Synthetic Oligonucleotides

Antisense oligonucleotides (ASOs) are short, 10- to 30-bp, chemically synthesized DNA molecules that are designed to be complementary to the coding sequence of a target RNA. ASOs can be introduced into cells simply by diffusion or complexed with liposomes, such as HVJ.32 Inside the cell, ASOs form double-stranded complexes with their complementary RNA and decrease translation of RNA. Mechanisms of antisense inhibition include interference with ribosome binding and processing of mRNA, interference with mRNA conformation or mRNA splicing, and RNase-H activation of mRNA digestion.39,40

ASOs are attractive agents for in vivo gene therapy. They can be chemically synthesized in large quantities, and they do not require a viral component for in vivo delivery. However, there are several limitations to their use in vivo. ASOs have a short half-life in vivo due to nuclease degradation. Chemical modifications, such as substitution of sulfur for one of the nonbridging oxygens of a phosphate group (phosphorothiorates), renders ASOs more stable to degradation in serum.44 Several nonspecific effects of ASOs have been noted that account in part for their biologic effect.39^42 ASOs can have nonspecific cytotoxic effects due to binding to intracellular and cell surface proteins. Some ASOs affect the expression of multiple genes in addition to the gene of interest. This effect is sequence-specific and cannot be controlled for by scrambled oligonucleotides. ASOs containing CpG dinucleotides have been shown to have nonantigen activation of the humoral immune system.43 Despite these relative limitations, ASOs are well suited to the treatment of diseases where transient reductions in gene expression are required. ASOs theoretically can be used to specifically reduce the expression of one or more genes. The uptake of ASOs can be enhanced by complexing the oligonucleotide with cationic liposomes25 or HVJ liposomes.34,35

Synthetic double-stranded oligonucleotides containing binding sites for transcription factors serve as "decoys" to block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation.4444 Morishita et al.45 have shown that a single administration of an E2F decoy (containing the E2F cis element) that binds the transcription factor E2F inhibits smooth muscle cell hyperplasia in a rat carotid balloon injury model45 (Fig. 8-2). The binding of E2F prevents it from transactivating the gene expression of cell cycle regulatory proteins such as PCNA, c-myc, and cdk2, thereby inhibiting vascular smooth muscle cell proliferation and subsequent neointimal formation in vivo.

Figure 8-2: Principal of E2F "decoy" strategy. TTTCGCGC is the consensus sequence for the E2F binding site. In the quiescent cell state, the transcription factor E2F is complexed with Rb (retinoblastoma gene product), cyclin A, and the cyclin-dependent kinase cdk2 (top). Phosphorylation of Rb releases free E2F, which binds to cis elements of the cell cycle regulatory genes, resulting in the transactivation of these genes (middle). The E2F decoy cis-element double-stranded oligonucleotide binds to free E2F, preventing E2F-mediated transactivation of cell cycle regulatory genes (bottom). (From Morishita et al.,45 with permission.)

Figure 8-2: Principal of E2F "decoy" strategy. TTTCGCGC is the consensus sequence for the E2F binding site. In the quiescent cell state, the transcription factor E2F is complexed with Rb (retinoblastoma gene product), cyclin A, and the cyclin-dependent kinase cdk2 (top). Phosphorylation of Rb releases free E2F, which binds to cis elements of the cell cycle regulatory genes, resulting in the transactivation of these genes (middle). The E2F decoy cis-element double-stranded oligonucleotide binds to free E2F, preventing E2F-mediated transactivation of cell cycle regulatory genes (bottom). (From Morishita et al.,45 with permission.)

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