Pilot Study Assessing the Impact of Intrathecal Administration of Variants AAV-PHP.B and AAV-PHP.eB on Brain Transduction in Adult Rhesus Macaques

FIGURE 1. Stronger widespread brain EGFP distribution after intrathecal injection of AAV-PHP. eB compared to variants AAV-PHP. B in non-human primates. (A–B) Representative coronal brain sections at two different rostrocaudal levels (top, “STR” = striatal level, AC0; bottom, “SN” = Substantia Nigra level, AC-8) of EGFP immunostaining (left) and illustrative photomicrographs (right) of EGFP immunostaining in the precentral gyrus (purple square), in the caudate nucleus (light blue square), in the putamen (green square), in the globus pallidus (dark blue square), in the white matter (orange square) and the substantia nigra (yellow square) in monkeys injected with either AAV-PHP. B (A) or AAV-PHP. eB (B). Scale bars = 5 mm (left, sections) and 40 µm (right, insets). (C) Quantitative analysis of GFP immunostaining measured by the surface threshold in all analyzed regions, including the precentral gyrus, the caudate nucleus, the putamen, the globus pallidus, the white matter, and the substantia nigra in AAV-injected monkeys. Data represent mean ± SEM.

FIGURE 2. Stronger widespread brain EGFP distribution after intrathecal injection of AAV-PHP. eB compared to variants AAV-PHP. B in the cerebellum of non-human primates. (A) Representative coronal brain sections at the cerebellum level (AC-17 mm) of EGFP immunostaining (left) and illustrative photomicrographs (middle and right) of EGFP immunostaining in the cerebellum in monkeys injected with either AAV-PHP. B (up) or AAV-PHP. eB (down). Scale bars = 2 mm (left, sections), 1 mm (middle, sections) and 100 µm (right, insets). (B) Quantitative analysis of EGFP immunostaining measured by surface threshold analyzed on four sections of the cerebellum in AAV-injected monkeys. Data represent mean ± SEM.

FIGURE 3. Stronger widespread brain EGFP distribution after intrathecal injection of AAV-PHP. eB compared to variants AAV-PHP.B. Representative images and quantification of EGFP expression analyzed across brain lysates of AAV-injected monkeys.

FIGURE 4. Neuronal cell-type specificity resulting from AAV-PHP. B and AAV-PHP. eB injections in the putamen of non-human primates. (A–B) Representative images (top) and quantifications (down) of striatal sections from AAV-PHP. B-injected monkey (A) and AAV-PHP. eB-injected monkey (B) immunofluorescently labeled with anti-GFP antibody (green) and either a neuronal (NeuN), microglia (Iba1), astrocytic (GFAP + S-100) or oligodendroglial (CNPase) marker (red). Merging of the signals produced colocalization, thus confirming cellular transduction. Scale bar: 20 µm; 10 µm (high magnification). Quantitative analysis of percentage of cell types counted for each viral vector in the putamen of injected monkeys (grey) and percentage of cells transduced (dark green) or not (light green) per cell type.

FIGURE 5. Assessment of the cell-type specificity resulting from AAV-PHP. B and AAV-PHP. eB injections in the hippocampus of non-human primates. (A–B) Representative images (up) and quantifications (down) of striatal sections from AAV-PHP. B-injected monkey (A) and AAV-PHP. eB-injected monkey (B) immunofluorescently labeled with anti-GFP antibody (green) and either a neuronal (NeuN), microglia (Iba1), astrocytic (GFAP + S-100) or oligodendroglial (CNPase) marker (red). Merging of the signals produced colocalization, thus confirming cellular transduction. Scale bar: 20 µm; 10 µm (high magnification). White arrows represent the AAV-GFP transduced cells. Quantitative analysis of percentage of cell types counted for each viral vector in the hippocampus of injected monkeys (grey) and percentage of cells transduced (dark green) or not (light green) per cell type.

FIGURE 6. Broad transduction of the spinal cord and liver resulting from AAV-PHP. B and AAV-PHP. eB injections in non-human primates. (A) Representative image of spinal cord sections at three levels (cervical, thoracic, lumbar) immunostained with EGFP in both injected monkeys. Scale bar: 1 mm. (B) Representative image of liver sections immunostained with a GFP antibody in both injected monkeys. Scale bar: 200 µm.