Eliciting protective mechanisms against alcoholism

描述(申请人提供)：在美国，三分之一的可预防的生命损失是由酒精中毒造成的。大约50%-60%的酗酒易感性是由基因决定的，允许和保护基因都已经被识别出来。两个与防止酗酒和酗酒密切相关的基因变异改变了乙醇的新陈代谢。这些基因编码的酶要么增加乙醛的产量，要么减少乙醛的消除，乙醛是乙醇代谢的第一个产物。在东亚人中发现的乙醛脱氢酶(ALDH2)的显性负等位基因编码一种不能有效代谢乙醛的无效ALDH2*2。这导致了循环乙醛水平的增加，并导致了一种令人厌恶的反应，可以防止65%到95%的酒精中毒。另一个保护性基因变体ADHIB*2编码的是一种酒精脱氢酶，它能将乙醇氧化成乙醛。这种针对酒精中毒的先天保护作用可以达到50%-60%。 双硫兰是一种用于治疗酒精中毒的ALDH抑制剂，它有两个主要缺点，这些缺点减少了它的使用；非特异性作用引起的显著毒性和患者对每日摄入量的依从性差。这项应用中的研究验证了一种假设，即ALDH2基因表达的减少或ADH基因转导减少了大鼠自愿的酒精摄入量，从而模拟了自然保护机制。这一假说将首先在大鼠肝癌细胞中进行体外测试，然后在大鼠体内进行进一步评估。ALDH2活性低所提供的保护机制将通过(I)针对ALDH2 mRNA的口服可吸收的第三代反义吗啉寡核苷酸，以及(Ii)用长效腺病毒载体阻断ALDH2 mRNA的短干扰RNAi分子来实现。(3)转导人ADHIB*2基因和用腺病毒载体过表达大鼠ADH可模拟ADH的高活性保护机制。作为“原则证明”，我们将(Iv)确定单点错配寡核苷酸在携带ADH基因的人肝癌细胞中产生ALDH2*2的Glu486Lys显性-负性点突变的能力，从而产生产生高水平乙醛的新细胞系。 总体而言，拟议的研究旨在诱导基于基因的保护机制，这些机制是特异的，可以用于酒精中毒的治疗。

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.