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Enhancing siRNA efficacy in vivo with extended nucleic acid backbones

Nature Biotechnology volume 43, pages 904–913 (2025)Cite this article

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Abstract

Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation. However, metabolic stabilization by phosphorothioate (PS), the only backbone chemistry used clinically, may be insufficient for targeting extrahepatic tissues. To improve oligonucleotide stabilization, we report the discovery, synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5′-C and 5′-OH of a nucleoside. exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone, provides stabilization against 3′ and 5′ exonucleases and is tolerated at multiple oligonucleotide positions. A combined exNA–PS backbone enhances resistance to 3′ exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone, improving tissue exposure, tissue accumulation and efficacy in mice, both systemically and in the brain. The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues, both with siRNA and with other oligonucleotides such as CRISPR guide RNA, antisense oligonucleotides, mRNA and tRNA.

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Fig. 1: Chemical structure of natural epigenetic modifications and exNA.
Fig. 2: Thermal stability of rxU-modified RNA–RNA and RNA–DNA duplexes.
Fig. 3: Nuclease resistance of exNA-modified oligonucleotides.
Fig. 4: Position-dependent impact of exNA replacement on siRNA activity in vitro.
Fig. 5: Systemic delivery of DCA-conjugated siRNA with 3′-exNA modification.
Fig. 6: Impact of 3′-exNA modification on siRNA efficacy in the CNS.

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Data availability

The data supporting the findings of this study are available from the corresponding authors upon reasonable request. The raw NMR data are included in the Supplementary Information.

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Acknowledgements

This project was funded by the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (R01 NS104022 to A.K.), S10 (OD020012 to A.K.), Maximizing Investigators’ Research Award (R35 GM131839 to A.K.), NIH CREATE (U01 NS114098 to N.A.), Cure Huntington’s Disease Initiative Foundation (RecID A-5038 to N.A.) and the Berman–Topper Fund (to A.K. and N.A.). The authors would like to thank the University of Massachusetts Chan Medical School Animal Medicine Department and veterinary technicians for contributing to the large-animal studies. Some icons in Figs. 5b and 6b were adapted from BioRender.com. The authors would also like to thank E. Haberlin for proofreading the manuscript, A. Ali for maintaining the infrastructure for nuclear magnetic resonance (NMR) and Y. Tan and S. Nguyen for measurement of high-resolution mass analysis.

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Authors and Affiliations

  1. RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Ken Yamada, Vignesh N. Hariharan, Jillian Caiazzi, Rachael Miller, Chantal M. Ferguson, Hassan H. Fakih, Qi Tang, Nozomi Yamada, Raymond C. Furgal, Joseph D. Paquette, Annabelle Biscans, Brianna M. Bramato, Nicholas McHugh, Ashley Summers, Clemens Lochmann, Bruno M. D. C. Godinho, Samuel Hildebrand, Dimas Echeverria, Matthew R. Hassler, Julia F. Alterman, Neil Aronin & Anastasia Khvorova

  2. Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Rachael Miller, Chantal M. Ferguson, Joseph D. Paquette & Neil Aronin

  3. Department of Neurology, Harvard Medical School and Mass General Institute for Neurodegenerative Disease, Charlestown, MA, USA

    Ellen Sapp & Marian DiFiglia

  4. Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Anastasia Khvorova

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Contributions

K.Y. and A.K. conceived the project. K.Y., V.N.H. and A.K. contributed to the experimental design for exNA studies in hepatic and extrahepatic tissues. K.Y., V.N.H., J.C., H.H.F., Q.T., A.B., R.C.F, A.S., C.L. and B.M.D.C.G. conducted the experiments. C.M.F., K.Y., R.M., E.S., J.F.A., M.D., N.A. and A.K. contributed to the experimental design for exNA studies in CNS. C.M.F., R.M., E.S. and J.D.P conducted experiments. K.Y. and N.Y. contributed to the exNA thermal stability and nuclease resistance study. K.Y., N.Y., B.M.B., N.M., S.O.J. and D.E. contributed to the oligonucleotide synthesis. S.H. contributed to the statistical analysis of exNA CNS studies. V.N.H. and M.R.H. gave inspirational intellectual contributions. K.Y. and A.K. wrote the manuscript.

Corresponding authors

Correspondence to Ken Yamada or Anastasia Khvorova.

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Competing interests

K.Y., V.N.H. and A.K. have filed patent applications for exNA platforms. A.K. discloses ownership of stocks in RXi Pharmaceuticals and Advirna, and is a founder of Atalanta Therapeutics and Comanche Biopharma. V.N.H. is an employee of Comanche Biopharma and owns stock options. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Eriks Rozners and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Synthesis of exNA phosphoramidites (6a and 6b) and exNA-modified oligonucleotide.

Reagent and conditions are as follows: (i) IBX/CH3CN, 85 °C, 1.5–2 h. (ii) CH3PPh3Br, tBuOK, THF, 0 °C then rt, overnight, 2a: 75% (2 steps), 2b: 67% (2 steps). (iii) 9-BBN, THF, 0 °C, overnight. (iv) NaBO3·4H2O, MeOH, THF, H2O, 0 °C then rt, overnight, 3a: 62% (2 steps), 3b: N.D. (v) DMTr-Cl, pyridine, rt, 2 h. (vi) 0.1 M TBAF, THF, rt, 1 h, 5a: 93% (2 steps), 5b: 12% (3 steps). (vii) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA, CH2Cl2, 0 °C then rt, 0.5 h, 6a: 86%, 6b: 81%. (viii) RNA synthesizer (Supplementary Information).

Supplementary information

Supplementary information

Supplementary Note (general remarks), Figs. 1–9, Tables 1–11, Scheme 1 and Data (synthetic oligonucleotide sequence information, synthesis procedures of compounds, LC–MS data and all raw NMR material data).

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Yamada, K., Hariharan, V.N., Caiazzi, J. et al. Enhancing siRNA efficacy in vivo with extended nucleic acid backbones. Nat Biotechnol 43, 904–913 (2025). https://doi.org/10.1038/s41587-024-02336-7

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