Cited by 235Virus-based gene therapy researchBarrier Structure and Function StudiesRNA Interference and Gene Delivery

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Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids

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Huang, Qin, Chan, Ken Y., Tobey, Isabelle G., Chan, Yujia Alina, Poterba, Tim, Boutros, Christine L., Balazs, Alejandro B., Daneman, Richard, Bloom, Jonathan M., Seed, Cotton, Deverman, Benjamin E.·2019·PLOS ONE

DOI 10.1371/journal.pone.0225206PMC PMC6855452

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Abstract

The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of the AAV-PHP.B vectors. Leveraging CNS tropism differences across 13 mouse strains, we systematically determined a set of genetic variants that segregate with the permissivity phenotype, and rapidly identified LY6A as an essential receptor for the AAV-PHP.B vectors. Interfering with LY6A by CRISPR/Cas9-mediated Ly6a disruption or with blocking antibodies reduced transduction of mouse brain endothelial cells by AAV-PHP.eB, while ectopic expression of Ly6a increased AAV-PHP.eB transduction of HEK293T and CHO cells by 30-fold or more. Importantly, we demonstrate that this newly discovered mode of AAV binding and transduction can occur independently of other known AAV receptors. These findings illuminate the previously reported species- and strain-specific tropism characteristics of the AAV-PHP.B vectors and inform ongoing efforts to develop next-generation AAV vehicles for human CNS gene therapy.

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Fig 1 The nonpermissive AAV-PHP.eB CNS tropism phenotype associates with Ly6 SNPs. (A) GFP within sagittal brain sections from C57BL/6J or BALB/cJ mice two weeks after intravenous administration of AAV-PHP.eB:CAG-NLS-GFP. (B) AAV capsid IHC within the cerebellum one hour after intravenous injection of AAV-PHP.eB. (C) Vector genome (vg) biodistribution of AAV-PHP.eB or AAV9 two hours after intravascular administration to C57BL/6J or BALB/cJ mice (n = 6/virus/line, mean ± s.e.m.; 2-way ANOVA). (D) The number of mouse lines predicted to be required to reduce the number of candidate variants associated with AAV-PHP.eB permissivity. The plotted lines depict the median number of simulated candidate variants; high (loss-of-function; blue) or high+medium (loss-of-function, missense, splicing variant; orange). Shaded regions represent 5–95th percentiles. (E) Native GFP fluorescence in the mouse thalamus two weeks after intravenous injection of 1011 vg/mouse CAG-NLS-GFP packaged into AAV9 (first two panels from top left) or AAV-PHP.eB. (F) Ly6a and Ly6c1 SNPs correlate with the nonpermissive phenotype. Missense SNPs relative to C57BL/6J are listed as the amino acid change. SRV, splice region variant; IV, intron variant; SDV, splice donor variant. (G) Expression data (mean fragments per kilobase-million ± S.D.) for Ly6a, Ly6c1, and the endothelial cell marker, Pecam1 [22].CC BY · PubMed Central

Fig 2 Brain Ly6a expression differs between C57BL/6J and BALB/cJ strains. (A) Representative images of LY6A IHC in sagittal brain sections from C57BL/6J or BALB/cJ mice. LY6A IHC in the striatum (B, white or C, green) with IHC for GLUT1 (magenta) to mark endothelial cells (see S2 Fig for images of LY6A IHC in brain sections from the 11 additional mouse lines). LY6C1 IHC in the cerebral cortex (D) of C57BL/6J or BALB/cJ mice. (E) Western blot from forebrain lysates reveals LY6A abundance and protein states in each mouse line (n = 4).CC BY · PubMed Central

Fig 3 Ly6a mediates enhanced binding and transduction by AAV-PHP.eB. (A) Binding of the indicated virus to HEK293T cells transfected with Ly6a or Ly6c1 or left untransfected (–). (B) Transduction measured by Luciferase assay normalized to AAV9 on mock transfected cells. (A, n = 3/group, ****p<0.0001; 2-way ANOVA, Tukey’s correction; B n = 3/group, ***p<0.001; 3-way ANOVA). (C) LY6A (magenta, top) and LY6C1 (magenta, bottom) immunostaining with nuclei (green, DAPI) in BMVECs. AAV9 and AAV-PHP.eB binding (D) and transduction (E) of BMVECs (n = 3/virus, mean ± S.D., ***p<0.0001; two-tailed t test). Binding was assessed by qPCR of the viral genome. Transduction was assessed by measuring Luciferase luminescence in relative light units (RLU). Binding (F; 2-way ANOVA, Dunnett’s multiple comparison test) and transduction (G; 1-way ANOVA, Sidak’s post test) by the indicated virus in cells treated with a vector containing an sgRNA to disrupt Ly6a or Ly6c1 or no sgRNA (n = 3/group, mean ± S.D., ****p<0.0001). Data in F and G are normalized to cells treated with a control vector lacking a sgRNA sequence. Each data point represents cells that received a different sgRNA. (H-I) AAV-PHP.eB-mediated transduction (Luciferase RLU) of BMVECs (H) or HEK293 cells mock transfected (-) or transfected with Ly6a (I) following the pre-incubation of cells with the indicated antibody (n = 2/group, H; or n = 3/group, I, #p = 0.023, ##p = 0.010, ***p = 0.001, ****p<0.0001, αLY6C vs. αLY6A, 2-way ANOVA, Tukey’s correction for multiple comparisons). (J) Virus overlay assay using lysates from HEK293T cells transfected with Ly6a cDNAs from C57BL/6J or containing one or both BALB/cJ SNPs. Panels show immunoblotting for AAV capsid proteins after overlaying with AAV-PHP.eB or AAV9. Bottom panel shows the αLY6A signal from the same blot previously overlaid with AAV-PHP.eB.CC BY · PubMed Central

Fig 4 Ly6a expression renders cells permissive to AAV-PHP.eB transduction in the absence of galactose or AAVR. (A-C) AAV-PHP.eB or AAV9 viruses were added to control Pro5 CHO cells, Lec2 CHO cells with excess galactose, or Lec8 CHO cells deficient for galactose transfer. (A) Quantification of AAV binding to CHO cell derivatives via qPCR for viral genomes. (B) Transduction of CHO cells as measured by Luciferase assay 48 hours after virus addition, normalized to values from Pro5 cells transduced with AAV9. (C) AAV-PHP.eB capsid immunostaining of CHO cells that were untransfected (top row) or transfected with Ly6a (bottom row). (D-F) WT or AAVR KO mice were intravenously injected with AAV-PHP.eB:CAG-NLS-GFP (1011 vg/mouse) and brain tissue was assessed via IHC for capsid binding (D), vector genome biodistribution analysis (E) at two hours, or transduction (F) at three weeks post-injection (D-F: n = 2 per group/per experiment, mean ± SD). (F) Representative images of GFP fluorescence (green) with LY6A IHC (magenta).CC BY · PubMed Central

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Authors: Huang, Q., Chan, K. Y., Tobey, I. G., Chan, Y. A., Poterba, T., Boutros, C. L., Balazs, A. B., Daneman, R., Bloom, J. M., Seed, C., & Deverman, B. E.
Affiliations: Broad Institute (US)
Ragon Institute of MGH, MIT and Harvard (US)
University of California San Diego (US)
Corresponding author: Deverman, Benjamin E.
ORCID: Huang, Qin
Chan, Ken Y.
Tobey, Isabelle G.
Chan, Yujia Alina
Boutros, Christine L.
Balazs, Alejandro B.
Daneman, Richard
Bloom, Jonathan M.
Deverman, Benjamin E.
Venue: PLOS ONE
Publisher: Public Library of Science (PLoS)
Published: 2019-11-14
Volume: 14
Issue: 11
Pages: e0225206
ISSN: 1932-6203 (ISSN-L)
DOI: 10.1371/journal.pone.0225206
PMC: PMC6855452
OpenAlex: W2986362275
Cited by: 234
References: 40
Citations by year: 2026: 28 · 2025: 33 · 2024: 39 · 2023: 30 · 2022: 34 · 2021: 38 · 2020: 28 · 2019: 4
FWCI: 15.67
Type: journal article
Language: en
Open access: gold
doi.org
Licence: cc-by
creativecommons.org/licenses/by/4.0/ (vor)
Locations: PLoS ONE (publishedVersion, cc-by)
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
PubMed Central (publishedVersion, cc-by)
PubMed (publishedVersion)
DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Figshare (submittedVersion, cc-by)
Funders: Stanley Center for Psychiatric Research, Broad Institute
National Institute of Neurological Disorders and Stroke (UG3NS111689)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords: blood-brain barrier, gene therapy vectors, receptor identification, mouse strain differences, brain endothelial cells, neurological disorders
MeSH terms: Dependovirus, Animals, Antigens, Ly, Blood-Brain Barrier, Brain, Cell Line, Genetic Vectors, Humans, Membrane Proteins, Neurons, Transduction, Genetic, Genetic Variation, Gene Transfer Techniques, Tropism, Transgenes, Mice

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Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids

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