Eliciting protective mechanisms against alcoholism

描述（由申请人提供）：在美国，酗酒占可预防生命损失的三分之一。大约50-60%的酗酒倾向是由基因决定的，并且已经确定了允许和保护基因。两种与防止酒精滥用和酒精中毒密切相关的基因变异改变了乙醇的代谢。这些基因编码的酶，要么增加生产或减少消除乙醛，乙醇代谢的第一个产品。在东亚人中发现的醛脱氢酶（ALDH 2）的显性负等位基因编码不有效代谢乙醛的无活性ALDH 2 *2。这会导致循环中乙醛水平的增加，并产生一种令人厌恶的反应，可以防止65%至95%的酒精中毒。另一种保护性基因变体ADHIB*2编码在将乙醇氧化成乙醛中具有高活性的醇脱氢酶。这种对酒精中毒的先天保护可以达到50- 60%。 双硫仑，一种用于治疗酒精中毒的ALDH抑制剂，有两个主要缺点，减少了它的使用;由于非特异性作用和患者对每日摄入量的依从性差而导致的明显毒性。本申请的研究验证了ALDH 2基因表达或ADH基因转导减少大鼠自愿乙醇摄入的假设，从而模拟了天然保护机制。该假设将首先在体外大鼠肝癌细胞中进行测试，并在大鼠体内进行进一步评价。由低ALDH 2活性提供的保护机制将通过（i）针对ALDH 2 mRNA的口服可吸收的第三代反义吗啉代寡核苷酸和（ii）与长效腺病毒载体一起施用的阻断ALDH 2 mRNA的短干扰RNAi分子来引发。高活性ADH保护机制将通过（iii）转导人ADHIB*2基因和用腺病毒载体过表达大鼠Adh来模拟。作为一个“原则证明”，我们将（iv）确定单点错配的寡脱氧核苷酸在携带ADH基因的人肝癌细胞中产生ALDH 2 *2的Glu 486 Lys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。 总的来说，拟议的研究旨在诱导基于基因的保护机制，这些机制是特异性的，可用于治疗酒精中毒。

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.