Gene therapy using adeno-associated virus serotype 9 in the sheep brain

Abstract

Large animal models are vital in the validation of gene therapies, acting in a translational capacity for human studies. Sheep are particularly advantageous in this role, possessing a human-like gyrencephalic brain of comparable size and physiology. Intracerebroventricular and intraparenchymal deliveries of recombinant adeno-associated virus serotype 9 (rAAV9), encoding green fluorescent protein (GFP), were examined in naïve, healthy juvenile sheep and transgene expression analysed by immunohistochemistry 30 days later. Following intraventricular delivery, transgene distribution was observed throughout the CNS, including the prefrontal and occipital cortices, and all along the spinal cord. Robust GFP expression after intraparenchymal infusion was localised to the injection sites but transduced cells were also observed in many distal regions, including the Purkinje cells of the cerebellum and projection neurons of the frontal association and motor cortices, probably from retrograde axonal transport. Co-immunolabeling with GFP and cell-type specific markers indicated dominantly neurotropic transduction. The neuronal tropism by both delivery methods and extensive protein expression in the sheep CNS provided proof of principle for the use of rAAV9 to deliver corrective genes to ovine models. Gene therapy trials of rAAV9 vectors containing corrective CLN5 and CLN6 injected into sheep affected with CLN5 and CLN6 Batten disease are underway.