Gene Transfer of Mutant Mouse Cholinesterase Provides High Lifetime Expression and Reduced Cocaine Responses with No Evident Toxicity

Abstract

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with 3H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3×1011 particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (1010 particles) to 20,000 fold (1013 particles), while the hdAD vector (1.7×1012 particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7×1012 particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose.

Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector-Delivered Cocaine Hydrolase

“…5) complements results of Geng et al (2013), who reported that mice maintained substantial plasma levels of CocH for 2 years or more. The present results also concur with Zlebnik et al (2014), who used the same form and dose (10 12 particles) of vector and reported CocH enzyme expression levels across 10 weeks postinjection that were similar to the present study.…”

“…This vector localizes in liver tissue, where its payload cDNA drives hepatocytes to synthesize and release CocH into the blood stream at high levels for long intervals. CocH vector-treated rodents showed enhanced cocaine metabolism for several months Brimijoin, 2004, 2006) to 2 years (Geng et al, 2013). In preclinical models of drug abuse, CocH-treated rats displayed no locomotor response to stimulatory doses of cocaine 30 days postinjection .…”

Plasma butyrylcholinesterase regulates ghrelin to control aggression

“…cDNAs encoding the following peptides and proteins were subcloned into AAV backbone vector: luciferase, mouse BChE wild type, mouse BChE mutant (A199S/S227A/S287G/ A328W/Y332G), human BChE wild type, human BChE mutant (A199S/S227A/S287G/A328W/Y332G), CocH-6 ΔT (human BChE E1-V529 with A199S/F227A/S287G/A328W/Y332G/E441D), mouse GOAT, and mouse ghrelin. The resulting transfer vectors were cotransfected into HEK293T cells with the helper vectors pHELP (Applied Viromics) and pAAV 2/8 (Department of Pathology and Laboratory Medicine, University of Pennsylvania) (8). Viruses in cell lysates were isolated by ultracentrifugation, and viral particles were determined by real-time PCR.…”

“…The experiments did not reveal cholinergic dysfunction, immunological reactions to enzyme, or liver damage from viral vector (6). They did confirm that gene transfer of cocaine-hydrolyzing BChE can suppress drug-seeking behavior by eliminating cocaine before it reaches the brain (7)(8)(9). Unexpectedly, the treatment also led to reduced fighting among mice that overexpressed BChE.…”

Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice

“…Over a range of species, this technology can drive CocH to extremely high levels, for years after a single treatment and without apparent toxicity (Murthy et al, 2014a,b). The result is a greatly accelerated cocaine hydrolysis and a near-complete suppression of physiologic, behavioral, and toxicological responses to cocaine (Gao and Brimijoin, 2004;Carroll et al, 2012;Brimijoin et al, 2013;Gao et al, 2013;Geng et al, 2013). We consider that a successful translation of this therapy to treatment-seeking cocaine users might aid them in becoming abstinent and, later, reduce their risk of relapse into drug taking.…”