Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Henriques, C.; Lopes, M. M.; Albuquerque, P.; Rufino-Ramos, D.; Gaspar, L. S.; Lobo, D.; Leandro, K.; Silva, A. C.; Baganha, R.; Duarte, S.; Maguire, C. A.; Santana, M.; Pereira de Almeida, L.; Nobre, R. J.

doi:10.1101/2023.05.30.542901

bioRxiv · 10.1101/2023.05.30.542901

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Henriques, C.·Lopes, M. M.·Albuquerque, P.·Rufino-Ramos, D.·Gaspar, L. S.·Lobo, D.·Leandro, K.·Silva, A. C.·Baganha, R.·Duarte, S.·Maguire, C. A.·Santana, M.·Pereira de Almeida, L.·Nobre, R. J.

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Abstract

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

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Henriques, C., Lopes, M. M., Albuquerque, P., Rufino-Ramos, D., Gaspar, L. S., Lobo, D., Leandro, K., Silva, A. C., Baganha, R., Duarte, S., Maguire, C. A., Santana, M., Pereira de Almeida, L., Nobre, R. J.. 2023-05-30. Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography. https://doi.org/10.1101/2023.05.30.542901

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