Probing aspects of extracellular vesicle associated AAV allows increased vector yield and insight into its transduction and immune-evasive properties

Figures

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  Figure 1. Characterization and quantitation of EV-AAV in producer cell media using iodixanol density gradient ultracentrifugation (A and B) Overview. 293T cells are transfected with AAV plasmids to generate the AAV serotype of choice (AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9). At a given time point, media is isolated and a density gradient ultracentrifugation performed. The gradient is fractioned and EV-AAV are predicted to be in the less dense fractions and free AAV in denser fractions. (C) Quantitation of tetraspanin positive EVs using the ExoView instrument. High concentrations of EVs are in less dense fractions. (D) Quantitation of AAV vector genomes (vg) on gradient (note y axis scale break). (E) VP1,VP2, and VP3 AAV6 capsid immunoblot. Arrow points to VP3 band detected in all fractions. F7 and F8 are putative empty capsid fractions. (F) Percent of vg on gradient in EV-AAV fractions 1–6. (G) Total vg on gradient in EV-AAV fractions 1–6. Error bars in all panels are the standard error from the mean. Two gradients per serotype were run, except for AAV5 which was performed once. Each data point in (F) and (G) represents an independent purification of the respective serotype of AAV. Two…

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  Figure 2. A substantial portion of AAV capsids and genomes in EV-AAV preparations are intraluminal and mediate transduction of cells and resistance to antibody neutralization (A) EV-AAV is purified from media and then extra-luminal AAV capsids are removed with anti-AAV9 conjugated beads. (B) Purified AAV9 or EV-AAV9 were incubated with control beads or anti-AAV9 conjugated beads. (C) Purified AAV1, AAV2, AAV6, or their cognate EV-AAV preparations were incubated with control or anti-AAV conjugated beads (AAVX antibody). In (B) and (C), the percent resistance to pulldown in the EV-AAV fraction is putative intraluminal AAV. (D) Transduction of HeLa cells with intraluminal EV-AAV9. (E) Intraluminal AAV resists neutralization to IVIg compared with AAV9. ns, not significant; ∗∗p = 0.009; ∗∗∗p < 0.001.

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  Figure 3. EV-AAV9 and intraluminal EV-AAV9 mediate transduction of neurons after direct low-dose intracranial injection in adult mice (A) Intracranial injection of C57BL/6 adult mice with AAV9, EV-AAV9, or intraluminal EV-AAV9. All vectors packaged an AAV-CAG-GFP cassette and were injected at 1.27 × 107 vg; n = 3 mice/vector group. (B) Whole hemisphere anti-GFP immunostaining for each group. Scale bar, 1 cm. (C) Zoom-in on transduced sections of representative mice from each vector group. (D) Quantitation of transduced area/group. The area of transduction was calculated using integrated density of pixel intensity. (E) Neuronal (NeuN) immunostaining shows colocalized with some of the transduced cells in each vector group. Scale bar, 100 μm.

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  Figure 4. MAAP8 trans-complementation enhances both free and EV-AAV9 yields (A) Experimental overview. AAV9 is produced with (1) AAV9 rep/cap plasmids with a deleted MAAP9 (AAV9 MAAP null), (2) AAV9 MAAP null + MAAP8, (3) AAV9 MAAP9, or (4) AAV9 MAAP9 + MAAP8. Media is harvested, purified on gradients, and EV-AAV and free AAV genomes quantitated by qPCR. (B) Day 3 post-transfection yields of EV-AAV9 genomes in each group. ∗p = 0.02; ∗∗p = 0.0055; ns, not significant. (C) Day 3 post-transfection yields of free AAV genomes quantitated by qPCR. ∗p = 0.042; ∗∗p = 0.016. (D) Ratios of EV-AAV to free AAV for each group. (E) Time course of purified EV-AAV9 genomes from cells transfected with AAV9 MAAP9 or AAV9 MAAP null + MAAP8. ∗∗p = 0.0068; ∗∗∗p < 0.0001. Experiments in (B), (C), and (D) were performed two independent times. Experiment in (E) was performed one time.

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  Figure 5. The AAV capsid is required for efficient transduction by EV-AAV (A) HeLa cells were transduced by either EV-AAV8-FLuc vector (gradient fractions 1–6) or EVs isolated from fractions 1–6, which were isolated from 293T cells transfected with AAV-FLuc plasmid but no AAV8 rep/cap plasmid. (B) Total AAV vector genomes isolated from gradient fractions 1–6 for either preparation. ∗∗p = 0.005. (C) Transduction efficiency on HeLa cells incubated with 6.66 × 103 vg/cell of either preparation. ∗∗∗∗p < 0.0001.