Exogenous application of ribozymes for inhibiting gene expression

Sequence-specific inhibition of gene expression is an attractive concept for the development of a new generation of therapeutics. Two alternatives can be envisaged for the introduction of ribozymes into cells: endogenous or exogenous delivery. In the latter, the ribozyme is prepared by chemical synthesis or transcription and delivered to the cell either unaided or with the help of liposomes. A problem with this approach is the abundance of RNases in the serum, and thus the stabilization of the ribozyme is necessary but without the impairment of catalytic efficiency. This has been achieved by several groups by 2'-modification of the pyrimidine nucleosides and the introduction of a few phosphorothioates at the termini. The selection of ribozyme-accessible sites on the target and the attachment of cholesterol and peptides to the ribozymes will be discussed. Examples of the application of these modified ribozymes in cell cultures will be presented, including the inhibition of expression of the multiple drug resistance gene, after unaided as well as liposome-aided delivery, and studies of animal models demonstrating the potential of this particular application strategy.