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Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides

Nature Biotechnology volume 35, pages 845–851 (2017)Cite this article

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Abstract

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3′-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

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Figure 1: The synthesis cycle of the SOSICS method.
Figure 2: The stereochemical preferences of RNase H1.
Figure 3: Stereochemistry affects ASO pharmacological properties in vitro.
Figure 4: An SSR code promotes RNase HC activity in vitro.
Figure 5: In vivo serum APOB and APOC3 protein kinetics in transgenic mice that were treated with mixtures or stereopure ASOs.

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Acknowledgements

We thank S. Mathieu, D. Boulay, S. Divakaramenon, K. Bowman, V. Vathipadiekal, M. Melkonian, J.C. Dodart, H. Yang, Y. (Benny) Yin, F. Desai and Z. Zhong of Wave Life Sciences for helpful discussions and support for experiments. We thank T. Wada of Tokyo University of Science for general discussions regarding the original oxazaphospholidine method. We thank U. Shigdel of Harvard University (present affiliation, Warp Drive Bio) for purifying the human RNase HC protein. We thank W. Yang of National Institute of Diabetes and Digestive and Kidney Diseases for providing the human RNase HC clone. While at RA Capital Management, A. Donner (present affiliation, The Chemical Probes Portal) provided expert assistance on production of the manuscript.

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Author notes

  1. Ivan Zlatev & Dinah W Y Sah

    Present address: Present addresses: Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA (I.Z.). and Voyager Therapeutics, Cambridge, Massachusetts, USA (D.W.Y.S.).,

  2. Naoki Iwamoto, David C D Butler and Nenad Svrzikapa: These authors contributed equally to this work.

Authors and Affiliations

  1. Wave Life Sciences, Cambridge, Massachusetts, USA

    Naoki Iwamoto, David C D Butler, Nenad Svrzikapa, Susovan Mohapatra, Ivan Zlatev, Dinah W Y Sah,  Meena, Stephany M Standley, Genliang Lu, Luciano H Apponi, Maria Frank-Kamenetsky, Jason Jingxin Zhang, Chandra Vargeese & Gregory L Verdine

  2. Department of Stem Cell and Regenerative Biology, Department of Chemistry and Chemical Biology, Harvard University and Harvard Medical School, Cambridge, Massachusetts, USA

    Gregory L Verdine

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Contributions

All authors contributed to the designing, planning and/or data collection of this project. N.I. designed and developed the SOSICS platform. N.I., D.C.D.B., I.Z., S.M.S. and G.L. carried out the synthesis and characterization of ASOs and reagents. N.I. and Meena conducted rat liver homogenate stability studies and rat serum stability studies. N.I., S.M., N.S. and Meena conducted RNase HC cleavage studies. N.S., L.H.A., M.F.-K. and J.J.Z. performed in vitro and in vivo biological analyses. N.I. and G.L.V. wrote the manuscript, and all authors refined the manuscript.

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Correspondence to Gregory L Verdine.

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Competing interests

Wave Life Sciences filed patent applications on this work. G.L.V. is one of the co-founders of Wave Life Sciences and serves as the chairman of the board of directors. D.W.Y.S. was a consultant under contract with Wave Life Sciences. N.I., D.C.D.B., N.S., S.M., I.Z., Meena, S.M.S., G.L., L.H.A., M.F.K., J.J.Z. and C.V. are or were employed by Wave Life Science.

Integrated supplementary information

Supplementary Figure 1 Synthesis of 3’-O-oxazaphospholidine monomers. All oxazaphospholidine monomers were synthesized according to published protocols with minor modifications.

DMTr, 4,4’-dimethoxytrityl; Et3N, triethylamine; Ph, phenyl; THF, tetrahydrofuran

Supplementary Figure 2 Reversed-phase HPLC data for dimers (Sp)-8a-i and (Rp)-8a-i and purified ASOs mipomersen and WV-1-7.

Sp dimers (left column, top to bottom: (Sp)-8a [(Sp)-d(AsT)], (Sp)-8b [(Sp)-d(GsT)], (Sp)-8c [(Sp)-d(5mCsT)], (Sp)-8d [(Sp)-d(CsT)], (Sp)-8e [(Sp)-d(TsT)], (Sp)-8f [(Sp)-A(MOE)sdT], (Sp)-8g [(Sp)-G(MOE)sdT], (Sp)-8h [(Sp)-5mC(MOE)sdT], (Sp)-8i [(Sp)-T(MOE)sdT]) and Rp dimers (center column, top to bottom: (Rp)-8a [(Rp)-d(AsT)], (Rp)-8b [(Rp)-d(GsT)], (Rp)-8c [(Rp)-d(5mCsT)], (Rp)-8d [(Rp)-d(CsT)], (Rp)-8e [(Rp)-d(TsT)], (Rp)-8f [(Rp)-A(MOE)sdT], (Rp)-8g [(Rp)-G(MOE)sdT], (Rp)-8h [(Rp)-5mC(MOE)sdT], (Rp)-8i [(Rp)-T(MOE)sdT]) were eluted using different gradients (see Methods). Dimers were synthesized to demonstrate stereochemical control of synthesis. Absorbance at 254 nm is shown with respect to elution time (min) for each dimer. Purified ASOs (right column, top to bottom: Mipomersen, WV-1, WV-2, WV-3, WB-4, WV-5, WV-6, WV-7), showing purity on C18 column with elution time (x axis, min) and Absorbance at 254 nm (y axis).

Supplementary Figure 3 Diastereomixture and stereochemically pure ASOs show different behaviors on reversed-phase and ion-exchange HPLC.

(a) Ion-exchange HPLC absorbance profiles of mipomersen and WV-1-6. (b) Reverse-phase HPLC data of mousomersen (the mouse sequence of mipomersen, see Supplementary Table 3) and WV-8-11. (c) Ion-exchange HPLC data of mousomersen and WV-8-11. Elution time (x axis) and Absorbance (Abs.) at 254 nm (y axis) are shown. Light green curves show the gradients of percentage of buffer B.

Supplementary Figure 4 Determination of initial velocities (V0) for WV-5 and mipomersen in RNase HC cleavage experiment.

Slope of the best fit line (V0) for WV-5 was 6.90 ± 0.54 μM/min and mipomersen was 2.37 ± 0.20 μM/min under the conditions described in Methods.

Supplementary Figure 5 Dose-response data for mipomersen and ASOs, WV-1-6, in cultured HepB3 cells.

APOB100 mRNA levels were quanitified by RT-PCR and standardized to GAPDH. There is no correlation between potency (IC50) in these assays and efficacy in mice (Fig. 5). Error bars indicate s.d. (n=3), and statistical analysis was performed in Graphpad Prism 6.01.

Supplementary Figure 6 Mipomersen dose-finding study.

Mipomersen was dosed by intraperitoneal injection on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, and 39. Human APOB serum protein (shown as a percentage compared with PBS control) dose-dependently decreases in response to mipomersen at days 17 and 24 (n=5 mice per group). We opted for the 10 mg/kg dose in subsequent experiments.

Supplementary Figure 7 Comparative efficacy and duration of action study data.

(a) Human APOB100 serum protein levels (shown as percentage compared with PBS control) decreases in response to 10 mg/kg mipomersen or its components, and recovers in the wash out period (Dosing days: 1, 4, 8, 11, 15, 18, 22, 25, 29, 32; blood collection days 17, 24, 31, 38, 45, 52, 60, 66; n=5 mice per group, error bars represent s.d.). (b) The concentration of WV-5, WV-6 and mipomersen persisting in mouse liver (μg/g) on days 3, 24 and 45 (n=4, error bars represent s.d.) is shown. The concentrations of the ASOs detected at each time point were not statistically significantly different. (c) LC-MS quantification of ASOs in the liver. Calibration curves for mipomersen, WV-5 and WV-6 were generated using the protocol described with a concentration range of 5-500 μg/g. The oligonucleotides were detected using selected reaction monitoring for the following transitions involving loss of phosphate from intact oligonucleotide: m/z 896.5 ◊ 94.9 for mipomersen, WV-5 and WV-6, and m/z 855.9 ◊ 94.9 for the internal standard.

Supplementary Figure 8 Chemical structure of WV-12 (top) and WV-13 (bottom).

(a) ASOs are GalNAc-conjugated, 20mer PS oligonucleotides composed of unmodified 10-nucleotide DNA cores and 2′-MOE-modified ends. C is 5MeC in the 2′-MOE modified ends. Complementary RNA strand (human APOC3 mRNA): rArUrArArArGrCrUrGrGrArCrArArGrArArGrCrU. (b) Ion-exchange HPLC data for WV-12 (left) and WV-13 (right). Absorbance at 254 nm is shown with respect to elution time (min) for each oligo.

Supplementary Figure 9 WV-12 (black) and WV-13 (pink) persists in mouse liver on day 78 (n=5, error bars represent s.d.).

The concentrations of the ASOs (μg/g) detected were not statistically significantly different.

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Iwamoto, N., Butler, D., Svrzikapa, N. et al. Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat Biotechnol 35, 845–851 (2017). https://doi.org/10.1038/nbt.3948

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