Ribozyme-mediated specific gene replacement of the α1-antitrypsin gene in human hepatoma cells

Backgroundl Aims: Some of the mutant forms of cellular proteins not only lose their function, but also cause diseases by their toxic effects. One of the challenging tasks in the field of gene therapy will be “gene replacement” accomplished by inhibiting mutant gene expression and providing normal function of the same gene, simultaneously. Although lung involvement in α1-antrypsin (α1-AT) deficiency is caused by the lack of α1-AT function, the liver involvement is due to the accumulation of the mutated α1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of α1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning α1-AT protein in the liver. Methods: For the inhibition of α1-AT gene expression, panels of α1-AT-specific hammerhead ribozymes designed to target different GUC sites in the α1-AT mRNA were evaluated in a human hepatoma cell-line, transduced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which contained a functional α1-AT ribozyme and was combined with a modified α1-AT gene, whose product was engineered to be resistant to the specific α1-AT ribozyme. This construct was transduced into target hepatoma cells. Results: The transduced hepatoma cells showed the effective expression of modified α1-AT, under the conditions where the endogenous α1-AT gene expression was inhibited. Conclusion: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of α1-AT deficiency.