Extended Data Fig. 5. AAV-PHP.AX characterization following single injection in mouse brain.

a, AAV-PHP.AX was engineered by substituting a 7-amino acid (AA) sequence of a glial homing peptide (green), at AA452-458 of AAV-PHP.eB (9-AA peptide shown in red). b, AAV9 and AAV-PHP.AX (each packaging a single-stranded CAG-eGFP genome) were delivered by retro-orbital injection to 8-week-old C57BL/6 J male mice (n ≥ 3 per group) at 3 × 10¹¹ vg/mouse. Transgene expression was evaluated three weeks later. c, Representative images of human U87 glioblastoma (astrocyte-like) cells transduced by AAV-PHP.AX or AAV-PHP.eB, packaging CAG-eGFP, 48 or 72 hours post-infection (n ≥ 3 wells per condition). d, e, AAV-PHP.AX transduces astrocytes more efficiently than AAV-PHP.eB when paired with an astrocytic promoter. AAV:GFAP-2xNLS (nuclear localization signal)-mTurq2 was delivered by retro-orbital injection to 8-week-old C57BL/6 J male mice (n = 3 per group) at 1.5 × 10¹¹ vg/mouse. Transgene expression was evaluated three weeks later. d, Brain sections were stained with the astrocyte marker S100β (red). Representative images of the cortex, hippocampus, and thalamus are shown. e, Quantification of the percentage of S100β + cells transduced by AAV-PHP.AX and AAV-PHP.eB (mean ± SD; two-way ANOVA, Tukey’s multiple comparisons test with adjusted p values; ***p = 0.0001 for cortex; ***p = 0.0006 for hippocampus; *p = 0.0217 for thalamus). f, AAV-PHP.AX efficiently transduces the brain after intravenous administration to C57BL/6 J, DBA2/J, and FVB mice. AAV-PHP.AX:CAG-2xNLS-eGFP was delivered by retro-orbital injection to 8-week-old male mice (n ≥ 5 per group) at 3 × 10¹¹ vg/mouse. Transgene expression was evaluated three weeks later. Representative images of the brain and liver are shown. DAPI staining for nuclei is shown in blue.