Biodistribution of adeno-associated virus type-2 in nonhuman primates after convection-enhanced delivery to brain

Abstract

A combination treatment of AAV2-hAADC with oral levodopa is a novel therapeutic approach that is being developed for late-stage Parkinson's disease. Biodistribution of AAV2-hAADC was assessed over a wide range of vector dose in 12 monkeys with parkinsonian syndrome, 6 months after intraputamenal infusion. Quantitative PCR (Q-PCR) from all the major neuroanatomical regions of the brain indicated a dose-dependent increase in vector DNA, with 99% being detected in the target site and other basal ganglia tissues. Within these tissues, the distribution varied widely between the putamen (PT) and the globus pallidus, and this was attributed to differences in vector transport. Q-PCR and immunocytochemistry were consistent with results reported earlier for various measures of transgene expression including aromatic L-amino acid decarboxylase (AADC) activity assays, behavioral response, and in vivo imaging with positron emission tomography (PET). Outside of the brain, trace amounts of vector DNA were detected in the spleens of animals in the two highest dose groups, but not in any other peripheral tissue, blood, or cerebrospinal fluid. Some increase in neutralizing antibody titers to adeno-associated virus type-2 (AAV2) capsid protein was observed in monkeys that received high doses of AAV2-hAADC or control AAV2-GFP. This study further validates convection-enhanced delivery (CED) as the preferred method of viral vector delivery to the brain, and supports a Phase I clinical testing of AAV2-hAADC in humans with Parkinson's disease.

Figure 1. Average density of human aromatic l-amino acid decarboxylase (hAADC)-positive cells in putamen

The average density of distribution of hAADC-positive cells (mean cell count (±SD) per mm²) that was detected by immunohistochemistry was estimated in the putamen for all animals, using National Institutes of Health Image software. The density of distribution was determined using a counting grid at six randomly selected sites in each section, and six sections (on average) were examined from each animal. Although AAV2-hAADC was infused into both hemispheres, only the side contralateral to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intracarotid artery administration was examined, because the ipsilateral side was processed for PCR and high-performance liquid chromatography analyses. The dose units are therefore represented as per hemisphere. *Control animals received 0 U AAV2-hAADC and 500 U AAV2-GFP. Avg, average; AAV2, adeno-associated virus type-2; GFP, green fluorescent protein.

Figure 2. Average area of AAV2-hAADC expression in putamen

Area of human aromatic l-amino acid decarboxylase (hAADC) distribution within the putamen was determined on the side of the brain contralateral to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intracarotid artery administration. Random sections through the putamen were selected and the area (mean mm² ± SD) in which hAADC-positive cells were present was calculated using National Institutes of Health Image software. Six sections, on an average, were evaluated from each animal, and n = 2 animals/group. The dose units are represented as per hemisphere. *Control animals received 0 U AAV2-hAADC and 500 U AAV2-GFP. AAV2, adeno-associated virus type-2; GFP, green fluorescent protein.

Figure 3. Immunohistochemical detection of AAV2-hAADC in escalating dose groups

Immunohistochemical staining of brain tissue from each dose group was performed with anti-hAADC primary antibodies followed by secondary antibodies labeled with immunoperoxidase. Images shown are (a,b) ×20 and (c,d) ×100 original magnification. Scale bars = 2 mm (a,b) and 100 μm (c,d). CN, caudate nucleus; GFP, green fluorescent protein; hAADC, human aromatic l-amino acid decarboxylase; PUT, putamen.