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

Liyi Geng, Yang Gao, Xiabin Chen, Shurong Hou, Chang Guo Zhan, Zoran Radic, Robin J. Parks, Stephen J Russell, Linh Pham, William Stephen Brimijoin

Research output: Contribution to journalArticle

27 Citations (Scopus)

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/<bold>S227</bold>A/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.

Original languageEnglish (US)
Article numbere67446
JournalPLoS One
Volume8
Issue number6
DOIs
StatePublished - Jun 28 2013

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Gene transfer
cocaine
cholinesterase
Cholinesterases
Cocaine
gene transfer
Toxicity
Hydrolases
toxicity
hydrolases
mutants
Butyrylcholinesterase
mice
Dependovirus
Genes
Adenoviridae
promoter regions
Apolipoproteins E
Viruses
Butyrylthiocholine

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Gene Transfer of Mutant Mouse Cholinesterase Provides High Lifetime Expression and Reduced Cocaine Responses with No Evident Toxicity. / Geng, Liyi; Gao, Yang; Chen, Xiabin; Hou, Shurong; Zhan, Chang Guo; Radic, Zoran; Parks, Robin J.; Russell, Stephen J; Pham, Linh; Brimijoin, William Stephen.

In: PLoS One, Vol. 8, No. 6, e67446, 28.06.2013.

Research output: Contribution to journalArticle

Geng, Liyi ; Gao, Yang ; Chen, Xiabin ; Hou, Shurong ; Zhan, Chang Guo ; Radic, Zoran ; Parks, Robin J. ; Russell, Stephen J ; Pham, Linh ; Brimijoin, William Stephen. / Gene Transfer of Mutant Mouse Cholinesterase Provides High Lifetime Expression and Reduced Cocaine Responses with No Evident Toxicity. In: PLoS One. 2013 ; Vol. 8, No. 6.
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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.",
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