Glial-derived neurotrophic factor gene transfer for Parkinson's disease: anterograde distribution of AAV2 vectors in the primate brain

Abstract

Delivery of neurotrophic factors to treat neurodegenerative diseases has not been efficacious in clinical trials despite their known potency for promoting neuronal growth and survival. Direct gene delivery to the brain offers an approach for establishing sustained expression of neurotrophic factors but is dependent on accurate surgical procedures to target specific anatomical regions of the brain. Serotype-2 adeno-associated viral (AAV2) vectors have been investigated in multiple clinical studies for neurological diseases without adverse effects; however the absence of significant clinical efficacy after neurotrophic factor gene transfer has been largely attributed to insufficient coverage of the target region. Our pre-clinical development of AAV2-glial-derived neurotrophic factor (GDNF) for Parkinson's disease involved real-time image guided delivery and optimization of delivery techniques to maximize gene transfer in the putamen. We have demonstrated that AAV2 vectors are anterogradely transported in the primate brain with GDNF expression observed in the substantia nigra after putaminal delivery in both intact and nigrostriatal lesioned primates. Direct midbrain delivery of AAV2-GDNF resulted in extensive anterograde transport to multiple brain regions and significant weight loss.

Fig. 1. Prediction of vector distribution by real-time MR imaging

Co-infusion of gadoteridol and AAV2-GDNF allows real-time monitoring of distribution at the site of infusion that is highly predictive of the area of GDNF expression. Representative MR images and immunohistochemical staining for GDNF after bilateral infusion into the NHP (A) thalamus or (B) putamen (2 sites per hemisphere). MR imaging immediately after completion of the infusions showed that the gadoteridol tracer was confined to the target structures with no leakage or backflow along the cannula tracts. The distribution of GDNF in the thalamus and putamen correlated exactly with the MR signal. In addition, anterograde transport of AAV2-GDNF to secondary brain regions was observed with GDNF present in multiple regions of the cortex after infusion into the thalamus, and basal ganglia nuclei after infusion into the putamen. Dual staining for GDNF (brown) and tyrosine hydroxylase (blue) shows some overlap between GDNF expression in the substantia nigra and TH-positive neurons in the pars compacta. However, many GDNF-positive neurons (arrows) were found in the TH-negative SNr. Abbreviations: GPe: Globus Pallidus external; GPi: Globus Pallidus internal; Put: Putamen; SNc: Substantia nigra pars compacta; SNr: Substantia nigra pars reticulata; VTA: ventral tegmental area.

Fig. 2. Anterograde transport by striatonigral neurons is not impaired by MPTP lesioning of the nigrostriatal neurons

Delivery of AAV2-GDNF into the putamen of severely MPTP lesioned NHP resulted in progressive recovery of motor function and partial restoration of dopaminergic activity. (A) High levels of GDNF were expressed in the putamen and globus pallidus after AAV2-GDNF delivery (150 μL per hemisphere). (B) Complete loss of TH staining in the right side caudate and putamen demonstrated the extensive degeneration of the nigrostriatal neurons caused by right side intracarotid artery administration of MPTP, whereas the left side is only mildly lesioned. (C, D) Dual staining for GDNF (brown) and tyrosine hydroxylase (blue) show identical distribution of GDNF in both hemispheres with the majority of GDNF localized in TH-negative areas including the substantia nigra pars reticulata (SNr) and subthalamic nucleus (STN), both of which receive direct and indirect axonal projections from the putamen.

Fig. 3. Distribution of GDNF after delivery of AAV2-GDNF to the midbrain

(A) Direct infusion of AAV2-GDNF into the substantia nigra of aged NHP resulted in extensive GDNF expression in many distal areas of the primate brain, including (B) caudate nucleus, medial putamen, (C) globus pallidus and (D) cerebral cortex. GDNF-positive neurons and fibers (E) were also found in the ventral thalamus, hypothalamus and subthalamic nucleus, none of which were directly targeted during AAV2-GDNF infusion. Within the targeted midbrain, extensive GDNF expression was present (F) in the substantia nigra, ventral tegmental area and red nucleus. No GDNF was observed in the contralateral hemisphere. IHC staining: tyrosine hydroxylase (blue), GDNF (brown).