Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation

Abstract

Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t_1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

Fig. 1. Strategy for labeling AAVs with a positron emitter.

a Table presents the number of AAVs systemically injected and the molar activity of positron emitters. b Solvent accessible surface of AAV9 capsid (PDB ID:3UX1) displayed by PyMOL software. Insets highlight a trimer around a threefold axis. Orange and green color represent lysine and cysteine residues, respectively. Solvent radius is set as 1.4 angstrom. AAV capsid is composed of 60 structurally identical viral protein subunits (VPs) with 1:1:10 ratio of VP1:VP2:VP3. c, d are the labeling schemes of AAV-PHP.eB, AAV9, and AAV9-TC. c Surface modification with multichelators (MC) on lysine residues in capsids. (NOTA)₈-TCO (incorporating a PEG₂₇ spacer) is employed for the radiolabeling of Tz-AAV9 or Tz-PHP.eB after reaction of Tz-NHS ester with AAV9 or PHP.eB. (i, tetrazine-PEG₅-NHS (Tz-NHS) ester, 1x PBS (pH 8), 4 °C, overnight dialysis in 20 kDa molecular weight cut-off (MWCO) membrane). d The site-specific radiolabeling on cysteine residues in AAV9-TC was employed with the multichelator-maleimide conjugate ((NOTA)₈-MI) incorporating Cu-64 after the reduction of tetracysteine with TCEP (ii, TCEP in 1x PBS (pH 7.0–7.5)). Asterisk indicates average molar radioactivity of Cu-64 from commercial vendor as used in this study.

Fig. 2. Transduction and labeling efficiency of surface modified AAVs.

a Fluorescence microscopy images of human embryonic kidney(HEK) 293T cells after 24 h incubation with intact AAVs (upper row, PHP.eB, AAV9, and AAV9-TC) and corresponding modified AAVs (lower row, Tz-PHP.eB, Tz-AAV9, and HS-AAV9-TC) at 1 × 10⁶ AAV/cell. b Percentage of green fluorescent positive (GF⁺) HEK293T cells 2 days after incubation with unmodified AAVs (PHP.eB, AAV9, and AAV9-TC, white bar with black circles) and the corresponding modified AAVs (gray bar with black squares), assessed by flow cytometry. The frequency of GF⁺ cells treated with unmodified and modified AAVs was similar and distinct from non-treated (NT, black triangles) cells (n = 4 per group). c Representative GFP images of sagittal brain sections from a C57BL/6 mouse at 3 weeks after tail vein administration of ⁶³Cu-PHP.eB, PHP.eB (1.5 × 10¹⁰ vg) or saline (negative control) and d mean fluorescence intensity (MFI) of sagittal brain sections (⁶³Cu-PHP.eB: gray bar with black squares, PHP.eB: white bar with black circles, saline: black triangles, n = 4). e SDS-PAGE of modified AAVs (Tz-PHP.eB, Tz-AAV9, and HS-AAV9-TC; lane 1) and radiolabeled AAVs (⁶⁴Cu-PHP.eB, ⁶⁴Cu-AAV9, and ⁶⁴Cu-AAV9-TC; lane 2 and 3). The three bands depict viral protein (VP) 1–3 (L: standard protein ladder). Lane 1–3 illustrate blue-stained VPs (lanes 1 and 2) and radiolabeled VPs (lane 3), respectively. f Illustration of AAV9 capsid with modified lysines. Left: full view of AAV9, middle and right: top and side views of trimer viral proteins, respectively. The K557 (yellow) and K567 (red) lysine residues are highlighted. g Field view of direct-electron cryoEM images of PEG(40 kDa)-AAV9 (left image) and enhanced projection images of selected PEG(40 kDa)-AAV9 capsids (six right images). White arrows mark the 40 kDa PEG molecules extended from the selected AAV capsids. Data are shown as mean ± SD. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparison test compares means (b, d). Significance is presented as n.s. (not significant), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. Whole gel and gel autoradiography images and P values are shown in the source data. Scale bars: 100 μm (a), 2 mm (c), 50 nm (g, left), 20 nm (g, right).

Fig. 3. PET and optical imaging-based assessment of AAV pharmacokinetics in C57BL/6 mice.

a Experimental setup for region of interest (ROI) analysis (0, 4, and 21 h) and biodistribution (21 h) of ⁶⁴Cu-PHP.eB, -AAV9, and -AAV9-TC. PET images are acquired at 0, 4 and 21 h after AAV tail vein administration. b Projected PET/CT images at 4 (left) and 21 h (right) (H heart, L liver, S spleen, B brain). c Time activity curves (over 21 h) and d maximum brain uptake (at 4 h) of ⁶⁴Cu-PHP.eB (magenta triangle), ⁶⁴Cu-AAV9 (black circle), and ⁶⁴Cu-AAV9-TC (turquoise square) from the ROI analysis of blood and brain (n = 3) after tail vein administration. e Representative projected PET/CT image at 4 h of ⁶⁴Cu-PHP.eB within the brain (B brain, JV jugular vein). f PK (left) and 21-h biodistribution (right) of PHP.eB (n = 3, black circle) and (NOTA)₈-A555-PHP.eB (n = 4, black squares) obtained by qPCR. g Sliced PET/CT, autoradiography and GFP images of sagittal section of mouse brain (CB cerebellum, M midbrain, Th thalamus, CC cerebral cortex, S striatum) acquired at 21 h, 21 h and 3 weeks, respectively, after tail vein injection of ⁶⁴Cu-PHP.eB for PET/CT and autoradiography and non-radioactive ⁶³Cu-PHP.eB for the GFP image. h ⁶⁴Cu-AAVs brain accumulation (n = 3 per group) measured 30 min after tail vein administration (left) and Logan plots (right) of brain uptake rate after AAV administration. i Representative confocal images of (NOTA)₈-A555-PHP.eB (red) on brain endothelium (green) acquired 4, 24, and 48 h after tail vein injection. White arrows indicate (NOTA)₈-A555-PHP.eBs (red). Data are shown as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test (c, d, and h (left)) compared means of the three groups. Multiple unpaired t-tests with the Holm-Sidak method with alpha = 0.05 compared the means in f. Significance: n.s. (not significant), *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. P values are shown in the source data. Intensity values in b, d, e, and (g, left) are percent injected dose per cubic centimeter (% ID/cc). Scale bars: 2 mm (g), 25 μm (i).

Fig. 4. Strain and neuraminidase-dependent pharmacokinetics of ⁶⁴Cu-PHP.eB.

a Time activity curves of PHP.eB obtained from region of interest (ROI) analysis of blood (left) and brain (right) from C57BL/6 (black circle), BALB/c (turquoise square), and neuraminidase-treated BALB/c (magenta triangle) mice over 21 h (n = 3 per group). Radioactivity from ROI analysis is presented as % ID/cc. b Representative PET/CT projection images (B brain, H heart, L liver) acquired over 30 min after tail vein injection of ⁶⁴Cu-PHP.eB to C57BL/6 and BALB/c mice. c Biodistribution (% ID/g) of ⁶⁴Cu-PHP.eB in brain (left) and blood (right) in C57BL/6 (gray bar with black squares) and BALB/c (white bar with black circle) mice at 21 h (n = 3). d Sliced PET/CT image (H heart, L liver) at 4 h after tail vein injection of ⁶⁴Cu-PHP.eB. e Time activity curve of ⁶⁴Cu-PHP.eB measured from C57BL/6 (black circle) and BALB/c (turquoise square) livers (n = 3). f Biodistribution (21 h) of ⁶⁴Cu-PHP.eB in blood (left), brain (middle), and lung (right) from BALB/c with no treatment white bar with black circle) versus BALB/c mice treated with neuraminidase (gray bar with black square) (n = 3). Data are shown as mean ± SD. For statistical analysis, a one-way ANOVA with Tukey’s multiple comparison test in a was performed to compare means of three groups (C57BL/6 vs BALB/c: turquoise, C57BL/6 vs BALB/c (neuraminidase): magenta, BALB/c vs BALB/c (neuraminidase): black) at each time point. Unpaired two-tailed Welch’s t-test was performed in c, (e, 0, 4, and 21 h) and f. Significance is presented as *P ≤ 0.05, **P ≤ 0.01. P values are shown in the source data. Maximum and minimum intensity values of PET/CT images in b, d are presented as percent injected dose per cubic centimeter (% ID/cc).