Alpha-l-Antitrypsin is a plasma 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 diffuse into the lung and protect this important organ from destruction by excessive neutrophil elastase. Genetic deficiency of alpha1-antitrypsin predisposes affected individuals to develop chronic obstructive pulmonary emphysema and there is no cure for this fatal condition at the present time. The deficient individuals have a general life expectancy of about 40-50 years, which can be shortened 10-20 years for cigarette smokers. The genetic deficiency is inherited as an autosomal co-dominant trait, and has a prevalence of about 1 in 3,000-4,000 Caucasians of Northern European ancestry. The protein in deficiency individuals has a lysine instead of a glytamate at residue 342 in the carboxyl region. Having cloned and analyzed the human alpha1-antitrypsin cDNA and its chromosomal gene, we demonstrated that the amino acid substitution is caused by a G to A transition at the corresponding position in the gene. The mutation information has permitted the development of analytical procedures for prenatal diagnosis and carrier detection of this genetic disorder. The full length cDNA was also introduced into retroviral vectors and used to transduce fibroblasts, hepatoma cells as well as primary mouse hepatocytes in culture. All virally transduced cells synthesized and secreted into culture media authentic human alpha1-antitrypsin which was fully capable of inactivating neutrophil elastase in vitro. More recently we have created transgenic mice on inbred lines that expressed high levels of human alpha1-antitrypsin in the liver, and used them as donors for hepatocyte transplantation into congenic recipients. We demonstrated that hepatocytes injected into the portal vein or the spleen of recipient mice migrated to the liver, embedded into the parenchyma, lived and continued to function as hepatocytes for the life of the recipients. In the current proposal we will attempt hepatic gene therapy by retroviral gene transduction followed by hepatocellular transplantation in laboratory animals. We will also attempt direct delivery of genes to the liver by forming DNA complexes with asialo-orosomucoid which is rapidly endocytosed into hepatocytes through the asialo- glycoprotein receptor. Finally, we will develop the adeno-associated virus as an alternative vector for in vitro as well as in vivo gene delivery to the liver and lung tissues in order to achieve gene therapy for alpha1-antitrypsin deficiency.
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