Gene delivery to the CNS is undermined by the lack of suitable vectors capable of delivering genes with sufficient specificity, efficacy and safety. In previous studies we have shown that bacteriophage particles, which lack tropism for mammalian cells, can be genetically modified to display specific ligands that allow binding, internalisation and cell transduction of receptor-bearing target cells. Moreover, phage particles can be forced to evolve using combinatorial techniques of phage display for cell specific targeting. In the studies described here, we took the first step towards the creation of a novel targeted vector system for gene delivery in the CNS by investigating whether EGF-targeted phage are internalised into cells after intraventricular injection. EGF-targeted or untargeted phage (1011cfu/ml) encoding GFP were injected into the cerebral lateral ventricle and brains processed for immunohistochemical analyses. By 24h, immunostaining for M13 coat protein (irM13) revealed that EGF-targeted phage was localised to the needle track. Strong irM13 was also found in endothelial cells and glia in the corpus callosum and striatum, in the choroid plexus, subependymal/ependymal cells and in the meninges. By 96h, irM13 persisted in all locations although at lower levels, indicating the high stability of the phage particles once internalised. In contrast, irM13 was restricted to the needle tract after untargeted phage injection, indicating that EGF-targeted phage internalisation occurred by receptor mediated endocytosis. irGFP, as a measure of cell transduction, was detected in selected endothelial cells and glia in the cortex and striatum, ependymal/subependymal and choroid plexus epithelial cells only after EGF-targeted phage injection. These results support the strategy of using phage-based vectors to discover CNS-related gene targeting ligands, in vivo that may aid the creation of novel gene delivery vectors.
01 - 03 Nov 2004
Society for Endocrinology