In Vitro Biological Activity of Anti-C Ⅱ TA Hammerhead Ribozyme——A Novel Approach for Autoimmune Diseases

This study investigated the feasibility of using an hammerhead ribozyme against C Ⅱ TA, a major regulator of MHC Ⅱ antigens, to repress the expression of MHC Ⅱ molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C Ⅱ TA and its target gene were transcribed, then mixed up and incubated in vitro . The cleavage products were analyzed by PAGE and silver staining. Rz464 was then inserted into the pIRES2 EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class Ⅱ MHC induction by recombinant human interferon gamma (IFN γ). mRNA of C Ⅱ TA was measured by RT PCR. Our results showed that Rz464 could exclusively cleave C Ⅱ TA RNA. When induced with IFN γ, the expression of HLA DR, DP, DQ on pRz464 + Hela was induced, and the mRNA content of C Ⅱ TA decreased too. It is concluded that Rz464 could inhibit C Ⅱ TA and thus the family of genes was regulated by C Ⅱ TA:MHC Ⅱ molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto immune diseases.

Construction and Cleavage Characterization of Single Targeted Multi-ribozyme Expression Systems in vitro

In order to clarify cleavage efficiency of a multi-ribozyme expression system on their substrates,plasmids containing 20 cis-acting hammerhead ribozymes for self-cleavage and 10 trans-acting hammerhead ribozymes targeted on MDR1 and MRP1 substrates,the target plasmids pGEM-MDR1 and pGEM-MRP1 were constructed by employing PCR and DNA recombination.The endonuclease＇s digestion and DNA sequencing confirmed exactness of the multi-ribozyme expression systems and the target plasmids.The repeated self-cleavage tests revealed that the cisacting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and trans-acting hammerhead ribozymes 196 Rz and 210 Rz were released.The cleavage efficiencies of liberated ribozymes on their targets were evaluated in vitro and the results indicate that 196 Rz and 210 Rz were able to cleave the MDR1 and MRP1 RNA substrates.The profile of cleavage reaction shows that the cleavage efficiencies were correlated with the numbers of transacting hammerhead ribozymes contained in the multi-ribozyme expression systems,as well as the multi-ribozyme system is much better than the single ribozyme.Test of various concentrations of Mg^2＋ reveals that ribozyme cleavage reactions were dependent on Mg^2＋ concentration.The plot of lg（kobs） vs.lg[Mg^2＋] displays a linear relationship in the concentration range observed（2.5―20 mmol/L）.