Journal: bioRxiv

Article Title: Suppressing microtubule detyrosination augments AAV2 endosomal escape and gene delivery

doi: 10.1101/2025.08.12.669862

Figure Lengend Snippet: (A–C) Three-color SIM imaging of AAV2 (A), detyrosinated tubulin (B), and tyrosinated tubulin (C) in Huh7 cells at 0, 2, 4, and 8 hours post-AAV2 treatment. (D–E) Quantification of tyrosinated and detyrosinated microtubules in Huh7 (D) and BS-C-1 cells (E) across time points post-AAV2 treatment (mean ± SD; n = 15 cells analyzed across three independent experiments). (F) Schematic model of AAV2-induced signaling via RTK leading to GSK3β inhibition and CLASP2-mediated microtubule detyrosination. (G–I) Immunostaining (G) and quantification of cytoplasmic (H) and nuclear (I) phospho-GSK3β (Ser9) in mock– and AAV2-infected Huh7 cells at 4 hours, showing increased Ser9 phosphorylation (n=125 cells, analyzed across three independent experiments). (J–K) Expression of constitutively active GSK3β S9A mutant (green) and quantification of cells with detyrosinated microtubules (magenta) under mock or AAV2-infected conditions (n=190 cells, analyzed across three independent experiments). (L–M) Immunostaining (L) and quantification (M) of endogenous CLASP2, showing increased microtubule association in infected cells (in triplicate). Arrows indicate CLASP2 localized to microtubules. (N–O) Modulation of detyrosinated microtubules in cells overexpressing CLASP2 WT (green) or CLASP2 mutant-9xS/A (green) with AAV2 treatment compared to mock controls (n=160 cells, analyzed across three independent experiments). In the graphs the grey line or bar represents the mean, and whisker denotes the standard deviation. Significance was analyzed using two paired two-sample t-test, ***p<0.001 and “ns” indicating non-significant. Scale bar: (A-C) is 10 µm, (G, J, L and N) is 20 µm.

Article Snippet: Cells were then incubated in primary antibodies for detyrosinated microtubule (ab48389; 1:100) or tyrosinated tubulin (ab6160; 1:100) for 2 hours at RT, or AAV2 (Fitzgerald 10R-A110a 1:100), or Phospho-GSK-3β (Ser9) (CST 5558; 1:200), or CLASP2 (Thermo Fisher PA5-109547; 1:100) overnight at 4° C, and then washed three times with washing buffer (0.2% BSA and 0.05% TritonX100) for 5min each.

Techniques: Imaging, Inhibition, Immunostaining, Infection, Phospho-proteomics, Expressing, Mutagenesis, Whisker Assay, Standard Deviation

Journal: bioRxiv

Article Title: Suppressing microtubule detyrosination augments AAV2 endosomal escape and gene delivery

doi: 10.1101/2025.08.12.669862

Figure Lengend Snippet: (A-A’) Two-color SIM images of Huh7 cells treated with AAV2 (green) and immune stained for detyrosinated microtubules (magenta) at 0-, 2-, 4-hour. Zoom of the respective insets (A’) given in (A). Arrowhead and arrows show AAV2 localized to and not localized to detyrosinated microtubules, respectively. (B-C) Quantification of the localization (B) and enrichment (C) of AAV2 on detyrosinated microtubules at different time points post-infection in Huh7 cells (10 cells analyzed for each time point; n=10). (D-F) Correlative live-cell and STORM imaging of AAV2 and detyrosinated microtubules in BS-C-1 cells. Trajectory of AAV2 motility (yellow) from the live-cell imaging (D). STORM image of the detyrosinated microtubules (magenta) from the same region (E). AAV2 trajectory from A overlaid with STORM image of detyrosinated microtubule from E (F). (G-G’) Color-coded AAV2 trajectory shows processive run phases in blue and non-processive pause phases in red (G) and their corresponding speed measurements (G’). (H-K) Quantitative analysis of speed (H), run length (I), number of pauses per track (J), and fraction of time spent pausing (pause duration) (K) of AAV2 motility on detyrosinated tubulin compared to tyrosinated tubulin (20 tracks analyzed per condition: n=20). In the graphs the grey line represents the mean and whisker denotes the standard deviation. Significance was analyzed using two paired two-sample t-test, ***p<0.001. Scale bar: (A) is 10µm, (A’) is 2 µm and (D-F) is 1 µm.

Article Snippet: Cells were then incubated in primary antibodies for detyrosinated microtubule (ab48389; 1:100) or tyrosinated tubulin (ab6160; 1:100) for 2 hours at RT, or AAV2 (Fitzgerald 10R-A110a 1:100), or Phospho-GSK-3β (Ser9) (CST 5558; 1:200), or CLASP2 (Thermo Fisher PA5-109547; 1:100) overnight at 4° C, and then washed three times with washing buffer (0.2% BSA and 0.05% TritonX100) for 5min each.

Techniques: Staining, Infection, Imaging, Live Cell Imaging, Whisker Assay, Standard Deviation

Journal: bioRxiv

Article Title: Suppressing microtubule detyrosination augments AAV2 endosomal escape and gene delivery

doi: 10.1101/2025.08.12.669862

Figure Lengend Snippet: (A-B) Live cell imaging and track displacement measurement of Cy3 tagged AAV2 in Huh7 cells in untreated control conditions and cells overexpressing VASH2-GFP or TTL-GFP or pretreated with parthenolide. Color-coded trajectories indicate blue for the start and red for the end of the tracks acquired for 60 seconds duration. (B) Quantification of AAV2 track displacement obtained from movies in A. Whiskers represent the standard deviation, and the grey line shows the mean for ∼500 trajectories from n=4 cells analyzed for each condition. (C-E) Time dependent spatial distribution analysis of AAV2 in Huh7cells. TIRF imaging of AAV2-Cy3 (white) in cells fixed at 1-, 2-, and 4-hour after AAV2 endocytosis (C). Schematic representation of the division of the cytoplasmic region into three equal zones: outer (near plasma membrane), inner (perinuclear) and middle (between zone 1 and 2), to analyze the spatial distribution of AAV2 within cells (D). Percentage of AAV2 distribution across the zones over time in Huh7 cells (E). Bars represent the mean and whisker represent the standard deviation for n= 10 cells analyzed in each condition. Statistical significance assessed using a two paired two-sample t-test, with ***p<0.001. Scale bar: (A) 10 µm and (C) 20 µm.

Article Snippet: Cells were then incubated in primary antibodies for detyrosinated microtubule (ab48389; 1:100) or tyrosinated tubulin (ab6160; 1:100) for 2 hours at RT, or AAV2 (Fitzgerald 10R-A110a 1:100), or Phospho-GSK-3β (Ser9) (CST 5558; 1:200), or CLASP2 (Thermo Fisher PA5-109547; 1:100) overnight at 4° C, and then washed three times with washing buffer (0.2% BSA and 0.05% TritonX100) for 5min each.

Techniques: Live Cell Imaging, Control, Standard Deviation, Imaging, Clinical Proteomics, Membrane, Whisker Assay

Journal: bioRxiv

Article Title: Suppressing microtubule detyrosination augments AAV2 endosomal escape and gene delivery

doi: 10.1101/2025.08.12.669862

Figure Lengend Snippet: (A-B) Time dependent colocalization analysis of AAV2 with late endosomes in Huh7 cells. Confocal imaging of Rab7-GFP (green) along with Cy-3 tagged AAV2 (magenta) at different time points in Huh7 cells in untread conditions and pre-treated with parthenolide (A). Percentage of AAV2 colocalized with Rab7 decorated late endosomes over time (n=10) (B). (C-D) Confocal imaging and Quantification of AAV2 (magenta) colocalization with lysosomes decorated by LAMP1 (green) (C). While untreated control cells show significant accumulation of AAV2 in lysosomes over time, parthenolide-treated cells show no significant change in lysosomal colocalization with time (n=10) (D). (E-G) AAV2 endosomal escape measurement using calcien fluorescence assay in Huh7 cells. Schematic representation of calcein fluorescence assay. Endosomal calcein fluorescence appears as a punctate signal whereas, AAV2 mediated endosomal rupture leads to diffused cytoplasmic calcein fluorescence (E). Live-cell imaging of calcein (white) in AAV2 infected cells at 1-, 2-, 4-, and 8-hour post AAV2 endocytosis in untreated control, VASH2-GFP overexpressed, TTL-GFP overexpressed, or parthenolide pretreated conditions (F). Quantification of cytoplasmic calcein fluorescence over time (n=22) (G). In the graphs, whiskers show the standard deviation, the grey line indicates the mean value. Statistical significance was determined using a two paired two-sample t-test, with ***p<0.001 indicating significance and “ns” indicating non-significance. Scale bars: (A-C) 10 µm and (D) 20 µm.

Article Snippet: Cells were then incubated in primary antibodies for detyrosinated microtubule (ab48389; 1:100) or tyrosinated tubulin (ab6160; 1:100) for 2 hours at RT, or AAV2 (Fitzgerald 10R-A110a 1:100), or Phospho-GSK-3β (Ser9) (CST 5558; 1:200), or CLASP2 (Thermo Fisher PA5-109547; 1:100) overnight at 4° C, and then washed three times with washing buffer (0.2% BSA and 0.05% TritonX100) for 5min each.

Techniques: Imaging, Control, Fluorescence, Live Cell Imaging, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Suppressing microtubule detyrosination augments AAV2 endosomal escape and gene delivery

doi: 10.1101/2025.08.12.669862

Figure Lengend Snippet: (A-B) Assessment of the impact of VASH and TTL overexpression on AAV2 transduction efficiency. Confocal imaging of Huh7 cells overexpressing VASH2 (A-upper panel, magenta) or TTL (A-lower panel, magenta) infected with AAV2 EGFP, where expression of EGFP transgene (green) indicates AAV2 transduction (A). Percentage of EGFP-positive cells among VASH2-or TTL-overexpressing populations, highlighting their influence on transduction. n=100 cells analyzed for each condition from triplicate (B). (C-D) Huh7 cells, untreated or pretreated with parthenolide, were infected with AAV2-EGFP at low (MOI:5×10³ vg/cell) and high (MOI: 2×10⁴ vg/cell) vector doses. Confocal imaging shows GFP expression (green) in infected cells, with topro staining nuclei (magenta) (C). Fluorescence intensity quantification confirmed enhanced AAV2-EGFP expression in parthenolide-treated cells at both MOIs (no. of images; n=18) (D). (E-G) Flow cytometry analysis (E) and quantification of GFP-positive cells (F) and normalized fluorescence intensity (MOI: 5×10³ vg/cell) (G), also indicate improved transduction efficiency with parthenolide treatment n=10000 cells were analyzed by flow cytometry for each condition in triplicate. (H-J) Schematic illustrates the in-vivo therapeutic evaluation of parthenolide combined with scAAV2-LP1-h.FIX in a hemophilia B mouse model (H). Parthenolide was administered from day –5 to day –1, followed by AAV2-FIX administration on day 1 and blood collection on day 30 for FIX activity analysis (no. of mice/group; n=5 to 7). Chromogenic assay (I) compared FIX activity across four groups: mock (untreated hemophilia B mice), PAR (parthenolide only), AAV2 (vector without drug), and PAR+AAV2 (combination therapy). Immunohistochemistry and confocal imaging of liver sections showed FIX expressions (red) in the PAR+AAV2 group compared to controls and healthy C57 mice (J). (K-N) Quantification of tyrosinated (K-L) and detyrosinated tubulin (M-N) in liver sections further revealed differences across groups, highlighting the impact of combination therapy. In graphs, the grey line represents the mean value, and whiskers indicate the standard deviation. Statistical significance was determined using two paired two-sample t-tests, with ***p<0.001 indicating significance. For in-vivo FIX expression significance was analyzed using two-way ANOVA, Scale bar: (A, J, K and M) 20 µm and (C) 100 µm.

Article Snippet: Cells were then incubated in primary antibodies for detyrosinated microtubule (ab48389; 1:100) or tyrosinated tubulin (ab6160; 1:100) for 2 hours at RT, or AAV2 (Fitzgerald 10R-A110a 1:100), or Phospho-GSK-3β (Ser9) (CST 5558; 1:200), or CLASP2 (Thermo Fisher PA5-109547; 1:100) overnight at 4° C, and then washed three times with washing buffer (0.2% BSA and 0.05% TritonX100) for 5min each.

Techniques: Over Expression, Transduction, Imaging, Infection, Expressing, Plasmid Preparation, Staining, Fluorescence, Flow Cytometry, In Vivo, Activity Assay, Chromogenic Assay, Immunohistochemistry, Standard Deviation