MR-guided parenchymal delivery of adeno-associated viral vector serotype 5 in non-human primate brain

Abstract

The present study was designed to characterize transduction of non-human primate brain and spinal cord with AAV5 viral vector after parenchymal delivery. AAV5-CAG-GFP (1 × 10¹³ vector genomes per milliliter (vg ml^-1)) was bilaterally infused either into putamen, thalamus or with the combination left putamen and right thalamus. Robust expression of GFP was seen throughout infusion sites and also in other distal nuclei. Interestingly, thalamic infusion of AAV5 resulted in the transduction of the entire corticospinal axis, indicating transport of AAV5 over long distances. Regardless of site of injection, AAV5 transduced both neurons and astrocytes equally. Our data demonstrate that AAV5 is a very powerful vector for the central nervous system and has potential for treatment of a wide range of neurological pathologies with cortical, subcortical and/or spinal cord affection.

Figure 1

GFP expression after bilateral delivery of AAV5-CAG-GFP into putamen. GFP immunostaining (brown) showed optimal coverage at the injection sites of both putamina (a). Gadolinium signal was mostly contained within target structures as shown in the 3D MRI reconstruction (b). Strong signal was found in the injection site (c), while no GFP signal was detected in other subcortical regions like caudate or thalamus (d). No cortical transport was found as revealed by the absence of GFP-transduced cells in different parts of the cortex (e) and other subcortical areas (f). Only substantia nigra pars reticulata (g) and globi pallidi (h) were found transduced. No transduced motor neurons were found in the spinal cord along cervical, thoracic or lumbar regions (i). Sections were processed with DAB immunostaining counterstained with Nissl (blue). Scale bars=5 mm for a and f; 100 μm for c–e; 500 μm for g and h; 2 mm for i. Cd, caudate nucleus; GP, globus pallidus; Occ, occipital cortex; Par, parietal cortex; PF, pre-frontal cortex; Tem, temporal cortex; Th, thalamus; TH, tyrosine hydroxylase.

Figure 2

GFP expression after bilateral delivery of AAV5-CAG-GFP into thalamus. DAB immunostaining (brown) illustrating GFP expression derived from bilateral vector delivery into the thalamus (a). Gadolinium signal was mostly contained within target structures as shown in the 3D MRI reconstruction (b). Strong cellular transduction occurred primarily at the brain nuclei receiving the AAV5 vector infusion (c). In contrast to putaminal infusion, GFP-positive cells were detected in distal brain regions such as caudate, putamen and subthalamic nucleus (d). Cortical transduction was also found along antero-posterior axis of the brain (e). Interestingly, positive GFP cells were found in substantia nigra reticulata (f) and globi pallidi (g), suggesting retrograde transport after thalamic infusion. Strong motor neuron transduction was found in the spinal cord along cervical, thoracic or lumbar regions (h). Sections were processed with DAB immunostaining counterstained with Nissl (blue). Scale bars=5 mm for a and e; 100 μm for c, f (bottom), g and h (bottom); 200 μm for d and f (top); 2 mm for h (top). Insets are digital magnification to show details of the areas pointed by white or black arrowheads.

Figure 3

GFP expression after delivery of AAV5-CAG-GFP into left putamen and right thalamus. Infusion of AAV5-CAG-GFP vector into the brain resulted in a widespread expression of the transgene throughout target structures for all animals. DAB immunostaining (brown) showed GFP expression into cortical and subcortical structures along prefrontal and occipital regions of the brain. Histology analysis of the anatomical targets showed a massive transduction both after thalamic (b) and putaminal infusions (c). Sections were processed with DAB immunostaining counterstained with Nissl (blue). Scale bars=5 mm for a.

Figure 4

Axonal transport after thalamic and putaminal combined delivery of AAV5-CAG-GFP. Delivery of AAV5-GFP vector in a combined approach resulted in a more extensive vector propagation along cortical and subcortical brain structures. Thalamic injection on the left hemisphere (a) showed GFP-positive cells in structures that receive thalamic projections like putamen and subthalamic nucleus (anterograde transport, blue arrows), and in brain nuclei that project to thalamus like globus pallidus and substantia nigra pars reticulate (retrograde transport, orange arrows). Putaminal injection on the right hemisphere (b) showed GFP-positive cells in structures that project to putamen like thalamus and SNpc (retrograde transport, orange arrows), while structures that receive putaminal projections like globus pallidus and substantia nigra pars reticulata showed GFP-positive fibers (anterograde transport, blue arrows). Scale bars=100 μm for a and b. Ctx, cortex; GP, globus pallidus; Put, putamen; SNpc, substantia nigra pars compacta; STN, subthalamic nucleus; Th, thalamus; TH, tyrosine hydroxylase.

Figure 5

Expression of GFP in spinal cord after thalamic/putaminal infusion of AAV5-CAG-GFP. GFP-positive motor neurons were present in the ventral horns at all levels of the spinal cord (a). GFP signal was found also in dorsal horns at all levels (b) and at the dorsal root ganglia entrance (c), being more pronounced at cervical than in the other levels. Scale bars=100 μm for a–c.