation coefficients were measured for colocalization of MAAP2 with SNAP23 (C) or STX7 (F) using NIH ImageJ.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Infection, Transfection, Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: SNAP23 or STX7 interacts with MAAP2 in cells but not in vitro (A and B) Co-IP. HEK293 cells were transfected with pCI-MAAP2 Flag or pCI-empty. At 2 dpt, cells were harvested and lysed. 90% of the lysates were used for immunoprecipitation with anti-Flag-conjugated magnetic beads. Western blotting was performed for detection of MAAP2 Flag and SNAP23, respectively (A), and for detection of MAAP2 Flag and STX7, respectively (B). β-actin is shown as a loading control. 10% of the lysates were loaded as the whole cell lysate (WCL). (C and D) In vitro pulldown assay. Approximately 2 μg of the purified GST-MAAP2 protein and negative control GST protein were employed as baits to pull down ∼2 μg prey proteins; purified SNAP23 (C) or STX7 (D) using glutathione agaroses. Western blotting was performed for detection of GST-MAAP2 and control GST using anti-GST (C and D), for the detection of SNAP23 using anti-SNAP23 (C), and for the detection of STX7 using anti-STX7 (D). ∼200 ng of the bait and pray proteins were loaded as inputs. Asterisks indicate the major detected GST-MAAP and GST.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Control, Purification, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: A proposed model of the role of SNARE proteins in AAV egress AAV capsids produced in the nucleus are egressed through the nuclear pore complex (NPC), the endoplasmic reticulum (ER), and the Golgi apparatus, where a portion of the vectors are associated with EVs (marked with exosome marker CD63), on which MAAP2 is attached. , These EV-associated or -carried AAVs are routed to late endosomes (Rab7+) or MVB through the egress pathway of EVs as exosomes to secrete EV-AAVs. A portion of the late endosomes/MVBs are captured or recognized by v-SNARE proteins (e.g., STX7). STX7 interacts with t-SNARE proteins, e.g., SNAP23, for the fusion of late endosomes with lysosome, where SNAP23 makes up the heterodimeric t-SNAREs required for lysosome exocytosis, leading to the formation of lysosome/endosome hybrid for degradation of the virions. On the other hand, the EV-associated AAV can reach early endosomes (Rab5+) and then traffic to recycling endosomes (Rab11+), where they can be released as microvesicles. Knockout of SNARE expression can block the formation of a lysosome/endosome hybrid, resulting in less virion degradation and more virion release/secretion out of the plasma membrane.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Produced, Marker, Knock-Out, Expressing, Blocking Assay, Membrane

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: STX7 or SNAP23 colocalizes with MAAP2 in both wtAAV2-infected cells and rAAV2-producing cells (A and D) wtAAV2 infection. HEK293 cells were mock-infected or infected with wtAAV2 followed by pHelper transfection. At 2 dpi, the cells were co-immunostained for MAAP2 and SNAP23 (A) or MAAP2 and STX7 (C). (B and E) rAAV2 production. HEK293 cells were mock or transfected with pR2C2, pHelper, and prAAV2. At 2 dpt, the transfected cells were co-immunostained for MAAP2 and SNAP23 (B) or MAAP2 and STX7 (D). SNAP23 (B) or STX7 (D) cells were co-immunostained with a secondary antibody conjugated with a far-red dye. Co-immunostained cells were observed under a Leica STED microscope with a 100× objective lens. Images captured in the far-red wavelength were pseudo-colored in red. The colors of confocal images correspond to blue for DAPI, green for MAAP, and red for SNAP23 or STX7 as indicated. Scale bar, 5 μm. Representative confocal images are shown. (C and F) Quantification of colocalization. Pearson’s correlation coefficients were measured for colocalization of MAAP2 with SNAP23 (C) or STX7 (F) using NIH ImageJ.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Infection, Transfection, Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: SNAP23 or STX7 interacts with MAAP2 in cells but not in vitro (A and B) Co-IP. HEK293 cells were transfected with pCI-MAAP2 Flag or pCI-empty. At 2 dpt, cells were harvested and lysed. 90% of the lysates were used for immunoprecipitation with anti-Flag-conjugated magnetic beads. Western blotting was performed for detection of MAAP2 Flag and SNAP23, respectively (A), and for detection of MAAP2 Flag and STX7, respectively (B). β-actin is shown as a loading control. 10% of the lysates were loaded as the whole cell lysate (WCL). (C and D) In vitro pulldown assay. Approximately 2 μg of the purified GST-MAAP2 protein and negative control GST protein were employed as baits to pull down ∼2 μg prey proteins; purified SNAP23 (C) or STX7 (D) using glutathione agaroses. Western blotting was performed for detection of GST-MAAP2 and control GST using anti-GST (C and D), for the detection of SNAP23 using anti-SNAP23 (C), and for the detection of STX7 using anti-STX7 (D). ∼200 ng of the bait and pray proteins were loaded as inputs. Asterisks indicate the major detected GST-MAAP and GST.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Control, Purification, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: Knockout of STX7 or SNAP23 increases rAAV vector secretion into the media, as well as total vector yields, during rAAV production (A and B) Generation of KO cell lines. (A) Western blotting. HEK293 cells were transduced with lentiviruses expressing STX7 -, SNAP23 -targeting, or scramble guide RNA (Scramble), followed by single-cell cloning for STX7-KO cell line generation. SNAP23-KO cell line was not single-cell cloned. Cells were analyzed for expression of STX7 or SNAP23, as indicated, using western blotting. β-actin serves as a loading control. (B) Cell viability. WT, Scramble, STX7-KO, and SNAP23-KO HEK293 cells were seeded in wells of six-well plates at equal cell number. Cells were trypsinized, and the viable cells were counted after staining with trypan blue at 24, 48, and 72 h, respectively. (C–F) Small-scale production of rAAV2 (C), rAAV1 (D), rAAV5 (E), and rAAV9 (F) in the gene knockout cells. WT, Scramble, STX7-KO, and SNAP23-KO HEK293 cells, as indicated, were transfected with pR2C2 (C), pR2C1 (D), pR2C5 (E), or R2C9 (F), together with pHelper and prAAV2. At 3 dpt, cells and media were harvested for subsequent treatments. DNase-digestion-resistant viral DNA was extracted from crude lysates of harvested cells (pellet) and media, respectively. Produced rAAV vectors in the pellet and media were quantified by qPCR using an mCherry probe. Left panel: Related bars represent total vector yields in vector genomic copies (vgc) in the pellet (cells), media and total (pellet plus media), respectively. Right panel: Bars indicate ratios of produced rAAV yield in the pellet (cells) vs. in media based on the data presented in the left panel. Means and SDs were calculated using data from three independent experiments ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001; and ns, no significant difference.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Knock-Out, Plasmid Preparation, Western Blot, Transduction, Expressing, Clone Assay, Control, Staining, Gene Knockout, Transfection, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: Large-scale production of rAAV5 in STX7-KO and SNAP23-KO HEK293 cells Scramble, STX7-KO, and SNAP23-KO HEK293 cells were transfected with pR2C5, pHelper, and prAAV2. (A) mCherry expression. Transfected cells were imaged for mCherry expression at 2 dpt by the ZOE Fluorescent Cell Imager (Bio-Rad). Representative images are shown. The intensity of mCherry expression of each group was displayed as fold changes relative to scramble control group. The relative fold changes of mCherry expression in the transfected cells were measured by ImageJ. (B) Titers of the purified rAAV5 vectors in the cells (pellet) and in the media. At 3 dpt, cells and media were harvested for vector purification. DNase-digestion-resistant viral DNA were extracted from the final purified vectors from the pellets and media and quantified by qPCR. Left panel: Data shown are total vector yields (vgc) in pellet, media or total (pellet plus media). Right panel: Bars indicate ratios of produced rAAV yield in the pellet (cells) vs. in the media based on the data presented in the left panel. (C) Transduction efficiency. HEK293 cells were transduced with rAAV5 purified from the cells and the media of the indicated HEK293 cell lines (x axis). Bars represent the intensity of luciferase activity of each group, shown as fold changes relative to the Scramble group (y axis). Means and SDs were calculated using data from three independent experiments ( n = 3). ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001; and ns, no significant difference.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Transfection, Expressing, Control, Purification, Plasmid Preparation, Produced, Transduction, Luciferase, Activity Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: A proposed model of the role of SNARE proteins in AAV egress AAV capsids produced in the nucleus are egressed through the nuclear pore complex (NPC), the endoplasmic reticulum (ER), and the Golgi apparatus, where a portion of the vectors are associated with EVs (marked with exosome marker CD63), on which MAAP2 is attached. , These EV-associated or -carried AAVs are routed to late endosomes (Rab7+) or MVB through the egress pathway of EVs as exosomes to secrete EV-AAVs. A portion of the late endosomes/MVBs are captured or recognized by v-SNARE proteins (e.g., STX7). STX7 interacts with t-SNARE proteins, e.g., SNAP23, for the fusion of late endosomes with lysosome, where SNAP23 makes up the heterodimeric t-SNAREs required for lysosome exocytosis, leading to the formation of lysosome/endosome hybrid for degradation of the virions. On the other hand, the EV-associated AAV can reach early endosomes (Rab5+) and then traffic to recycling endosomes (Rab11+), where they can be released as microvesicles. Knockout of SNARE expression can block the formation of a lysosome/endosome hybrid, resulting in less virion degradation and more virion release/secretion out of the plasma membrane.

Article Snippet: Recombinant GST-fused MAAP2 His (GST-MAAP2 His ) was expressed and purified as previously described., Purified human STX7 (#14692-H07H) and SNAP23 (#pro-659) proteins were purchased from Sino Biological (Wayne, PA) and Prospec Bio (East Brunswick, NJ), respectively.

Techniques: Produced, Marker, Knock-Out, Expressing, Blocking Assay, Membrane