60. Engineering AAV Vector for the Delivery of Human BuChE to Protect Against Exposure to Organophosphates

Abstract

Human butyrylcholinesterase (hBuChE) is a widely-distributed enzyme that may play a role in nerve conduction, can hydrolyze various toxic esters (e.g., cocaine), and acts as a potent scavenger of organophosphates (OP) including pesticides and chemical warfare agents. Major limitations prohibiting the development of purified hBuChE as a mainstream biologic drug include: limited availability of human plasma, limited scalability of manufacturing, high cost of purified human-derived product, and the need for intravenous administration. In addition, the preventive uses of hBuChE are limited by its relatively short half-life. In an attempt to address some of these limitations, several methods for the production of recombinant hBuChE in vitro (cell lines) and in vivo (transgenic animals, plants) were developed; however, the resulting recombinant proteins presented with unfavorable pharmacokinetic profiles making them unsuitable for preventive uses. Alternatively, adenovirus-mediated delivery of hBuChE is very effective, but is not a candidate for development due to the inherent immunogenicity of the vector. Expression of hBuChE peaks at around 4 days post IV administration of the adenoviral vector, and the peak is followed by a rapid decline, with minimal expression being detectable 10 days post-administration.AAV vectors present a radically different, non-immunogenic hBuChE delivery platform, which is not reliant on protein purification and has the flexibility to deliver the product into both liver and muscle. AAV technology engineers the recipient's own cells to produce sustainable pharmacologic amounts of hBuChE on demand and/or replenish the exhausted scavenger via a single dose of hBuChE-expressing vector under the control of a constitutive promoter. Through the systematic optimization of AAV-hBuChE vectors, we achieved not only rapid onset of hBuChE expression in mice, but also sustainable, long-term expression for at least 2 months with no decline. AAV-hBuChE expressed at 100 units/ml of serum at 24 hours post vector administration into mouse muscle, with steady-state expression levels of up to 30,000 units/ml of serum at 1 month. The optimization parameters included selection of the protein isoform, tetramerization partner, coding sequence, as well as promoters and other regulatory elements. Overall, approximately 40 vectors were evaluated in comparative studies. Based on previous findings, the levels of hBuChE expressed by our platform are predicted to be protective in OP challenges, which we plan to perform in future studies. In the future, the use of our platform can be expanded to include populations that are occupationally exposed to pesticides. This approach may also be applicable for the delivery of other protective human enzymes, such as the use of rhodanese to protect against cyanides or glutathione against 2-chloro-acetophenone.