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. 2018 Feb 7;26(2):510-523.
doi: 10.1016/j.ymthe.2017.10.017. Epub 2017 Oct 26.

Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier

Blake H Albright  1 , Claire M Storey  2 , Giridhar Murlidharan  1 , Ruth M Castellanos Rivera  2 , Garrett E Berry  1 , Victoria J Madigan  1 , Aravind Asokan  3
Affiliations

Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier

Blake H Albright et al. Mol Ther. .

Abstract

Effective gene delivery to the CNS by intravenously administered adeno-associated virus (AAV) vectors requires crossing the blood-brain barrier (BBB). To achieve therapeutic CNS transgene expression, high systemic vector doses are often required, which poses challenges such as scale-up costs and dose-dependent hepatotoxicity. To improve the specificity and efficiency of CNS gene transfer, a better understanding of the structural features that enable AAV transit across the BBB is needed. We generated a combinatorial domain swap library using AAV1, a serotype that does not traverse the vasculature, and AAVrh.10, which crosses the BBB in mice. We then screened individual variants by phylogenetic and structural analyses and subsequently conducted systemic characterization in mice. Using this approach, we identified key clusters of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice. Through rational design, we mapped a minimal footprint from AAVrh.10, which, when grafted onto AAV1, confers the aforementioned CNS phenotype while diminishing vascular and hepatic transduction through an unknown mechanism. Functional mapping of this capsid surface footprint provides a roadmap for engineering synthetic AAV capsids for efficient CNS gene transfer with an improved safety profile.

Keywords: AAV; blood-brain barrier; neurotropic.

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Figures

Figure 1
Figure 1
Phylogenetic and Structural Analyses of the AAV1/rh.10 Domain Swap Capsid Library (A) Schematic of the panel of representative AAV1/rh.10 chimeric capsids isolated from the library, demonstrating sequence diversity, at the amino acid level, of domain swaps obtained through shuffling. Individual parental or chimeric capsids are listed vertically, whereas the VP1 capsid sequence with different domain swaps is displayed horizontally, from the N terminus on the left to the C terminus on the right. AAV1-derived residues are shown in gray, AAVrh.10 residues in green-cyan, and consensus residues between the two in black. (B) Neighbor-joining phylogeny of the VP1 capsid sequences of AAV1/rh.10 chimeric capsids. Capsid amino acid sequences were aligned with ClustalW, the phylogeny was generated using a neighbor-joining algorithm, and a Poisson correction was used to calculate amino acid distances, represented as units of the number of amino acid substitutions per site. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the tree. Bootstrap values were calculated with 1,000 replicates, and the percentage of replicate trees in which the associated taxa clustered together are shown next to the branches. (C) Three-dimensional surface models of capsid subunit trimer/3-fold symmetry axis regions of parental and representative chimeric AAV capsid mutants selected for in vivo screening. Amino acid residues derived from AAV1 are shown in gray, whereas residues derived from AAVrh.10 are shown in green-cyan. Structural models were visualized and generated using PyMOL.
Figure 2
Figure 2
In Vivo Screen Yields AAV1/rh.10 Chimeras Capable of Crossing the Blood-Brain Barrier following Intravenous Administration Shown are scans of immunostained brain sections showing GFP expression in the cerebral cortex mediated by parental or chimeric vectors packaging a CBh-scGFP transgene 3 weeks after administration via tail vein injection at a dose of 5 × 1011 vg (or PBS in the case of mock treatment). The global coronal brain section (top) indicates the boxed region of the cortex seen in the insets shown for each parental or chimeric vector, representing their individual transduction profiles. Scale bars, 100 μm.
Figure 3
Figure 3
CNS Transduction Profile of AAV1R6 and AAV1R7 Compared with Parental AAV1 and AAVrh.10 in the Brain (A–H) Transduction profiles 3 weeks after tail vein injection of AAV vectors packaging a CBh-scGFP transgene at a dose of 5 × 1011 vg for parental serotypes, AAV1 and AAVrh.10 (left columns), and for AAV1R6 and AAV1R7 (right columns) across various brain regions, including the (A) motor cortex, (B) cerebral (somatosensory) cortex, (C) hippocampus, (D) dentate gyrus, (E) thalamus, (F) hypothalamus, (G) striatum, and (H) amygdala. Scale bars, 100 μm.
Figure 4
Figure 4
Quantitative Comparison of Neuronal and Glial Transduction Levels for Parental and Chimeric Capsid Variants (A–H) Relative neuronal transduction levels 3 weeks after administration of vectors packaging a CBh-scGFP transgene via tail vein injection at a dose of 5 × 1011 vg were quantified by counting the number of transduced neurons and glia, identified based on morphology, for each brain region per 50 μm coronal section. Transduction of parental serotypes, AAV1 and AAVrh.10, are shown alongside the chimeric variants, AAV1R6 and AAV1R7, across various regions of the brain, including the (A) motor cortex, (B) cerebral (somatosensory) cortex, (C) hippocampus, (D) dentate gyrus, (E) thalamus, (F) hypothalamus, (G) striatum, and (H) amygdala. Error bars represent SD (n > or = 3). An unpaired two-tailed t test with Welch’s correction and one-way ANOVA were used to demonstrate statistical significance for neuronal and glial transduction by each group relative to AAV1 and that differences between the means were statistically significant, respectively. ns, not significant; *p < 0.05.
Figure 5
Figure 5
Relative Cardiac and Liver Transduction by AAV1R6 and AAV1R7 Compared with Parental Capsids Either parental (AAV1 or AAVrh.10) or chimeric (AAV1R6 or AAV1R7) vectors packaging a CBh-scGFP transgene were administered at a dose of 5 × 1011 vg via tail vein injection. 21 days after injection, heart and liver sections were immunostained for GFP and imaged. (A) GFP fluorescence for cardiac (top) and liver (bottom) tissues in mice treated with either PBS (Mock), AAV1, AAVrh.10, AAV1R6, or AAV1R7. (B) Transduction levels for heart (top) and liver (bottom), measured by quantifying relative fluorescence across multiple images taken for each treatment. The bars represent the range from lowest to highest values, with the center line representing the average across samples. Relative fluorescence was normalized to mock-treated tissues. Error bars represent SD (n = 3). One-way ANOVA and unpaired two-tailed t test with Welch’s correction for each group were carried out, and significance relative to AAVrh.10 is shown. *p < 0.05.
Figure 6
Figure 6
Rational Design and Functional Mapping of a Minimal AAVrh.10 Footprint for Crossing the BBB (A) The sequence alignment of AAV1RX with AAV1 and AAVrh.10 shows the numbered amino acid residues within and adjacent to the neurotropic footprint. Conserved residues are highlighted in yellow, the residues composing the footprint are highlighted in cyan, and non-conserved residues are highlighted in either green or white. Structural models of the engineered AAV1RX chimeric capsid were generated using SWISS-MODEL software for homology-based modeling, and VIPERdb was used to generate oligomers. (B–F) PyMOL was used to generate surface-rendered models of the AAV1RX VP3 subunit (B) monomer, (C) trimer dimer/2-fold axis of symmetry, (D) trimer/3-fold axis of symmetry, (E) pentamer/5-fold axis of symmetry, and (F) the full capsid (60-mer). AAV1-derived amino acids and those homologous between AAV1 and AAVrh.10 are represented in gray, whereas the residues comprising the neurotropic footprint from AAVrh.10 for crossing the BBB are depicted in green-cyan. (G) The stereographic roadmap projection shows the neurotropic footprint as viewed down the 3-fold symmetry axis on the AAV1RX capsid and was generated using RIVEM. Only surface-exposed amino acid residues are shown, with the boundaries between each delineated with black lines. The green regions depict the topological protrusions at the 3-fold axis of symmetry, whereas the blue regions represent topological depressions. The key amino acid residues of this footprint are colored magenta (AAVrh.10 VP1 numbering). (H) CNS transduction profile of AAV1RX packaging a CBh-scGFP transgene administered via tail vein injection at a dose of 5 × 1011 vg. Sections taken 21 days after injection were immunostained and imaged. Scale bars, 100 μm.
Figure 7
Figure 7
Peripheral Tissue Transduction and Biodistribution of AAV1R6, AAV1R7, and AAV1RX following Intravenous Administration (A and B) Mice were injected via the tail vein with PBS or with either AAV1, AAVrh.10, AAV1R6, AAV1R7, or AAV1RXvectors packaging a CBA-Luc reporter transgene at a dose of 1 × 1011 vg. 2 weeks post-injection, (A) luciferase reporter transgene expression levels and (B) biodistribution of viral genome copies across various tissues were quantified. Luciferase expression levels were normalized to grams of tissue lysate and are represented as relative light units. vg copies are normalized to mouse lamin B (mLamB) as an endogenous housekeeping gene and are represented as vg copies per cell. Error bars represent SD (n = 3 for luciferase assays, n = 4 for biodistribution). One-way ANOVA and an unpaired two-tailed t test with Welch’s correction for each group were carried out, and significance relative to AAVrh.10 is shown. *p < 0.05.
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