Ribozyme-mediated specific gene replacement of the alpha 1-antitrypsin gene in human hepatoma cells

Background/Aims: Some of the mutant forms of cellular proteins not only los e their function, but also cause diseases by their toxic effects, One of th e challenging tasks in the field of gene therapy will be "gene replacement" accomplished by inhibiting mutant gene expression and providing normal fun ction of the same gene, simultaneously, Although lung involvement in alpha 1-antrypsin (alpha 1-AT) deficiency is caused by the lack of alpha 1-AT fun ction, the liver involvement is due to the accumulation of the mutated alph a 1-AT protein. Therefore, one possible approach to prevent and treat the d isease manifestations of alpha 1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning alpha 1-AT pr otein in the liver, Methods: For the inhibition of alpha 1-AT gene expression, panels of alpha 1-AT-specific hammerhead ribozymes designed to target different GUC sites i n the alpha 1-AT mRNA were evaluated in a human hepatoma cell-line, transdu ced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which con tained a functional alpha 1-AT ribozyme and was combined with a modified al pha 1-AT gene, whose product was engineered to be resistant to the specific alpha 1-AT ribozyme. This construct was transduced into target hepatoma ce lls. Results: The transduced hepatoma cells showed the effective expression of m odified alpha 1-AT, under the conditions where the endogenous a alpha 1-AT gene expression was inhibited. Conclusion: This ribozyme-mediated, specific gene replacement is a first st ep in the gene therapy of alpha 1-AT deficiency.