SAFETY AND TOLERABILITY OF MRI-GUIDED INFUSION OF AAV2-hAADC INTO THE MID-BRAIN OF NON-HUMAN PRIMATE

Abstract

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of non-human primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3 or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects.

Keywords: AADC deficiency; AAV2-hAADC; MRI-guided; pressurized infusion.

Figure 1

Reproducibility of magnetic resonance (MR) image-guided infusion of AAV2-hAADC into the primate mid-brain. Top panel shows representative coronal MR images of AAV2-hAADC after pressure-driven delivery in each animal. Please note there was slight reflux in only 2 out of 36 infusion performed (V002458 and V002254). Summary table below presents diffusion volume/infusion volume ratio (Vd/Vi, mean ± SD) for pilot and toxicology groups.

Figure 2

Surgery and vector-related histological findings. Independent evaluation of hematoxylin and eosin (H&E) staining of coronal sections containing the cannula tract revealed normal gliosis related to cannula insertion in all experimental groups (arrowheads). Higher magnification images (right column) were taken close to the cannula tip (black arrows in left column). H&E staining also showed perivascular cellular infiltrates in AAV2-hAADC-treated, but not PBS-treated, animals regardless of survival time. Although incidence and severity of perivascular cuffs was increased with AAV2-hAADC vector dose, these were not considered adverse. Note that there were also many vessels with no perivascular cuffing close to the infusion site (white arrows in left column) and that perivascular cellular infiltrates were not present in the pilot AAV2-hAADC animal. Scale bars: left column: 1 cm; right column: 100 μm.

Figure 3

AADC expression in the mid-brain of (a) pilot and (b) toxicology study animals. AADC staining of mid-brain revealed good correlation between infusate (gadolinium) signal in MRI and AAV2-hAADC expression (left and middle columns). AADC signal (brown) in SNpc and VTA that colocalized with endogenous tyrosine hydroxylase signal (blue, middle column). Although transgenic AADC in these nuclei was indistinguishable from endogenous AADC, AAV2-hAADC gene product was easily visible in SNpr when compared to naïve animal images. Higher magnification images show transgenic AADC staining in cells and fibers (right column). Scale bars: Left and middle columns: 1 cm; right column: 200 μm.

Figure 4

Axonal transport of AADC protein through the nigrostriatal pathway. Double immunohistochemical staining for TH (blue) and AADC (brown) in the striatum demonstrated the presence of the transgenic AADC in nigrostriatal terminals that widely covered both caudate and putamen nuclei. High-magnification images show the intensity of AADC staining in transduced fibers in AAV2-hAADC-treated animals compared to endogenous signal in PBS control group. Black squares indicate the area shown in higher magnification images. Notice the similar fiber density for both low and high AAV2-hAADC doses. Scale bar: 100 μm.