Adeno-associated viral vectors for modeling Parkinson’s disease in non-human primates

Figures

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  Figure 1. Lewy body pathology in Parkinson’s disease. Intracellular inclusions observed in idiopathic Parkinson’s disease (Hoehn and Yahr stage 5). Neuromelanin-pigmented dopaminergic neurons often exhibit two types of intracellular inclusions, namely Lewy bodies (LBs; intracytoplasmic) and Marinesco bodies (MBs; intranuclear). Intracytoplasmic LBs (arrows) are ring-shaped structures with an intense peripheral halo and a paler center and are positive for phosphorylated alpha-synuclein, P62, and ubiquitin. Multivesicular LBs are also quite often observed (asterisk). In contrast, MBs are inclusions of smaller sizes (arrowheads) that are considered indirect indicators of advanced aging. Scale bar: 25 µm. Unpublished data.

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  Figure 2. The MPTP model of Parkinson’s disease in NHPs. Systemic delivery of the dopaminergic neurotoxin MPTP results in severe damage to the nigrostriatal system together with the appearance of clinical symptoms that typically characterize PD. Upon bypassing the blood–brain barrier, MPTP is converted into its active metabolite MPP+. By taking advantage of the dopamine transporter receptor DAT, MPP+ penetrates dopaminergic neurons and induces a blockade of the mitochondrial respiratory chain, causing severe degeneration of dopaminergic neurons in the substantia nigra. Compared to control animals (panels A–A’’), MPTP-treated macaques (panels B–B’) exhibit a profound cell loss in the substantia nigra, resulting in a severe depletion of dopaminergic terminals reaching the caudate and putamen nuclei. This can be seen using immunoperoxidase detection of tyrosine hydroxylase. Scale bars: 4000 µm in panels A & B; 2000 µm in panels A’ & B’, and 800 µm in panels A’’ & B’’. Unpublished data. DAT: Dopamine transporter receptor; MPP: 1-methyl-4-phenylpyridinium iodide; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NHP: non-human primate; PD: Parkinson’s disease.

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  Figure 3. Transduction of neurons in the dorsal root ganglia upon AAV delivery into the cisterna magna. Intra-CSF delivery of AAV9-CAG-GFP (1 mL of the viral suspension, titrated 1 × 1013 gc/mL) at the level of the cisterna magna resulted in widespread transduction of primary sensory neurons 4 weeks post-viral injection. Potential toxicity of AAVs at the level of the dorsal root ganglia has been reported without inducing a sensory deficit (Hordeaux et al., 2020). Images were taken from the left dorsal root ganglion at the spinal cord level of C7. Scale bars: 1500 µm in panel A and 750 µm in panel B. Unpublished data. AAV: Adeno-associated viral vector; CSF: cerebrospinal fluid; GFP: green fluorescent protein.

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  Figure 4. Transduction of neurons in the substantia nigra upon AAV delivery into the lateral ventricle. Intra-CSF delivery of AAV9-CAG-mCherry (1 mL of the viral suspension, titrated 1 × 1013 gc/mL) at the level of the right lateral ventricle of the frontal lobe resulted in a robust transduction of neurons in the substantia nigra four weeks post-viral administration. Images were taken at different magnifications from a coronal section through the ventral mesencephalon of a cynomolgus macaque (Macaca fascicularis), where mCherry was detected by immunoperoxidase stain. Scale bars: 2000 µm in panel A, 400 µm in panel B, and 200 µm in panel C. Unpublished data. AAV: Adeno-associated viral vector; CSF: cerebrospinal fluid.

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  Figure 5. Systemic delivery of a BBB-penetrant AAV9 capsid variant in mice. Sagittal section through mouse brain (C57BL/6) showing widespread CNS transduction following delivery of AAV9-P31-CMV-GFP into the retro-ocular vein (100 µL of the viral suspension, titrated 1.16 × 1013 gc/mL). AAV9-P31 is a BBB-penetrant capsid variant developed by Voyager Therapeutics (Lexington, MA; see Nonnenmacher et al., 2021) by taking advantage of the so-called TRACER platform (Tropism Redirection of AAV by cell-type-specific expression of RNA). Immunoperoxidase detection of GFP revealed extensive, brain-wide transduction four weeks after intravenous administration of the viral suspension. Scale bar: 2000 µm. Unpublished data. AAV: Adeno-associated viral vector; BBB: blood–brain barrier; CMV: cytomegalovirus; CNS: central nervous system; GFP: green fluorescent protein.

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  Figure 6. Disseminated synucleinopathy following the intraputaminal delivery of an AAV9 vector encoding a mutated SNCA gene. Coronal sections of the NHP brain (Macaca fascicularis) taken from the superior frontal gyrus (panel A) and the ventral mesencephalon (panels B & B’) showing cortical and nigral neurons expressing mutated alpha-synuclein with a survival time of 4 weeks post-AAV delivery. AAV9-hSyn1-SynA53T was delivered into the putamen (3 sites, 25 µL each, titrated 7.85 × 1013 gc/mL). Corticostriatal and nigrostriatal neurons innervating the putamen become transduced with mutated alpha-synuclein by taking advantage of the circuit-specific retrograde spread of AAV9 capsid (Rico et al., 2024). Scale bar: 100 µm in panels A & B’, and 1000 µm in panel B. Unpublished data. AAV: Adeno-associated viral vector; NHP: non-human primate.

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  Figure 7. The neuromelanin model of PD in NHPs. The delivery of AAV1-CMV-hTyr into the left SNc (2 sites, 10 µm each, titrated 1.7 × 1013 gc/mL) resulted in progressive pigmentation of SNc dopaminergic neurons, reaching levels similar to those observed in older adult humans 4 months post-AAV delivery (panel A). Although still visible to the naked eye, pigmentation levels were reduced after a survival time of 8 months (panel A’), reflecting underlying dopaminergic cell death. While expression of human tyrosinase is ubiquitous, pigmentation is only observed in dopaminergic cells that are equipped with the required machinery to accumulate NMel. In other words, the obtained selective pigmentation is driven by the encoded gene (human tyrosinase) instead of being promotor-related. Pigmented neurons in the ventral tier of the SNc are shown in panel B, together with microglial cells in peri-vascular locations. Most importantly, intracytoplasmic inclusions positive for standard markers of Lewy bodies (α-syn and P62) are typically observed in pigmented TH+ neurons (panel C) (Chocarro et al., 2023). Scale bars: 500 µm in panels A & A’, 100 µm in panel B, and 5 µm in panel C. Unpublished data. AAV:…

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  Figure 8. The “retropigmented” model of PD in NHPs. Four months after delivery of AAV9-hSyn1-hTyr into the left putamen (2 sites, 25 µL each, titrated 1.18 × 1013 gc/mL), clear pigmentation was observed in the left SNc (panel A). Nigro-putaminal projection neurons located in the ventral tier of the SNc exhibited NMel accumulation (panel B; Masson-Fontana stain, counterstained with neutral red). Intracytoplasmic inclusions positive for P62 and α-syn were found in pigmented TH+ neurons. Thus, the retrograde spread of AAV9 through the nigrostriatal pathway following intra-putaminal deliveries allowed the induction of NMel accumulation in dopaminergic neurons of the SNc, without the need for directly injecting into the SNc. Scale bar: 750 µm in panel A, 100 µm in panel B, and 5 µm in panel C. Unpublished data. AAV: Adeno-associated viral vector; NHP: non-human primate; NMel: neuromelanin; PD: Parkinson’s disease; SNc: substantia nigra pars compacta; TH: tyrosine hydroxylase.