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Towards a therapy for Angelman syndrome by targeting a long non-coding RNA

Nature volume 518, pages 409–412 (2015)Cite this article

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Abstract

Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia1,2. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase3,4,5. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS)6,7,8. Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression9,10. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.

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Figure 1: Unsilencing of the Ube3a paternal allele by Ube3a-ATS-targeted ASOs in cultured mouse neurons.
Figure 2: A single administration of Ube3a-ATS ASOs resulted in paternal UBE3A unsilencing for 4 months.
Figure 3: Widespread distribution of paternal UBE3A unsilencing throughout the brain.
Figure 4: ASO administration in adult Angelman syndrome mice unsilenced paternal UBE3A and ameliorated abnormal phenotypes.

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Acknowledgements

We thank T. Cooper for suggesting the collaboration between L.M., A.L.B. and Isis Pharmaceuticals. L.M. thanks D. Liu, X. Jun, H. Zheng, J. Rosen, C. Spencer and X. Zhai for technical support and reagent sharing, and M. Costa-Mattioli, J. Jankowsky and Y. Elgersma for discussions. This research was supported by funding from National Institutes of Health grant R01 HD037283 (A.L.B.), the Angelman Syndrome Foundation (A.L.B. and L.M.), and Baylor College of Medicine Intellectual and Developmental Disability Research Center grant P30HD024064.

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Author notes

  1. Linyan Meng and Amanda J. Ward: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Human Genetics, Baylor College of Medicine, and Texas Children’s Hospital, Houston, 77030, Texas, USA

    Linyan Meng & Arthur L. Beaudet

  2. Department of Core Antisense Research, Isis Pharmaceuticals, Carlsbad, 92010, California, USA

    Amanda J. Ward, Seung Chun, C. Frank Bennett & Frank Rigo

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  1. Linyan Meng
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  2. Amanda J. Ward
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  3. Seung Chun
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  5. Arthur L. Beaudet
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Contributions

L.M. and A.J.W. designed and performed experiments, analysed data, and wrote the paper (equal contribution). S.C. performed ASO delivery for Figure 2. C.F.B., A.L.B. and F.R. supervised the project. All authors discussed the experimental results.

Corresponding authors

Correspondence to Arthur L. Beaudet or Frank Rigo.

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Competing interests

A.J.W., C.F.B. and F.R. are employees of Isis Pharmaceuticals.

Extended data figures and tables

Extended Data Figure 1 ASOs targeting Snord116 reduced Ube3a-ATS pre-mRNA.

a, Top, schematic of the ASO-binding sites and location of qRT–PCR primer and probe sets. Bottom, qRT–PCR from wild-type primary neurons treated with ASO A or ASO116 (72 h) using primer and probe sets to the indicated regions of Ube3a-ATS pre-mRNA and mRNA. b, Nascent transcripts were isolated from wild-type primary neurons incubated with 5-ethynyl uridine (see Methods) for the indicated time. qRT–PCR for pre-mRNA and mature mRNA within the Snord116 region. The red line indicates the 30 min delay between the appearance of pre-mRNA and mature mRNA. Assuming a transcription elongation rate of 4 kb min−1, it would take RNAPII 80 min to transcribe the 332 kb distance from the last copy of Snord116 to the ASO-binding site. HG, host gene. n = 2 per group, mean ± absolute deviation.

Extended Data Figure 2 ASOs complementary to two regions of Ube3a-ATS differed in their ability to unsilence paternal Ube3a.

PatYFP primary neurons were treated with ASOs that bind Ube3a-ATS 5′ of Ube3a (non-overlap ASOs, n = 15) or that bind to the gene body region (overlap ASOs, n = 12) for 72 h. The level of Ube3aYFP-ATS reduction and Ube3aYFP upregulation was analysed by qRT–PCR and normalized to untreated control (UTC) neurons. Data are shown as mean ± s.e.m.

Extended Data Figure 3 In vivo ASO administration was well tolerated.

a, Left, body weight of individual wild-type C57BL/6 female mice (2 months old) treated with PBS or ASO measured weekly for 4 weeks after treatment. Right, change in body weight at each time point relative to body weight at time of treatment. n = 4 per group, mean ± s.e.m. b, Per cent change in body weight of PatYFP mice 4 weeks after treatment relative to pre-treatment. n = 3–4, mean ± s.e.m. c, Microglial activation was measured by Aif1 qRT–PCR 4 weeks after treatment. CTX, cortex; HIP, hippocampus; SC, thoracic spinal cord. *P < 0.05, two-tailed t-test, n = 3–4 per group, mean ± s.e.m. d, Immunohistochemistry for AIF1 and GFAP on sagittal brain sections from wild-type C57BL/6 female mice treated with PBS or ASO for 2 weeks.

Extended Data Figure 4 Snord116 was not reduced in the hypothalamus.

qRT–PCR on RNA isolated from PatYFP mice 4 weeks after treatment with PBS or ASO B.

Extended Data Figure 5 UBE3A unsilencing persisted 4 months after treatment.

ASO and YFP immunofluorescence on brain sections of cortex and cerebellum in PatYFP mice 4 months after treatment with ASO A.

Extended Data Figure 6 UBE3A–YFP was upregulated throughout the brain.

Whole-brain image of YFP fluorescence in PatYFP mice treated with PBS or ASO 4 weeks after treatment.

Extended Data Figure 7 Imaging of unsilenced UBE3A–YFP in specific brain regions.

a–l, Immunofluorescence for ASO, UBE3A–YFP and NeuN 4 weeks after treatment in MatYFP or PatYFP mice of the amygdala (a), hippocampus CA2 and CA3 layers (b), dentate gyrus (c), striatrum (d), thalamus (e), hypothalamus (f), medulla (g), third ventricle (h), motor cortex (i), somatosensory cortex (j), auditory cortex (k) and visual cortex (l).

Extended Data Figure 8 Intrahippocampal injection of ASO A in PatYFP mice resulted in near complete unsilencing of paternal UBE3A–YFP.

YFP immunofluorescence on brain sections from PatYFP mice treated with Ctl ASO, 100 μg ASO A via intrahippocampal injection, or 700 μg ASO A via ICV injection. A MatYFP mouse treated with PBS was included for comparison.

Extended Data Figure 9 ASO treatment in Angelman syndrome mice upregulated Ube3a.

a, RNA levels of Ube3a-ATS and Ube3a were determined by qRT–PCR in wild-type (WT) mice treated with PBS and Angelman syndrome (AS) mice treated with Ctl ASO or ASO A. n = 2–3 per group, mean ± s.e.m. b, UBE3A immunofluorescence on brain sections was performed 2 to 8 weeks after treatment. c–h, ASO treatment in adult Angelman syndrome mice did not reverse some disease-associated phenotypes. c, Total distance travelled in the open field assay. d, Vertical activity in the open field assay. e, Stereotype activity in the open field assay. f, Marble burying test. The y axis represents the number of marbles at least 50% buried. g, Accelerating rotarod test during eight trials. h, Post-shock and cued response measured during the fear conditioning assay. n = 13–15 per group *P < 0.05, ***P < 0.001 (one-way ANOVA with Newman–Keuls post-hoc analysis). NS, not significant. i, Growth curve of age-matched female mice. Each line represents weight measurements of a single mouse over a 5-month time course post-injection, n = 5 per group. Tx age, age of mouse at time of treatment.

Extended Data Table 1 qRT–PCR primer sequences
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Meng, L., Ward, A., Chun, S. et al. Towards a therapy for Angelman syndrome by targeting a long non-coding RNA. Nature 518, 409–412 (2015). https://doi.org/10.1038/nature13975

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