Advancing AADC deficiency therapy through MR-guided multisite delivery of AAV2-hAADC to dopaminergic and serotonergic pathways in the brainstem

Figures

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  Figure 1. Reconstruction of infusion volumes and anatomical regions associated with contrast enhancement after multi-target infusion (A) Representative coronal sections of a macaque brain at the level of the midbrain (left) and brainstem (right), displaying regions of transduction indicated by gadolinium contrast, following AADC gene therapy. (B) Gadolinium-enhanced territories were segmented to derive distribution volumes (Vd) for each NHP, transformed into MNI Macaque space, and aggregated to generate an N-map (gold) reflecting the cumulative overlap of infusion volumes across the cohort. The 3D reconstruction features the relationship of Vd with atlas structures derived from the D99 atlas, associated with the dopaminergic (green), serotonergic (blue), and noradrenergic (red) systems. (C) Target coverage analysis showing the percentage of intersection between Vd and Gad signal. (D) N-map featuring the distribution of Vd within the midbrain and brainstem and relationship to atlas structures associated with neurotransmitter systems. Brighter regions represent areas of greater overlap, with the white outlines indicating regions of interest. The N-map is featured in sagittal planes spanning…

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  Figure 2. Safety assessment following surgical intervention and gene delivery All animals showed no adverse events following the multi-target delivery. (A) Body weight remained stable throughout the study. (B) Elevated neurofilament light chain levels were detected only at 1 month post-surgery, coinciding with the parenchymal intervention, and returned to baseline at subsequent time points until necropsy, suggesting a transient neuronal trauma likely caused by mechanical tissue disruption from the cannula insertion. (C–E) Histopathological analysis across all brain targets (C, substantia nigra/ventral tegmental area; D, dorsal raphe nucleus; E, locus coeruleus) showed no evidence of brain edema or cytotoxic response. Only minor interstitial cellular infiltration was observed at the injection site in all the targets (black arrowheads), consistent with subclinical mechanical tissue disruption and displacement associated with cannula insertion and convective delivery. Scale bars: 5 mm in low magnification and 500 μm in high magnification.

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  Figure 3. AAV2-hAADC-mediated AADC expression (A) Immunohistochemistry revealed a distinct distribution profile of AADC with increased and widespread nigral AADC expression in AAV2-hAADC-treated animals compared to the PBS group. Insets depict a digital magnification to show the nigral cells. (B) A distinct AADC distribution pattern was also detected in the dorsal raphe nucleus and (C) cell bodies of the locus coeruleus in AAV2-hAADC-treated animals compared to the PBS group. Scale bar, 5 mm

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  Figure 4. Immunohistochemical assessment of inflammation following AAV2-hAADC delivery at the midbrain level Consecutive coronal sections of the SN/VTA were stained against ionized calcium-binding adaptor molecule 1 (Iba1, A) and GFAP (B). Sections from a representative PBS-injected (left) and AADC-injected (right) animal showing (A) focal activated microglia at the injection sites and (B) mild astrocytic hyper-reaction. Scale bars: 5 mm in low magnification and 500 μm in high magnification.

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  Figure 5. Immunohistochemical assessment of inflammation following AAV2-hAADC delivery at the midline level of the brainstem Consecutive coronal sections of the DRN (A and B) and LC (C and D) were also stained against Iba1 (A) and GFAP (B). Sections from a representative PBS-injected (left) and AADC-injected (right) animal showing (A) focal activated microglia at the injection sites and (B) astrocytic hyper-reaction. Scale bars: 5 mm in low magnification and 500 μm in high magnification.