Catalytic Nucleic Acids
Concepts for gene therapy were developed almost 30 years ago and have been transformed into clinical reality. Currently, the strategies of gene therapy are aimed at either the replacement of defective genes or suppression of a pathological gene target. Strategies exemplified by the use of antisense RNA or oligonucleotides, expression of mutant structural or regulatory genes with dominant repressor activity and over-expression of competitive RNA sequences vary widely in their degree of specificity and serve primarily to block gene expression by interfering with RNA transcription or translation. Unique among these approaches is the use of catalytic nucleic acids, ribozymes or oligonucleotides capable of cleaving a target RNA molecule in a highly sequence-specific manner.
Ribozymes
Ribozymes, the most extensively studied of the catalytic nucleic acids, exist in a range of distinct categories of naturally occurring catalytic RNA. These include a series of small ribozymes important for the rolling circle replication of viroid genomes, such as hammerhead and hairpin ribozymes, the group I introns, the RNA component of RNase P, and hepatitis delta virus ribozyme. Ribozymes catalyze sequence-specific RNA processing. The specificity is determined by Watson-Crick base-paring between ribozymes and nucleotides near the cleavage site of the target RNA. By altering substrate recognition sequences, several intramolecular cis-cleaving ribozymes have been engineered to cleave target RNA in trans. Theoretically, the mRNA coding of any proteins associated with a disease can be selectively cleaved by ribozymes. Thus, ribozymes have become potentially valuable tools for the inhibition of virus replication, modulation of tumor progression, and analysis of cellular gene function.
Figure 1. Mechanism of action of the ribozyme.
Therapeutic Oligonucleotides
Advances in nucleic acid chemistry have led to progress in ribozyme synthesis, which allows the incorporation of modified ribonucleotide analogs with enhanced nuclease resistance. This usually involves the substitution of the 2'-hydroxyl moieties with some other chemicals such as 2'-deoxy, 2'-O-methyl, 2'-amino, or 2'-fluoro derivatives. The substitution of DNA into the helix-forming motifs and specific unpaired positions of the catalytic domain has been shown to produce a substantial improvement in biological stability. By comparison, antisense DNA oligonucleotides with their uninterrupted DNA composition have a much greater half-life in vivo. The natural biological stability of DNA compared with RNA can also be readily supplemented by chemistry, which provides even greater resistance to nuclease digestion; the more commonly used modification involves the replacement of phosphodiester linkages in the backbone with phosphorothioate or methylphosphonate moieties.
Figure 2. Mechanism of antisense oligonucleotides.
Delivery of Oligonucleotides
The most challenging aspect of the use of catalytic nucleic acids in a pharmaceutical role is delivering these molecules to their site of action, the RNA target located within the cytoplasm or nucleus. Delivery requires that the oligonucleotide survive local or systemic administration long enough to bind to the target cells, cross the cytoplasmic membrane or become released from an endosomal/ lysosomal vesicle, pass through the nuclear membrane, and be able to functionally hybridize to the target RNA. A range of viral vector constructs have been designed to express ribozymes endogenously within the target, and potentially successful ex vivo applications have been described. Studies of exogenously delivered oligonucleotides have been dominated for several years in studies of antisense oligonucleotide therapy. The pharmacological issues critical to the delivery of catalytic DNA and RNA can be extrapolated from the wealth of data collected from studies of the pharmacology of antisense drugs. By drawing on this wide body of data and including recent studies of ribozyme delivery, it is possible to discuss how issues such as stability, toxicity, immunology, in vivo and cellular pharmacokinetics, and the employment of delivery agents are challenges common to all forms of oligonucleotide-based therapy.
Reference
- Sun, L. Q.; et al. (2000). Catalytic nucleic acids: from lab to applications. Pharmacological reviews. 52(3): 325-348.
