Extracellular vesicles enhance pulmonary transduction of stably associated adeno-associated virus following intratracheal administration

Abstract

Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy.

Keywords: Extracellular vesicles; adeno-associated virus; airway mucus; cellular entry; inhaled gene therapy.

FIGURE 1

EVAAV6 prepared in HEK293 cells and harvested from the cell culture supernatant exhibits internal and/or external association of AAV6 with EV. (a) Western blot analysis of AAV6, EVs, and EVAAV6. Bands for VP1, 2 and 3 of AAV6 capsid and for syntenin‐1 (Syn‐1) and CD9 of EVs are shown. (b) ExoView analysis demonstrating that exosomes are a primary population similarly in EVs and EVAAV6. (c) Hydrodynamic diameters of AAV6, EVs, and EVAAV6 measured by DLS. (d) Representative transmission electron micrographs of AAV6, EVs, EVAAV6 and EV+AAV6. Scale bar = 50 nm.

FIGURE 2

EVAAV6 exhibits efficient penetration through human airway mucus and enhanced transduction of human bronchial epithelial (HBE) cell line. (a) Median MSD values of EVs and EVAAV6 in sputum samples spontaneously expectorated by CF patients. MSD is a square of distance traveled by an individual particulate matter within a predetermined time interval (i.e., time scale; τ = 1 s) and thus is directly proportional to the particle diffusion rate. The red dashed line indicates the MSD value of AAV6 previously measured in CF sputum (Gyorgy et al., 2014). respectively. n.s.: no significance (two‐tailed Student's t‐test) (b) Luciferase activity measured in lysates of HBE cells (16HBE14o‐) treated with EVs, EVAAV6 or EV+AAV6. n.s.: no significance, ****p < 0.0001 (one‐way ANOVA).

FIGURE 3

EVAAV6 provides widespread and enhanced reporter transgene expression in mouse lungs following intratracheal administration. (a) Representative confocal images demonstrating reporter transgene expression throughout the whole left lung lobes of mice intratracheally treated with normal saline, AAV6, EVAAV6 or EV+AAV6. Green: YFP, Blue: nuclei. Image‐based quantification of (b) coverage and (c) intensity of YFP transgene expression in whole lung lobes treated with normal saline, AAV6, EVAAV6 or EV+AAV6 (n = 5 mice per group). (d and e) Luciferase activity measured in lysates of the whole mouse lungs treated with normal saline, AAV6, EVAAV6 or EV+AAV6. Mice treated with EVAAV6 or EV+AAV6 with (d) lower and (e) higher EV content (n = 5 mice per group). n.s.: no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one‐way ANOVA).

FIGURE 4

EVAAV6 does not induce local toxicity following intratracheal administration. (a) Representative histological images and (b) a blinded histopathological scoring (n ≥ 3 mice per group) of H&E‐stained lung tissues from mice treated with normal saline, AAV6, EVAAV6, or EV+AAV6.

FIGURE 5

EVAAV6 provides enhanced reporter transgene expression in mucus‐secreting ALI cultures of primary wild‐type (WT) or cystic fibrosis (CF) HBE cells following apical administration. (a) Representative confocal images demonstrating reporter transgene expression in ALI cultures of primary WT or CF HBE cells treated with normal saline, AAV6, EVAAV6, or EV+AAV6. Green: YFP, Blue: nuclei. Image‐based quantification of (b) coverage and (c) intensity of YFP transgene expression in the ALI cultures of primary WT (black) and CF (red) HBE cells treated with normal saline, AAV6, EVAAV6, or EV+AAV6. n.s.: no significance, ***p < 0.001, ****p < 0.0001 (two‐way ANOVA).