Development of a Viral Based Gene Delivery System for Chemical Agent Bioscavengers and Biological Agent Vaccines

In recent years, exogenous administration of cholinesterases have been successfully used as safe and efficacious prophylactic treatments due to their capacity to scavenge nerve agents toxins inhibition of the targeted endogenous acetylcholinesterase can occur. Based on availability, broad spectrum efficacy, stability and safety, homologous native butyrylcholin-esterase (BChE) represents to date the best candidate in terms of develop-ing a human treatment. However, the currently used methods of native enzyme isolation from frozen human plasma are time consuming and expensive and highlight the importance of developing, as an alternate and parallel treatment strategy, a viral vector mediated gene therapy based approach to produce human BChE as an in vivo antidote. The goal of this proof of concept proposal is to determine the feasibility of developing a recombinant AAV2/9 transcapsidated vector for the in vivo transfer of HuBChE genes and in vivo production of bioscavenger sufficient to protect against exposure to neurotoxins. The study specifically aims to: (i) Construct bicistronic expression cassettes encoding HuBChE-EGFP for insertion into AAV-2 vector plasmid. (ii) Generate, produce and purify infectious replication-incompetent transcapsidated AAV2/9 viral particles using a triple transient transfection system in order to deliver the bicistronic HuBChE-EGFP genes and (iii) Express the rHuBChE gene in vitro by transduction of the IB3 airway epithelial cell and the C2C12 muscle cell lines. The AAV2/9 chimaera is relatively new and was chosen because of its good tropism for both airway epithelial (lung) cells (IB3) and muscle cells; both of which are important as in vivo sites of nerve agent exposure and injury. In Phase I, gene delivery by vectors encoding the expression of MaBChE? in addition to the HuBChE enzyme, is also planned due to the importance of using an homologous macaque system in Phase II to accurately assess levels of enzyme expression and to confirm the anticipated absence of anti-BChE antibodies and other adverse side effects following gene delivery.