Figure 1.

Exo-AAV Outperforms Conventional AAV in Hair Cell Transduction in Culture

(A) Standard (conventional) AAV and exo-AAV production workflow. AAV was purified from HEK293T cell lysate, whereas exo-AAV was isolated from the culture medium of the cells. Cryoelectron microscopy shows AAV1 capsids associated with exosomes. White arrowheads show AAV capsids, whereas the black arrowhead indicates the lipid membrane. Scale bars, 50 nm. (B) Transduction of cochlear whole mount cultures with AAV1-CBA-GFP or exo-AAV1-CBA-GFP. Cochleas were explanted from CD1 mice at P1. Vectors were added (10¹¹ GCs) the following day and incubated overnight. Organs were cultured for 3 more days. Exo-AAV1-GFP shows efficient transduction of IHCs and OHCs. Hair cells were labeled with anti-myosin VIIa antibody. Scale bar, 20 μm. (C) Proportion of GFP-positive hair cells in cochleas transduced with 1 × 10¹¹ GCs of conventional AAV1 or exo-AAV1. Numbers in the bars represent the number of cochleas. Three images were taken for each cochlea (base, middle, and apex; fields chosen by distance). Mean ± SEM; ***p < 0.001, **p < 0.01, one-tailed t test. (D) Proportion of GFP-positive hair cells in different regions of the cochlea (basal, middle, and apical turns) transduced with conventional AAV1 or exo-AAV1. n = 6 cochleas for each data point; **p < 0.01, one-tailed t test. Mean ± SEM. (E) GFP-positive hair cells in cochleas transduced with 1 × 10¹¹ GCs of conventional AAV9 or exo-AAV9. Mean ± SEM; ***p < 0.001, *p < 0.05, one-tailed t test.