Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

Abstract

Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9's cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of syndecans in AAV9's cellular entry. The ubiquitously expressed isoform, syndecan-4 proved its superiority in facilitating AAV9 internalization among syndecans. Introducing syndecan-4 into poorly transducible cell lines enabled robust AAV9-dependent gene transduction, while its knockdown reduced AAV9's cellular entry. Attachment of AAV9 to syndecan-4 is mediated not just by the polyanionic heparan-sulfate chains but also by the cell-binding domain of the extracellular syndecan-4 core protein. Co-immunoprecipitation assays and affinity proteomics also confirmed the role of syndecan-4 in the cellular entry of AAV9. Overall, our findings highlight the universally expressed syndecan-4 as a significant contributor to the cellular internalization of AAV9 and provide a molecular-based, rational explanation for the low gene delivery potential of AAV9 into the CNS.

Keywords: AAV9; cellular entry; gene delivery; heparan sulfate proteoglycans; syndecans.

Figure 1

SDC4 contributes to AAV9’s entry into hCMEC/D3 cells. (A–F) Imaging flow cytometry assessment of SDC4 expression and AAV9-mediated gene (GFP) delivery in wild-type (WT) and SDC4 KD hCMEC/D3 cells. SDC4 KD was performed using SDC4-specific shRNA plasmids. WT and SDC4 KD hCMEC/D3 cells were treated 4 × 10⁴ vg/cell AAV9-GFP for 72 h at 37 °C. SDC4 and GFP expression levels were measured with imaging flow cytometry, as shown by the representative histograms and cellular images of three independent experiments. Scale bar = 20 μm. The effect of SDC4 KD on SDC4 and GFP expression expressed as percent inhibition. The bars represent the mean ± SEM of three independent experiments. Statistical significance vs. WT was assessed with analysis of variance (ANOVA). ** p < 0.01; *** p < 0.001. (G) SDS-PAGE showing VP1-3 immunoprecipitated with SDC4 from AAV9-treated (4 × 10⁴ vg/cell AAv9 for 6 h at 37 °C) hCMEC/D3 cells’ extracts. Lane 1: 10⁶ vg recombinant AAV9; lane 2–3: immunoprecipitates of AAV9-treated WT hCMEC/D3 and SDC4 KD cells; lane 4: MW marker; lane 5–6: immunoprecipitates of WT and SDC4 KD hCMEC/D3 cells untreated with AAV9 (i.e., controls). Standard protein size markers are indicated on the right.

Figure 2

AAV9 colocalizes with SDC4 during cellular internalization. hCMEC/D3 cells were incubated with AAV9 (4 × 10⁴ vg/cell) for 6 h at 37 °C. The cells were then trypsinized, fixed, permeabilized and treated with AF488-labeled AAV9 (green) and APC-labeled SDC4 (red) antibodies. AAV9’s colocalization with SDC4 was analyzed with confocal microscopy. Representative images of three independent experiments are shown. Scale bar = 10 μm. The MOC ± SEM and PCC ± SEM for the overlap and colocalization of SDC4 with AAV9 are indicated below the images.

Figure 3

AAV9-mediated GFP transduction into SDC transfectants. WT K562 cells and SDC transfectants were incubated with AAV9-GFP vectors (4 × 10⁴ vg/cell) for 72 h at 37 °C. After incubation, GFP expression was analyzed with imaging flow cytometry and confocal microscopy. (A,B) Representative flow cytometry histograms and fluorescent images showing the intracellular fluorescence of AAV9-GFP-treated WT K562 cells and SDC transfectants. Scale bar = 20 μm. (C) Detected fluorescence intensities were normalized to AAV9-GFP-treated WT K562 cells as standards. The bars represent the mean ± SEM of seven independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Confocal microscopic visualization of AAV9-GFP-treated WT K562 cells and SDC transfectants. Representative images of three independent experiments are shown. Scale bar = 10 μm.

Figure 4

Contribution of the various parts of the SDC4 ectodomain to AAV9 uptake. GFP-tagged SDC4 mutants incubated with AAV9 vectors at 4 × 10⁴ vg/cell for 6 h were fixed, permeabilized, and treated with specific primary AAV9 and AF 633-labeled secondary antibodies. The cellular uptake of AAV9 was then analyzed with imaging flow cytometry and confocal microscopy. (A,B) Representative fluorescent images and flow cytometry histograms showing the intracellular fluorescence of AAV9-treated SDC4 mutants. Scale bar = 20 μm. The indicated BDS values of AAV9 and SDCs represent the mean + SEM of eight independent experiments. Statistical significance was assessed with ANOVA. (C) Detected fluorescence intensities were normalized to AAV9-treated transfectants expressing GFP-labeled WT SDC4 as standards. The bars represent mean + SEM of eight independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; *** p < 0.001. (D) Confocal microscopic visualization of AAV9-treated SDC4 mutants. Scale bar = 10 μm. MOC ± SEM for the overlap of AAV9 with the mutants was calculated by analysis of 15 images with ~10 cells in each image (from three separate samples). Statistical significance vs. AAV9-treated transfectants expressing WT SDC4 (standards) was assessed with ANOVA. *** p < 0.001.

Figure 5

The effect of undersulfation on SDC4 expression and AAV9-mediated gene transduction. Stable SDC4 transfectants (created in K562 cells) were preincubated with or without NaClO₃ for 48 h. Effect of NaClO₃ preincubation on HS and SDC4 expression was measured with imaging flow cytometry by incubating the cells with HS- and SDC4-specific antibodies. SDC4 transfectants preincubated with or without NaClO₃ were then treated with AAV9-GFP vectors at 4 × 10⁴ vg/cell. After 72 h of incubation with AAV9-GFP, GFP expression was measured with imaging flow cytometry. (A–D) Representative flow cytometry histograms and fluorescent cellular images showing HS, GFP, and SDC4 expression of cells preincubated with or without NaClO₃. Scale bar = 20 μm. (E) Detected HS, GFP, and SDC4 expression levels in SDC4 transfectants were normalized to those untreated with NaClO₃ (controls). The bars represent the mean + SEM of six independent experiments. Statistical significance was assessed with ANOVA. *** p < 0.001. (F) Linear regression between SDC4 expression and AAV9-mediated GFP transduction.

Figure 6

SDC4 overexpression increases AAV9-mediated GFP transduction in SH-SY5Y cells. SDC4 transfectants (created in SH-SY5Y cells) and WT SH-SY5Y cells were incubated with AAV9-GFP (4 × 10⁴ vg/cell) at 37 °C for 72 h. GFP expression was then analyzed with imaging flow cytometry. (A–C) Representative flow cytometry histograms and fluorescent images showing the GFP and SDC4 expression levels in WT SH-SY5Y cells and SDC4 transfectants treated with AAV9-GFP. Scale bar = 20 μm. (D,E) Detected SDC4 and GFP expression levels of AAV9-treated SDC4 transfectants were normalized to that of WT SH-SY5Y cells as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01.

Figure 7

AAV9-mediated GFP transduction is more efficient in U-87 MG than in SH-SY5Y cells. WT U-87 MG and SH-SY5Y cells were incubated with AAV9-GFP (4 × 10⁴ vg/cell) for 72 h. GFP expression was then analyzed with imaging flow cytometry. (A–C) Representative flow cytometry histograms and fluorescent images showing the SDC4 and GFP expression levels in SH-SY5Y and U-87 MG cells treated with AAV9-GFP. Scale bar = 20 μm. (D,E) Detected SDC4 and GFP expression levels of AAV9-treated U-87 MG cells were normalized to that of SH-SY5Y cells as standards. The bars represent the mean + SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01.