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Endothelial-Tropic AAVs for Genetic Access to Whole-Brain Vasculature in Wild-Type Mouse Strains Following Non-Invasive Systemic Delivery

Chen, Xinhong and Wolfe, Damien A. and Kumar, Sripriya Ravindra and Miles, Timothy F. and Sullivan, Erin E. and Hori, Acacia M. and Ding, Xiaozhe and Gradinaru, Viviana (2021) Endothelial-Tropic AAVs for Genetic Access to Whole-Brain Vasculature in Wild-Type Mouse Strains Following Non-Invasive Systemic Delivery. Molecular Therapy, 29 (4, S1). p. 25. ISSN 1525-0016. doi:10.1016/j.ymthe.2021.04.019.

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The neurovascular unit (NVU) is a vital yet understudied component of the nervous system. Malfunction of non-neuronal cell types within the NVU, including endothelial cells, can facilitate the progression of neurological disorders (Yu et al, Frontiers in Neuroscience, 2020), but limited options for cell-type specific transgene delivery hamper its study. Adeno-associated virus (AAV) vectors for gene delivery to the brain are commonly administered via intra-cranial injections, resulting in tissue damage and limited, uneven spatial coverage. Systemic AAV delivery provides a non-invasive, brainwide alternative for genetic access. Having engineered vectors that efficiently cross the blood-brain-barrier (BBB) with broad tropism in rodents (e.g. AAV-PHP. eB), we turned our focus to engineering cell-type-specific vectors that could access vasculature without targeting other components of the NVU. Using M-CREATE directed evolution (Kumar et al, Nature Methods, 2020), we identified a family of endothelial-enriched capsid variants, including one named AAV-CAP.X1. Following intravenous (I.V.) injection, AAV-CAP.X1 targets vasculature with high cell-type specificity and efficiency throughout the body, including the brain. After injecting 3E11 vg total of AAV-CAP.X1 packaging CAG-GFP into adult C57BL/6J mice, 97% (+/- 0.8%) of the GFP+ area in the hippocampus are CD31+ (demonstrating specificity), and 73% (+/- 9.1%) of the CD31+ area in the hippocampus is GFP+ (proving efficiency; note that an increased dosage of 1E12 vg per mouse resulted in even greater CD31+ labeling without losing specificity). As AAV-CAP.X1 vascular infectivity in the periphery may complicate applications that focus on brain-specific endothelial transduction, we introduced point mutations on the AAV-CAP.X1 capsid and incorporated microRNA target sites into the cargo genome that successfully de-target AAV-CAP.X1 from the liver without impairing brain transduction. AAV-CAP.X1 can be used across multiple genetically diverse mouse strains, with efficient labeling of both capillaries and arteries in the brains of C57BL/6J, FVB/NJ, CBA/J, and BALB/cJ mice following I.V. administration. We also observed a significant increase in transduction compared to its parent capsid AAV9 on multiple human-derived cell lines in vitro. In its brain-targeted form, AAV-CAP.X1 could be paired with pre-clinical therapeutic cargo both to probe vascular contributions to neurological disease and to inform intervention strategies. More broadly, gene delivery via endothelial-tropic AAV capsids could, in principle, be applied to study diverse pathologies that may benefit from vascular remodeling. Our evolving knowledge regarding vascular pathology in COVID-19 that could underlie generalized organ dysfunction demonstrates the timeliness and potential importance of such vectors.

Item Type:Article
Related URLs:
URLURL TypeDescription
Chen, Xinhong0000-0003-0408-0813
Miles, Timothy F.0000-0001-6591-3271
Hori, Acacia M.0000-0001-5868-348X
Ding, Xiaozhe0000-0002-0267-0791
Gradinaru, Viviana0000-0001-5868-348X
Additional Information:© 2021 American Society of Gene & Cell Therapy. Available online 27 April 2021.
Issue or Number:4, S1
Record Number:CaltechAUTHORS:20210528-081450340
Persistent URL:
Official Citation:ASGCT Annual Meeting Abstracts, Molecular Therapy, Volume 29, Issue 4, Supplement 1, 2021, Pages 1-427, ISSN 1525-0016, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109291
Deposited By: Tony Diaz
Deposited On:28 May 2021 15:22
Last Modified:16 Nov 2021 19:35

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