TARGETING RNA FOR DEGRADATION WITH A (2'-5')OLIGOADENYLATE-ANTISENSE CHIMERA

Antisense oligonucleotides hold considerable promise both as research tools for inhibiting gene expression and as agents for the treatment of a myriad of human diseases. However, targeted destruction of RNA has been difficult to achieve in a versatile, efficient, and reliable manner. We have developed an effective strategy for cleaving unique RNA sequences with 2-5A-dependent RNase, an endoribonuclease that mediates inhibitory effects of interferon on virus infection and is activated by 5'-phosphorylated 2'-5'-linked oligoadenylates known as 2-5A [p(n)5'A2'(p5'A2')(m)p5'A], resulting in the cleavage of single-stranded RNA predominantly after UpUp and UpAp sequences. To direct 2-5A-dependent RNase to cleave unique RNA sequences, p5'A2'p5'A2'p5'A2'p5'A was covalently linked to an antisense oligonucleotide to yield a chimeric molecule (2-5A:AS). The antisense oligonucleotide component of 2-5A:AS bound a specific RNA sequence while the accompanying 2-5A component activated 2-5A-dependent RNase, thereby causing the cleavage of the RNA in the targeted sequence. This strategy was demonstrated by inducing specific cleavage within a modified human immunodeficiency virus type 1 vif mRNA in a cell-free system from human lymphoblastoid cells. Because 2-5A-dependent RNase is present in most mammalian cells, the control of gene expression based on this technology-including therapies for cancer, viral infections, and certain genetic diseases-can be envisioned.