In vitro evaluation of a 'stealth' adenoviral vector for targeted gene delivery to adult mammalian neurones

Abstract

Background: Polymer coating of adenovirus type 5 (Ad5) particles produces a 'stealth' Ad5 (sAd5) that confers protection from immune recognition, blocks receptor-mediated uptake, and favours uptake into pinocytic cells.

Methods: In mixed cultures of primary adult rat dorsal root ganglion neurones (DRGN), rat C6 glioma cells, A9 non-Coxsackie and Ad Receptor (CAR)- and CAR-expressing fibroblasts, reporter gene expression after sAd5 pinocytotic uptake was monitored using the green fluorescent protein (gfp) gene, and viral particle trafficking and polymer coat dismantling was followed using Yoyo-1 tagged Ad5 DNA and Texas Red (TR) to label the coat.

Results: sAd5.gfp was pinocytosed by significantly higher proportions of neurones, than other cells, but GFP was not expressed. The TR-labelled coat remained co-localised with tagged viral DNA within transfected DRGN, showing that sAd5 did not uncoat and viral DNA did not traffic to the nucleus. Noncoated Ad5 transduced non-neuronal DRG cells more efficiently than DRGN, whereas A9(CAR) cells were more significantly transduced than any other cell type. Retargeting of the sAd5.gfp with either fibroblast growth factor-2 or nerve growth factor (NGF) enhanced internalisation by DRGN into endocytic vesicles allowing uncoating and thus GFP expression. Retargeting with NGF resulted in significantly higher numbers of DRGN expressing GFP than non-neuronal DRG cells.

Conclusions: These findings indicate that DRGN pinocytose atropic genetic particles at higher levels than non-neuronal DRG cells and the environment of pinocytic vesicles is not conducive to sAd5 uncoating and capsid dismantling, requiring reformulation of sAd5 with either a neurone specific ligand or a self-dismantling coat to target sAd5 transgene expression to neurones.