Background:  Many regions of the HIV-1 genome have been targeted by ribozymes (Rz) and general-purpose RNA-cleaving DNA enzymes (Dz) earlier to interfere with the expression of HIV-1 genes with varying degree of success. To achieve more potent inhibition of HIV-1 gene expression, we sought to exploit the synergistic effects of a catalytic RNA (hammerhead ribozyme) and a catalytic DNA (10 to 23 Dzs) for the first time for this purpose.

Methods:  Since HIV-1 genome consists of multiple overlapping exons, we selected a region that overlapped the second exons of TAT and rev gene to design novel Rz and a Dz. Since this short region lacks the conventional GUX ribozyme cleavage sites, we synthesized 2 hammer-head motif-containing Rz that recognized target sites other than GUX, namely UUC located at potion 8366 (Rz-8366) and CUC at position 8377 (Rz-8377), respectively. A di-Rz (Rz-77-66) was also constructed by joining the 2 mono-Rz in direct tandem such that Rz-8366 was cloned downstream of the Rz-8377. To compare the efficacy of a Dz with these Rz, a 10- to 23-catalytic motif containing Dz was synthesized that was targeted to cleave between A and U nucleotides (Dz-8381).

Results:  We show that both Rz and the Dz cleaved the target RNA under a variety of experimental conditions with greatly varying efficiencies. Di-Rz cleaved the target RNA at both target sites more efficiently than the 2 mono-Rz individually. Both Rz-8377 and Dz-8381 inhibit HIV-1-specific gene expression efficiently when used in extremely small amounts. The extent of inhibition by either of them correlated with their in vitro cleavage activities.

Conclusions:  Potential for using both Rz and Dz simultaneously for cleaving the target RNA simultaneously and interfering with HIV-1 replication was explored for the first time. The potential therapeutic usefulness of such a combinatorial approach using novel Rz and Dz is discussed.

Keywords: Therapeutic vaccine and immune based therapeutics, vaccines, Antiretroviral therapy