The influence of arm length asymmetry and base substitution on the activity of the 10-23 DNA enzyme.

A small oligodeoxyribonucleotide derived from in vitro selection has been shown to be capable of efficient sequence-specific cleavage of RNA at purine-pyrimidine junctions. As the reaction readily takes place under simulated physiologic conditions, this molecule described as the 10-23 general purpose RNA-cleaving DNA enzyme, has potential as a therapeutic agent. To further explore the character of this prototype, we examined the influence of base substitution and binding arm length asymmetry on its RNA cleaving activity. Surprisingly, substitution of the proximal nucleotide on the 3'-arm, to allow nonstandard Watson-Crick interactions, was found in some instances to improve the cleavage reaction rate. Although the identity of the unpaired purine in the RNA substrate cleavage site was found to have only a subtle influence on the rate of catalysis, with a slight decrease observed when a G at this position was changed to an A, nucleotide substitution (G to C) in the core motif at position 14 was found to completely abolish catalysis. The effect of arm length reduction varied with RNA substrate sequence and extent of helix asymmetry. Where the cleavage rate of one substrate was impaired by truncation of the deoxyribozymes 5'-arm (6 bp), the same modification in reactions with a different sequence produced a rate enhancement. Truncation of the 3'-arm, however, had no effect on the reaction rate of the one substrate tested yet nearly halved the cleavage rate in another substrate.

Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme.

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262-4266) and is known as the "10-23" general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3'-3'-internucleotide inversion at the 3'-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity.

Target site selection for an RNA-cleaving catalytic DNA.

A small catalytic DNA, known as the 10-23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.

Preclinical characterization of an anti-tat ribozyme for therapeutic application.

A hammerhead ribozyme retroviral construct, denoted RRz2, targeting the coding region of the human immunodeficiency virus type 1 (HIV-1) tat gene, has shown itself to be effective in a range of test systems. Inhibition of the replication of HIV-1 IIIB and primary drug-resistant strains in pooled transduced CEMT4 cells was consistently found to be more than 80% compared with the control-vector transduced cells, whereas a mutant RRz2 gave approximately 45% inhibition. A multiple HIV-1 passage assay showed the absence of emergence of mutations within the specific viral RNA ribozyme target sequences. This lack of generation of ribozyme "escape mutants" occurred despite the almost complete disappearance of a HIV-1 quasi-species in the testing virus. When RRz2 was tested in peripheral blood lymphocytes (PBLs) from HIV-1-infected patients, paired analysis showed that cell viability in the ribozyme-transduced HIV-1-infected PBLs was significantly higher than that in the vector-transduced cells. This difference in viability (vector versus RRz2) was not observed in PBLs from non-HIV-1-infected donors. Taken together, these results indicate that the transfer of an anti-HIV-1 ribozyme gene into human T lymphocytes could have major impact on viral replication and T cell viability in the HIV-1-infected individual.

Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.

Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.