Exosome-associated AAV vector as a robust and convenient neuroscience tool

Abstract

Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill, and time intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared to standard AAV. Here we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation, results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing 3-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex-vivo serial 2-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

“…exo-AAV8 vectors showed preferential tropism and superior transduction efficiency in the liver compared with standard AAV8 vectors (approximately twofold and approximately fourfold increase in male and female animals, respectively; Figure 2B). In agreement with previous publications, 16 an increase in transduction of brain was observed in mice treated with exo-AAV8 ( Figure 2B); skeletal muscle also seemed to be transduced at higher levels in exo-AAV8-treated mice (supplemental Figure 3).…”

“…13 The natural release of a fraction of recombinant AAV vectors associated with exosomes (exosome-associated AAVs [exo-AAVs]) in the cell medium during vector production has been recently described. 14 The use of exo-AAV vectors has been demonstrated to enhance transduction in the setting of gene transfer to the retina, 15 the nervous system, 16 and the inner ear. 17 In the present study, we tested the ability of exo-AAV vectors to transduce the liver and drive efficient correction of hemophilia B.…”

Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV

“…Nevertheless, we have previously reported low levels of gene transfer to inner ear hair cells after intravenous injection of exo-AAV in adult mice, suggesting that it may be possible to transduce both ears under certain conditions. 19 The average peak 2 amplitudes at 90 dB SPL were 0.88 ± 0.18 and 0.84 ± 0.12 mV (mean ± SEM) at 8 and 11 kHz, respectively, which is approximately 25% of those in normal heterozygotes ( Figure 6C). Latencies of peak 1 and peak 2 were not significantly increased in rescued animals compared to wild-type (WT) animals at the same SPL, except at 11 kHz for peak 1 ( Figure 6C; p < 0.01, two-tailed t test).…”

“…Exo-AAV is relatively easy to purify, 19 allowing rapid production of several different constructs for experimental testing. In contrast, purification of conventional AAV is a more complicated and timeconsuming process.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…Moreover, the coating with EV membranes potentiated the AAV's ability to cross the BBB. The mechanism behind this phenomenon is currently unknown [162]. Overall, the above mentioned features denote that this hybrid system is a potentially interesting therapeutic tool, combining the best of two worlds.…”