Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo

Abstract

Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377 ± 162 U/ml for full-length rHu BChE and 574 ± 143 U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9 U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.

Introduction

Nerve agents are organophosphorus (OP) compounds that are among the most toxic substances known. A recent approach to treating OP poisoning is the use of enzymes to sequester these compounds in circulation before they reach their physiological target, acetylcholinesterase (AChE). Of these, plasma-derived human butyrylcholinesterase (Hu BChE; accession # M16541) is the most viable candidate for human use [1], [2], [3], [4], [5], [6], [7]. Hu BChE is a stoichiometric scavenger in that one mol of enzyme binds and inactivates one mole of OP nerve agent [8]. Native Hu BChE is a tetrameric glycoprotein consisting of four identical subunits with a combined molecular weight of 340 kDa [9], [10]. The molecular weight of each subunit is 85 kDa, of which 65 kDa is for the protein and 20 kDa (24–26%) for the carbohydrate [11], [12]. A dose of 200 mg of Hu BChE is envisioned as a prophylactic treatment in humans to protect from up to 2× LD₅₀ of soman [13]. The purification and production of native Hu BChE in quantities sufficient to administer to hundreds and thousands of military personnel and civilian responders during deliberate or accidental nerve agent release requires large quantities of plasma. Therefore, methods for producing recombinant (r) Hu BChE were the focus of more recent investigations.

There are a variety of potential sources of rHu BChE, to include transgenic plants [14], transfected insect larva [15], algae [16], mammalian cells [17], [18], [19] and transgenic goats [20]. Although the catalytic and inhibitory properties of recombinant enzyme are similar to those for native Hu BChE, a major difference was observed when it was injected into animals; rHu BChE cleared rapidly from the circulation while plasma-derived Hu BChE showed a mean residence time of 40 h [18], [19], [21].

An alternate approach to introducing rHu BChE is via gene delivery using adeno- or adeno-associated viruses or in the form of naked DNA. In this method, the delivered gene enters the body's cells and turns them into small factories to produce the therapeutic protein. Four recent reports show the feasibility of a gene therapy approach to introduce candidate bioscavengers namely, human paraoxonase-1 [22], AChE [23], and a double mutant Ala328Trp/Tyr332Ala rHu BChE which exhibits enhanced cocaine hydrolase activity [24]. However, the systemic bioscavenger levels in serum achieved in these studies were rather low, for example a 60% increase for paraoxonase-1 [22], and only a 5–15% increase for AChE [23]. A 1-fold-increase in human paraoxonase-1 was also achieved in mice injected with naked plasmid DNA as compared to animals injected with the null plasmid [25].

In this study, we investigated the ability of highly efficient adenoviral vector (Ad) type V to transduce very high levels of full-length and truncated rHu BChE in BChE knockout mice. The rationale for using two constructs was (1) to determine if the full-length construct induced the expression of tetrameric rHu BChE, and (2) to investigate whether monomeric rHu BChE expressed by the truncated construct cleared faster from circulation than tetrameric rHu BChE expressed by the full-length construct. Previous studies suggested that the mean residence time of CHO cell expressed tetrameric rHu BChE is 3- to 4-fold greater than that for monomeric rHu BChE [18], [19], [21]. We found that the Ad gene therapy system was able to induce persistent and high-level expression (400–600 U/ml in mouse plasma) of both full-length and truncated rHu BChE in mice. This amount of rHu BChE is sufficient to fully protect mice against multiple LD₅₀'s of OP nerve agents. We also found that the expression profiles rHu BChE induced by full-length and truncated constructs were similar suggesting that both constructs induced the expression of rHu BChE with sufficient in vivo stability to function as effective nerve agent bioscavengers.