Eliciting protective mechanisms against alcoholism

引发针对酗酒的保护机制

• 批准号: 7125618
• 负责人: EMANUEL RUBIN
• 金额: $ 30.44万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125618
• 关键词: alcoholism /alcohol abuse; alcoholism /alcohol abuse chemotherapy; alcoholism /alcohol abuse prevention; alcoholism antagonist; aldehyde dehydrogenases; behavioral /social science research tag; disease /disorder model; enzyme activity; enzyme inhibitors; enzyme therapy; gene expression; genetic transduction; green fluorescent proteins; high performance liquid chromatography; laboratory rat; mass spectrometry; matrix assisted laser desorption ionization; molecular cloning; nonhuman therapy evaluation; point mutation; regulatory gene; small interfering RNA; transfection /expression vector; western blottings

DESCRIPTION (provided by applicant): Alcoholism is responsible for one third of preventible loss of life in the United States. About 50-60% of the predisposition to develop alcoholism is genetically determined, and both permissive and protective genes have been identified. Two gene variants strongly associated with protection against alcohol abuse and alcoholism modify the metabolism of ethanol. These genes code for enzymes that either increase the production or reduce the elimination of acetaldehyde, the first product of ethanol metabolism. A dominant negative allele of aldehyde dehydrogenase (ALDH2) found in East Asians codes for an inactive ALDH2*2 that does not metabolize acetaldehyde efficiently. This results in increased levels of circulating acetaldehyde and in an aversive reaction that protects against alcoholism by 65 to 95%. Another protective gene variant ADHIB*2 codes for an alcohol dehydrogenase with a high activity in oxidizing ethanol into acetaldehyde. This inborn protection against alcoholism can reach 50-60%. Disulfiram, an ALDH inhibitor used in the treatment of alcoholism has two main drawbacks that have reduced its use; marked toxicity due to nonspecific action and poor patient compliance with daily intake. Studies in this application test the hypothesis that a reduction ofALDH2 gene expression or ADH gene transduction decrease voluntary ethanol intake in rats, thus mimicking the natural protective mechanisms. The hypothesis will be tested initially in vitro in rat hepatoma cells and further evaluated in vivo in rats. The protective mechanism afforded by a low ALDH2 activity will be elicited by (i) orally-absorbable third generation antisense morpholino oligonucleotides directed against ALDH2 mRNA, and (ii) short interference RNAi molecules blocking ALDH2 mRNA administered with long-lasting adenoviral vectors. The high-activity ADH protective mechanism will be mimicked by (iii) transduction of the human ADHIB*2 gene and overexpression of the rat Adh with adenoviral vectors. As a 'proof of principle' we will (iv) determine the ability of single-point mismatched oligodeoxynucleotides to generate the Glu486Lys dominant-negative point mutation of ALDH2*2 in human hepatoma cells that carry the ADH gene, thus generating new cell lines that produce high levels of acetaldehyde. Overall, the proposed studies aim at inducing gene-based protective mechanisms that are specific and could be used for the treatment of alcoholism.

描述（由申请人提供）：酒精中毒负责美国预防生命的三分之一。大约50-60％的发展酒精中毒易感性是遗传确定的，并且均已鉴定出允许和保护性基因。两种基因变异与防止酗酒和酒精中毒密切相关，改变了乙醇的新陈代谢。这些基因代码用于增加产量或消除乙醛代谢的第一个产物乙醛的酶。在东亚人中发现的醛脱氢酶（ALDH2）的主要负等位基因代码为不活跃的ALDH2*2，该Aldh2*2不能有效地代谢乙醛。这导致循环乙醛的水平增加，并且厌恶反应可预防酒精中毒65％至95％。另一种保护性基因变体ADHIB*2代码醇脱氢酶，在将乙醇氧化成乙醛中具有较高活性。这种天生的防止酒精中毒可以达到50-60％。 用于治疗酒精中毒的ALDH抑制剂二硫仑有两个主要缺点，减少了其使用。由于非特异性动作和患者对日常摄入量的依从性不佳而引起的毒性明显。在此应用中的研究检验了一个假说，即降低了daldh2基因表达或ADH基因转导减少大鼠的自愿乙醇摄入，从而模仿了自然保护机制。该假设最初将在大鼠肝癌细胞中进行体外检验，并进一步在大鼠体内评估。低ALDH2活性提供的保护机制将由针对ALDH2 mRNA的（I）口服的第三代反义莫菲诺寡核苷酸引起，（II）短暂干扰RNAi分子阻断了用长期lasting adenoviral adenoviral adenoviral vectors构造的ALDH2 AldH2 mRNA。通过（iii）人类adhib*2基因和用腺病毒载体的大鼠ADH的过表达，将模仿高活动性ADH保护机制。作为“原理证明”，我们（iv）将确定单点不匹配的寡脱氧核苷酸生成Aldh2*2在人类肝癌细胞中的glu486lys显性阴性点突变的能力，这些突变是在携带ADH基因的人类肝癌细胞中，从而产生了产生高水平acetaldehyde的新细胞系的能力。 总体而言，拟议的研究旨在诱导特定的基因保护机制，可用于治疗酒精中毒。