Background/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 alpha1-antrypsin (alpha1-AT) deficiency is caused by the lack of alpha1-AT function, the liver involvement is due to the accumulation of the mutated alpha1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of alpha1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning alpha1-AT protein in the liver.
Methods: For the inhibition of alpha1-AT gene expression, panels of alpha1-AT-specific hammerhead ribozymes designed to target different GUC sites in the alpha1-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 alpha1-AT ribozyme and was combined with a modified alpha1-AT gene, whose product was engineered to be resistant to the specific alpha1-AT ribozyme. This construct was transduced into target hepatoma cells.
Results: The transduced hepatoma cells showed the effective expression of modified alpha1-AT, under the conditions where the endogenous alpha1-AT gene expression was inhibited.
Conclusion: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of alpha1-AT deficiency.