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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 β-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.

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Figure 1: The EGFP-654 splicing assay.
Figure 2: Transgene expression in EGFP-654 and EGFP-WT transgenic mice.
Figure 3: Ex vivo treatment of EGFP-654 mouse primary cells with 2′-O-methyl-antisense oligonucleotides.
Figure 4: Upregulation of EGFP expression in oligomer-treated EGFP-654 mice.
Figure 5: Visualization and quantification of splicing correction.

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Acknowledgements

We are grateful to Dr. Masaru Okabe, Osaka University, Japan, for a gift CXEGFP plasmid. We thank J. Summerton and P. Morcos for morpholino oligomers. We would like to thank Y. McNeill, K. Powell, C. Wilson, B. Spencer, D. Banner, M. Cameron, and E. Smith for their invaluable technical contributions, and F. Bennett, R. Griffey, and S. Crooke for critical review of this manuscript. This work was supported by the National Institutes of Health (PO1-GM59299) and GlaxoSmithKline grants to R. K.

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Correspondence to Ryszard Kole.

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R.K. is a founder of Ercole BioTech Inc., a startup company that is involved in modification of splicing by antisense oligonucleotides.

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Sazani, P., Gemignani, F., Kang, SH. et al. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol 20, 1228–1233 (2002). https://doi.org/10.1038/nbt759

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