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A high-efficiency AAV for endothelial cell transduction throughout the central nervous system

Abstract

Endothelial cells have a crucial role in nervous system function and mounting evidence points to endothelial impairment as a major contributor to a wide range of neurological diseases; however, tools to genetically interrogate these cells in vivo remain limited. Here, we describe AAV-BI30, a capsid that specifically and efficiently transduces endothelial cells throughout the central nervous system. At relatively low systemic doses, this vector transduces the majority of arterial, capillary and venous endothelial cells in the brain, retina and spinal cord vasculature of adult C57BL/6 mice. Furthermore, we show that AAV-BI30 robustly transduces endothelial cells in multiple mouse strains and rats in vivo and human brain microvascular endothelial cells in vitro. Finally, we demonstrate the capacity of AAV-BI30 to achieve efficient and endothelial-specific Cre-mediated gene manipulation in the central nervous system. This combination of attributes makes AAV-BI30 well suited to address outstanding research questions in neurovascular biology and aid the development of therapeutics to remediate endothelial dysfunction in disease.

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Fig. 1: AAV-BI30 enables efficient transduction of brain endothelial cells across species.
Fig. 2: AAV-BI30 efficiently transduces brain endothelial cells across the arterio–venous axis.
Fig. 3: AAV-BI30 targets endothelial cells throughout the retina and spinal cord vasculature.
Fig. 4: AAV-BI30 can be leveraged to achieve efficient endothelial-specific genetic manipulation.

Data availability

Statistical data used to generate the graphs presented throughout the manuscript are provided in the form of source data and supplementary statistical data files. The kiCAP-AAV-BI30, NLS-GFP-miR122-WPRE-pA and pAAV-CAG-Cre-miR122-WPRE-pA plasmids generated in this study have been deposited in the Addgene repository under identification numbers 183749, 183775 and 183776, respectively.

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Acknowledgements

We thank members of the Gu and Deverman laboratories for their insightful feedback throughout the study and their comments on the manuscript during drafting. This research was supported by a National Science Foundation Graduate Research Fellowship (grant no. DGE1745303) (T.K.), an HMS Mahoney Postdoctoral Fellowship (L.K.), an award from the National Institutes of Health (NIH) Common Fund and the National Institute of Neurological Disorders and Stroke through the Somatic Cell Genome Engineering Consortium (UG3NS111689) (B.D.), a Brain Initiative award funded through the National Institute of Mental Health (UG3MH120096) (B.D.), the Stanley Center for Psychiatric Research (B.D.), a Fidelity Biosciences Research Initiative (C.G.), an Allen Distinguished Investigator Award (C.G.), an AHA-Allen Initiative in Brain Health and Cognitive Impairment Award (C.G.), NIH R35NS116820 (C.G), NIH RF1DA048786 (C.G.) and NIH R01 HL153261 (C.G.). The research of C.G. was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute. C.G. is an investigator of the Howard Hughes Medical Institute. Imaging was in part performed in the Neurobiology Imaging Facility at Harvard Medical School. This facility is supported in part by the HMS/BCH Center for Neuroscience Research as part of an NINDS P30 Core Center grant (NS072030).

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Authors

Contributions

T.K., K.C., C.G. and B.D. conceived the study and interpreted the results. T.K., K.C., L.K., Q.H., J.W., Q.Z., T.B., I.T., S.P., Y.C. and D.R. performed experiments. V.K. and A.C. assisted with data processing. T.K., K.C., L.K., J.W., Y.C. and B.D. prepared the figures. T.K. wrote the manuscript with input from K.C., B.D. and C.G. and editing from L.K. and Y.C. C.G. and B.D. supervised the project. All authors discussed the experiments and read and approved the manuscript.

Corresponding authors

Correspondence to Chenghua Gu or Benjamin E. Deverman.

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Competing interests

K.C., Q.H. and B.D. are inventors on a provisional patent application filed by the Broad Institute (applicant). The specific aspects of the manuscript covered include modified AAV vectors and methods of making and using the same. B.D. is a scientific founder and scientific advisor of Apertura Gene Therapy. B.D. receives research funding from Apertura Gene Therapy, which was used to generate some of the data in this manuscript. B.D. is on the scientific advisory board of Tevard Biosciences. The remaining authors declare no competing interests.

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Integrated supplementary information

Extended Data Fig. 1 AAV-BI30 is more efficient at transducing hCMEC/D3 cells than AAV9 across a wide range of doses.

hCMEC/D3 cells were grown to confluence in a 24-well plate format. AAV9 or AAV-BI30 carrying a CAGNLS-GFP-miR122-WPRE genome was applied to cells at 0, 500, 1,000, 5,000, 10,000, or 50,000 vg/cell. 4 days post-treatment the cells were analyzed for GFP expression via flow cytometry. The profiles show data from a single sample from each condition but are representative of n = 2 replicates.

Extended Data Fig. 2 Comparison of the tropism of AAV-BI30 and AAV9.

AAV-BI30 or AAV9 vectors carrying a CAG-NLS-GFP-WPRE genome were intravenously administered to adult C57BL/6 mice at 1 × 1011 vg/animal and transduction was assessed after 7 days. Panels show endogenous NLS-GFP transgene expression in organs throughout the body. Scale bar shown is 200 μm. Images are representative of n = 3 animals per group.

Extended Data Fig. 3 Characterization of the peripheral tropism of AAV-BI30.

AAV-BI30:CAG-NLS-GFP-miR122-WPRE was intravenously administered to adult C57BL/6 mice at 1 × 1011 vg/animal and transduction was assessed after three weeks. Representative images of AAV-BI30 transduction throughout the periphery; high-zoom colocalization of GFP with endothelial markers is shown in the rightmost column. With the notable exception of lung, AAV-BI30 rarely transduced endothelial cells in the microvasculature of peripheral organs – a striking contrast to efficient transduction seen throughout the CNS vasculature. Note residual NLS-GFP expression in hepatocytes of the liver persisting in the presence of miR122 repeats in the viral genome, illustrating AAV-BI30’s potent transduction of this cell type. Transduction observed in kidney glomeruli was non-endothelial; GFP+ cells are most likely mesangial cells. Relatively strong endothelial transduction in the interlobular vessels of the renal medulla and the aorta suggest that AAV-BI30 may achieve transduction of large-diameter arteries and veins throughout the systemic circulation. Scale bars are as follows: 100 μm in fourth column from left, 15 μm in rightmost column, and 25 μm in aorta panel. Images are representative of n = 3 animals.

Extended Data Fig. 4 AAV-BI30 transduces endothelial cells throughout the BALB/cJ and rat brain.

AAV-BI30:CAG-NLS-GFP-miR122-WPRE was intravenously administered at 1 × 1011 vg/animal (BALB/cJ; top) or 1.42 × 1013 vg/kg (rat; bottom). Transduction was assessed after three (BALB/cJ) or four (rat) weeks. Images demonstrate endothelial expression of NLS-GFP transgene throughout brains of each species. Scale bars shown are 200 μm (BALB/cJ third row from left) or 100 μm (BALB/cJ rightmost row & rat). Images are representative of n = 3 BALB/cJ mice, n = 1 rat.

Extended Data Fig. 5 Endothelial transduction by AAV-BI30 is consistent across brain regions.

AAV-BI30:CAG-NLS-GFP-miR122-WPRE was intravenously administered to adult C57BL/6 mice at 1 × 1011 vg/animal and transduction was assessed after three weeks. Images demonstrate high endothelial expression of NLS-GFP transgene throughout the brain. Region-specific endothelial transduction efficiency was as follows: 86 ± 6% in cortex, 81 ± 5% in hippocampus, 85 ± 3% in thalamus, 83 ± 2% in cerebellum (mean ± s.e.m.; n = 3 animals). Compare to 84 ± 4% efficiency measured across entire brain. Scale bars are as follows: third row from left 100 μm; rightmost row 50 μm.

Source Data

Extended Data Fig. 6 The transduction profile of AAV-BI30 within the brain is highly endothelial-specific.

AAV-BI30 or AAV-BR1 vectors carrying a CAG-NLS-GFP-miR122-WPRE genome were intravenously administered to adult C57BL/6 mice at 1 × 1011 vg/animal and transduction was assessed after three weeks. Left: Representative images of rare instances of neuronal and astrocytic transduction observed following AAV-BI30 administration at the 1 × 1011 vg/animal dose; cell types of interest are demarcated with red arrowheads. Scale bar shown is 50 μm. Right: Quantification of AAV transduction in non-endothelial (GFP+ ERG-) cells per mm2 of cortex using 18 μm sagittal sections of brain. An average of 0.6 ± 0.3 and 10.8 ± 3.0 cells / mm2 (mean ± s.e.m.; n = 3 animals per group) were identified in AAV-BI30 and AAV-BR1 administered cohorts, respectively. Consistent with previous reports, neurons constituted the majority of non-endothelial cells transduced by AAV-BR1. The data presented were compared using an unpaired, two-tailed t-test (t4 = 3.37; P = 0.0281).

Source Data

Extended Data Fig. 7 AAV-BI30 efficiently transduces the brain’s largest arteries.

AAV-BI30:CAG-NLS-GFP-miR122-WPRE was intravenously administered to an adult C57BL/6 mouse at 5 × 1011 vg/animal and transduction was assessed after 3 weeks. Robust, endothelial-specific transduction was observed throughout the cerebral arteries, Circle of Willis, and the head of the basilar artery. Scale bar shown is 100 μm. Images are representative of n = 1 mouse.

Extended Data Fig. 8 AAV-BI30-mediated gene transfer enables long-term transgene expression in CNS endothelial cells.

AAV-BI30:CAG-NLS-GFP-miR122-WPRE was intravenously administered to adult C57BL/6 mice at 1 × 1011 vg/animal and transduction was assessed after approximately 5 months (152 days). Vascular counterstains displayed are as follows: ICAM2 in brain parenchyma and spinal cord; isolectin in retina; and ERG in pia vasculature. High-zoom colocalization with GFP shown in rightmost column. Scale bars are as follows: middle row 200 μm; rightmost row 50 μm. Images are representative of n = 3 mice.

Extended Data Fig. 9 AAV-BI30-mediated Cre delivery achieves efficient endothelial-specific gene deletion.

A 1 × 1011 vg/animal dose of AAV-BI30:CAG-Cre-miR122-WPRE or saline was intravenously administered to adult Cav1fl/fl mice, and Caveolin-1 protein levels were assessed after four weeks. Representative images of the brain microvasculature show strongly reduced endothelial Caveolin-1 protein levels in AAV-BI30 injected animals. Note lack of Caveolin-1 loss in the smooth muscle cell layer of arterioles, demarcated with red arrowheads. High-zoom colocalization of Caveolin-1 and ICAM2 shown in rightmost column. Scale bars are as follows: second column from right 100 μm; rightmost column 50 μm. Images are representative of n = 3 mice per group.

Extended Data Fig. 10 Comparison of engineered AAV capsids with enhanced CNS endothelial cell transduction.

The heptameric peptides used to generate the listed capsids were uniformly inserted within the surface exposed hypervariable region VIII of the AAV sequence67. Specifically, heptamers are inserted between amino acids 587 & 588 in AAV-PPS and 588 & 589 in AAVs BI30, BR1, PHP.B, PHP.V1, and PHP.V2. AAV-PHP.eB shares AAV-PHP.B’s TLAVPFK insert and additionally contains an AQ to DG mutation at amino acids 587-588 (bracketed sequence).

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Krolak, T., Chan, K.Y., Kaplan, L. et al. A high-efficiency AAV for endothelial cell transduction throughout the central nervous system. Nat Cardiovasc Res 1, 389–400 (2022). https://doi.org/10.1038/s44161-022-00046-4

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