Alpha-1-antitrypsin is a protease inhibitor that accounts for 90% of the total anti-protease activity in blood. Although synthesized mainly in the liver, its physiological function is to defuse into the lung and protect this important organ from destruction by excessive polymorphonuclear leukocyte elastase. Genetic deficiency of Alpha1-antitrypsin predisposes affected individuals to development of chronic obstructive pulmonary emphysema. The deficiency is inherited as an autosomal recessive trait, and there is no cure for the genetic disorder at the present time. The protein in deficient individuals has a lysine instead of a glutamate at residue 342 in the carboxyl region. Having cloned and analyzed the human Alpha1-antitrypsin cDNA and its corresponding gene, we demonstrated that the amino acid substitution is caused by a G to A transition at the corresponding position in the gene. Specific oligonucleotides were then designed and used to determine the genotypes of random individuals and in several instances the gene mapping methodology has been applied for prenatal diagnosis of the genetic disorder. The current proposal deals with the elucidation of regulatory mechanisims for the expression of the human Alpha1-antitrypsin gene in specific tissues. The cis-acting elements in the gene that specify its liver and macrophage/monocyte-specific expression will be identified and characterized by introduction of various recombinant constructs of the gene into cultured human hepatoma cells and monocytes followed by expression analysis. These results will also be extended to intact animals by introduction of the gene into mouse embryos to generate transgenic mice. Whether the cis-acting elements in the gene will be sufficient for its specific expression in the liver and macrophage versus other tissues and its potential for acute phase response to a number of physiological and pathological stimuli will be investigated. The cis-acting elements will be used to identify trans-acting factors in liver cells and monocytes that interact with them followed by their purification and characterization. The purified factors will then be used to investigate their potential roles in the regulation of tissue-specific expression of the gene at the molecular level. Finally, the human Alpha1-antitrypsin gene and its cis-acting elements will be engineered into recombinant retroviruses for efficient transfer into cultured hepatoma cells, primary hepatocytes and whole animals as a model for exploiting the potential of somatic gene therapy of the deficiency syndrome.
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