Eduardo Karahanian1, Paula Ocaranza, Yedy Israel. 1. Faculty of Health Sciences, Diego Portales University, Faculty of Chemical and Pharmaceutical Sciences, and Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, University of Chile, Santiago, Chile.
Abstract
BACKGROUND: Individuals carrying the Glu487Lys coding mutation in the gene for mitochondrial aldehyde dehydrogenase (ALDH2) have a diminished capacity to metabolize acetaldehyde. This deficiency leads to increases in blood acetaldehyde levels when they consume ethanol, which results in an aversion to alcohol and in marked protection against alcoholism. In the present studies, we aimed to mimic the high-acetaldehyde low-ALDH2 activity phenotype in a rat hepatoma cell line by inhibiting Aldh2 gene expression by an Aldh2 antisense-coding gene carried by an adenoviral vector. METHODS: We designed and produced elevated titers of adenoviral vectors (10 virions/ml) carrying Aldh2 cDNA cloned in the reverse orientation preceded by a CMV promoter and followed by a poly-A termination signal. Rat hepatoma cells were infected with these vectors. RESULTS: Studies showed that 1) the antisense gene is actively transcribed in the cells and high levels of antisense mRNA are attained, 2) the antisense gene reduced ALDH2 activity by 65%, and 3) when incubated with 10 mM ethanol, acetaldehyde accumulation by cells increased 8-fold to levels (80-90 microM) known to be aversive to animals and humans. CONCLUSIONS: Data presented show that antialcohol drugs that inhibit Aldh2 gene expression can be generated endogenously in liver cells infected by an adenoviral vector carrying an antisense-coding gene, thus mimicking the high-acetaldehyde phenotype that exists in humans carrying the Glu487Lys mutation who are protected against alcoholism.
BACKGROUND: Individuals carrying the Glu487Lys coding mutation in the gene for mitochondrial aldehyde dehydrogenase (ALDH2) have a diminished capacity to metabolize acetaldehyde. This deficiency leads to increases in blood acetaldehyde levels when they consume ethanol, which results in an aversion to alcohol and in marked protection against alcoholism. In the present studies, we aimed to mimic the high-acetaldehyde low-ALDH2 activity phenotype in a rathepatoma cell line by inhibiting Aldh2 gene expression by an Aldh2 antisense-coding gene carried by an adenoviral vector. METHODS: We designed and produced elevated titers of adenoviral vectors (10 virions/ml) carrying Aldh2 cDNA cloned in the reverse orientation preceded by a CMV promoter and followed by a poly-A termination signal. Rathepatoma cells were infected with these vectors. RESULTS: Studies showed that 1) the antisense gene is actively transcribed in the cells and high levels of antisense mRNA are attained, 2) the antisense gene reduced ALDH2 activity by 65%, and 3) when incubated with 10 mM ethanol, acetaldehyde accumulation by cells increased 8-fold to levels (80-90 microM) known to be aversive to animals and humans. CONCLUSIONS: Data presented show that antialcohol drugs that inhibit Aldh2 gene expression can be generated endogenously in liver cells infected by an adenoviral vector carrying an antisense-coding gene, thus mimicking the high-acetaldehyde phenotype that exists in humans carrying the Glu487Lys mutation who are protected against alcoholism.
Authors: Yuki Matsumura; Katie M Stiles; Jasmine Reid; Esther Z Frenk; Samantha Cronin; Odelya E Pagovich; Ronald G Crystal Journal: Mol Ther Methods Clin Dev Date: 2019-08-26 Impact factor: 6.698