Fig. 2: Transduction and labeling efficiency of surface modified AAVs.
From: Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation
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).
