Author: Anthony Neil Pollard
Pollard, Anthony Neil, 2009 INTRANASAL DELIVERY OF MACROMOLECULES TO THE RODENT BRAIN VIA OLFACTORY PATHWAYS, Flinders University, School of Medicine
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One of the major limitations in drug development and gene therapy for brain diseases is the natural defensive structure called the blood brain barrier (BBB), which prevents therapeutic polypeptide drugs and viral vectors from entering the brain. Intranasal delivery of therapeutic gene products into the brain offers a non-invasive alternative towards a feasible gene and protein therapy for neurological diseases. From recent studies involving axonal transport, it is tempting to speculate that therapeutic macromolecules including neurotrophic factors and viral vectors can be delivered into the brain by peripheral neurons, such as olfactory receptor neurons (ORNs), which span the BBB. It is thought that the nasal pathway into the brain involves two general mechanisms; intracellular (intraneuronal) or extracellular routes of transport. However the pathways involved have not yet been fully characterized. In this study I firstly investigated the temporal and spatial localisation pattern of both biotinylated and I125 labelled ciliary neurotrophic factor (CNTF) following nasal delivery into Sprague-Dawley rats. Results showed that intranasal delivered CNTF was transported to several brain regions by both intracellular axonal pathway through ORNs and the extracellular trigeminal pathway. Excess unlabelled CNTF competed for receptor binding in the olfactory mucosa confirming receptor mediated intracellular transport to the olfactory bulb via ORNs. Denervation of the olfactory mucosa prior to CNTF delivery failed to prevent CNTF transport to trigeminal and hypothalamic brain regions. Intranasal delivered CNTF was biologically active, resulting in activation of the STAT3 signalling pathway in the thalamus and hypothalamus. To examine the functional activity of intranasal delivered CNTF, I conducted a weight loss trial using an obese Zucker rat (OZR) model to test whether CNTF treatment caused body weight loss. Intranasal administration of CNTF resulted in reduced body weight in the CNTF treated OZR group compared to the BSA control group during the 12 day trial and for 3 days after. Intranasal delivery of CNTF may be a valuable method for the treatment of obesity. In the second study, I investigated the temporal and spatial expression of Enhanced Green Fluorescent Protein (EGFP) transferred by a single nasal delivery of either a recombinant adenovirus vector (Ad5CMV-EGFP) or an adeno-associated virus vector (AAV2-EGFP) into Sprague-Dawley rats. Adenovirus mediated EGFP expression was localized in ORNs throughout the olfactory epithelium after 24 hours. EGFP in the ORNs appeared to be anterogradely transported along their axons to the olfactory bulb and transferred in glomeruli to second-order neurons. EGFP was transferred to several brain regions including the cortex, hippocampus, and brainstem after 7 days. EGFP expression co-localized with Olfactory Marker Protein and was confirmed with EGFP immunofluorescence labelling and western blotting. AAV expressed EGFP localized in similar olfactory and brain regions 6 weeks after delivery. mRNA levels suggested that the AAV-EGFP construct was only incorporated into olfactory mucosa cells and the viral vector was not present in olfactory bulb and brain regions. In conclusion, this simple and non-invasive polypeptide and gene delivery method provides ubiquitous macromolecule distribution throughout the rodent brain and may be useful for the treatment of neurological disorders.
Keywords: intranasal delivery,CNTF,olfactory,obesity,BBB,AAV,adenovirus
Subject: Medicine thesis, Human Physiology thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Prof. Xin-Fu Zhou