Systemically delivered antisense oligomers upregulate gene expression in mouse tissues

Abstract

Systemically injected 2'-O-methoxyethyl (2'-O-MOE)-phosphorothioate and PNA-4K oligomers (peptide nucleic acid with four lysines linked at the C terminus) exhibited sequence-specific antisense activity in a number of mouse organs. Morpholino oligomers were less effective, whereas PNA oligomers with only one lysine (PNA-1K) were completely inactive. The latter result indicates that the four-lysine tail is essential for the antisense activity of PNA oligomers in vivo. These results were obtained in a transgenic mouse model designed as a positive readout test for activity, delivery, and distribution of antisense oligomers. In this model, the expressed gene (EGFP-654) encoding enhanced green fluorescence protein (EGFP) is interrupted by an aberrantly spliced mutated intron of the human beta-globin gene. Aberrant splicing of this intron prevented expression of EGFP-654 in all tissues, whereas in tissues and organs that took up a splice site-targeted antisense oligomer, correct splicing was restored and EGFP-654 expression upregulated. The sequence-specific ability of PNA-4K and the 2'-O-MOE oligomers to upregulate EGFP-654 provides strong evidence that systemically delivered, chemically modified oligonucleotides affect gene expression by sequence-specific true antisense activity, validating their application as potential therapeutics.

Antisense oligonucleotide-based therapeutics for cancer

“…PNA has been investigated as an antisense agent, but these efforts have generally been frustrated by the poor cellular penetration and in vivo pharmacokinetic properties of PNA (Larsen et al, 1999;McMahon et al, 2002). Recently, a four-lysine peptide conjugated to a PNA was found to provide robust in vivo activity when targeted to a splice junction (Sazani et al, 2002). This data is highly encouraging, as it may provide a path to realizing the promise of PNA as an antisense therapeutic agent.…”

“…Regulation of RNA processing is another efficient mechanism in which oligonucleotides can be utilized to regulate gene expression. Studies have been published documenting that antisense oligonucleotides can be used to regulate RNA splicing in both cellbased assays and in rodent tissues (Dominski and Kole, 1993;Taylor et al, 1999b;Sazani et al, 2001;Mercatante et al, 2002;Sazani et al, 2002). An increasing number of alternatively spliced transcripts are recognized to encode functionally antagonistic proteins, and so this represents a viable approach to reversibly 'switch' protein function.…”

RNA repair restores hemoglobin expression in IVS2–654 thalassemic mice

“…Furthermore, the resulting double-stranded structures must not be recognized by RNase H, which degrades RNA in RNA-DNA duplexes (13). These conditions are satisfied in cell culture and in vivo by modified oligomers with various backbones, including 2Ј-Omethoxyethyl phosphorothioate (MOE), locked nucleic acid (LNA), peptide nucleic acid conjugated with four lysines (PNA-4K), and phosphorodiamidate morpholino oligomers (14)(15)(16). However, MOE and LNA SSOs were ineffective at correcting aberrant splicing of IVS2-654 pre-mRNA when delivered ex vivo to cultured erythroid progenitor cells from IVS2-654 murine bone marrow cells (data not shown).…”

Artificial (Pseudo)peptides for Molecular Recognition and Catalysis

“…Most notably, Kole and coworkers reported on the antisense activity of PNA in a transgenic mouse containing the gene EGFP-654 encoding for the enhanced green f luorescent protein (EGFP) [56]. This gene was, however, interrupt by an aberrantly spliced mutated intron of the human β-globin gene.…”