title: 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 (3)H-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 × 10(11) 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 (10(10) particles) to 20,000 fold (10(13) particles), while the hdAD vector (1.7 × 10(12) 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 × 10(12) 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.
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, United States of America.
authors.forename: Liyi
authors.initials: L
authors.lastname: Geng
authors.forename: Yang
authors.initials: Y
authors.lastname: Gao
authors.forename: Xiabin
authors.initials: X
authors.lastname: Chen
authors.forename: Shurong
authors.initials: S
authors.lastname: Hou
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.forename: Zoran
authors.initials: Z
authors.lastname: Radic
authors.forename: Robin J
authors.initials: RJ
authors.lastname: Parks
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Russell
authors.forename: Linh
authors.initials: L
authors.lastname: Pham
authors.forename: Stephen
authors.initials: S
authors.lastname: Brimijoin
mesh_terms: D000256:Adenoviridae; D000818:Animals; D001057:Apolipoproteins E; D002091:Butyrylcholinesterase / Q000235:genetics; D002802:Cholinesterases / Q000235:genetics*; D003042:Cocaine / Q000009:adverse effects*; D019970:Cocaine-Related Disorders / Q000628:therapy*; D015870:Gene Expression / Q000235:genetics*; D022761:Gene Transfer, Horizontal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006867:Hydrolases / Q000097:blood; D007700:Kinetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001057:Apolipoproteins E; D006867:Hydrolases; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D003042:Cocaine