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Systematic screening identifies therapeutic antisense oligonucleotides for Hutchinson–Gilford progeria syndrome

Nature Medicine volume 27, pages 526–535 (2021)Cite this article

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An Author Correction to this article was published on 01 June 2021

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Abstract

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, invariably fatal childhood premature aging disorder caused by a pre-messenger RNA (mRNA) splicing defect in the LMNA gene. We used combined in vitro screening and in vivo validation to systematically explore the effects of target sequence, backbone chemistry and mechanism of action to identify optimized antisense oligonucleotides (ASOs) for therapeutic use in HGPS. In a library of 198 ASOs, the most potent ASOs targeted the LMNA exon 12 junction and acted via non-RNase H-mediated mechanisms. Treatment with an optimized lead candidate resulted in extension of lifespan in a mouse model of HGPS. Progerin mRNA levels were robustly reduced in vivo, but the extent of progerin protein reduction differed between tissues, suggesting a long half-life and tissue-specific turnover of progerin in vivo. These results identify a novel therapeutic agent for HGPS and provide insight into the HGPS disease mechanism.

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Fig. 1: ASOs screen for human lamin A RNA target.
Fig. 2: In vitro ASO screen.
Fig. 3: In vivo screen to identify efficient LMNA ASO.
Fig. 4: Efficient reduction of progerin mRNA and protein in LMNAG/G mice treated with LMNA ASO.
Fig. 5: Sex-specific LMNA ASO effect in LMNAG/G mice.

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Data availability

No large-scale, minable datasets were generated in this study. Accession codes for genes are indicated. Primary data are available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank M. Erdos and F. Collins for providing HGPS mice and U. Tavarez for technical help. Lonafarnib was provided by Merck through The Progeria Research Foundation’s Pre-Clinical Drug Supply Program. Research in the Misteli lab was supported by funding from the Intramural Research Program of the National Institutes of Health, National Cancer Institute and Center for Cancer Research (1-ZIA-BC010309) and the Progeria Research Foundation (grants PRF 2012-40, 2012-45 and 2016-66). L.G. was funded by the Progeria Research Foundation.

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Authors and Affiliations

  1. National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Madaiah Puttaraju, Asaf Shilo & Tom Misteli

  2. Ionis Pharmaceuticals, Carlsbad, CA, USA

    Michaela Jackson, Stephanie Klein, C. Frank Bennett & Frank Rigo

  3. Division of Genetics, Department of Pediatrics, Hasbro Children’s Hospital and Warren Alpert Medical School of Brown University, Providence, RI, USA

    Leslie Gordon

  4. Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Leslie Gordon

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  1. Madaiah Puttaraju
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Contributions

M.P. performed in vitro experiments and analysis and RNA and protein analysis of in vivo experiments. M.J. conduced in vivo experiments and performed histological staining and body weight and lifespan analysis. A.S. performed LMNA minigene experiments. S.K. performed histological and pathological analysis. M.P., L.G., C.F.B., M.J., F.R. and T.M. planned experiments, interpreted and discussed results and wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Tom Misteli.

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Competing interests

M.P., A.S., T.M. and L.G. declare no competing interests. C.F.B., F.R., K.S. and M.J. are employees of Ionis Pharmaceuticals and hold stock in the company.

Additional information

Peer review information Nature Medicine thanks Thomas Glover and the other, anonymous, reviewer for their contribution to the peer review of this work. Joao Monteiro was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Enhanced effect of progerin-targeted ASOs upon relaxation of inhibitory downstream RNA structure.

A, Schematic representation of the LMNA-GFP minigene reporter used to study the effect of RNA structure on alternative splicing as previously described27. The minigene consist of LMNA exons 9–12 and intron 11/exon12. Splicing events are represented by dotted lines. Forward primer F and reverse primer R are, indicated by arrows, allow for amplification of only the minigene RNA, not endogenous LMNA RNA. B, Exon 11 contains a potential dimerization region between the 5’ SS region and downstream sequence. Blue boxes indicate double strand complex region. Green Box indicates the progerin point mutation. Red box indicates tri-nucleotide C-G mutations which disrupt dimerization region and opens up the region in the MUT\5’SS-Open minigene. C, SHAPE-MaP diagram of LMNA (HGPS mutation) indicating the target location of ASOs A841 and A846 (green, blue lines, respectively). The progerin 5’ splice site (SS) is indicated by an arrow. The position of the C>G mutations in the MUT\5’ SS-Open minigene are indicated by double arrows. D, RT-PCR analysis for LMNA and progerin expression. 293T cells were transfected with 500 ng of the indicated mini-gene plasmids and 24 h later with the indicated ASOs. RNA was isolated at 48 h post the later transfection. Table show quantification of mRNA isoforms expressed as relative usage of the normal vs. alternative 5′ SS. E, Quantitative PCR results for LMNA and progerin expression levels for the same samples tested in (D). Expression levels are normalized to expression of TBP. Data are expressed as means ± SEM. For details of constructs and methods see ref. 27.

Extended Data Fig. 2 Combination treatment of LMNA-specific ASO and lonafarnib (FTI).

LMNAG/G mice (n = 6–8 per group) were treated with PBS, control scrambled ASO SCR760 (50 mg/kg) or LMNA specific ASO L-B143 (17 mg/kg) for 6.5 months. ASOs were administered as described in Methods. Mice treated with lonafarnib alone or combination treatment were singly housed to accurately measure food consumption. Lonafarnib (300 mg/kg) was mixed with transgenic dough (BioServ, Inc.,) and was orally administered. 5 g (once a day) or 10 g (once in 2 days) of transgenic dough was fed to each mouse and the leftover dough was recorded to asses food consumption. Animals were started on the diet and drugs at approximately 1 month of age. Western blot analysis of total protein extract from liver (top panel), and heart (bottom panel) using anti-Lamin A/C monoclonal antibody (sc376248) to detect lamin A and progerin. The data was normalized to housekeeping mouse β-actin and expressed relative to control (PBS) treatment. Values represent individual animals.

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Supplementary Figs. 1–9 and Supplementary Tables 1–4.

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Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

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Puttaraju, M., Jackson, M., Klein, S. et al. Systematic screening identifies therapeutic antisense oligonucleotides for Hutchinson–Gilford progeria syndrome. Nat Med 27, 526–535 (2021). https://doi.org/10.1038/s41591-021-01262-4

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