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G2C4 targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in human C9orf72 ALS/FTD induced pluripotent stem cell derived neurons

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Abstract

The G4C2 repeat expansion in the C9orf72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Many studies suggest that dipeptide repeat proteins produced from this repeat are toxic, yet, the contribution of repeat RNA toxicity is under investigated and even less is known regarding the pathogenicity of antisense repeat RNA. Recently, two clinical trials targeting G4C2 (sense) repeat RNA via antisense oligonucleotide failed despite a robust decrease in sense-encoded dipeptide repeat proteins demonstrating target engagement. Here, in this brief report, we show that G2C4 antisense, but not G4C2 sense, repeat RNA is sufficient to induce TDP-43 dysfunction in induced pluripotent stem cell (iPSC) derived neurons (iPSNs). Unexpectedly, only G2C4, but not G4C2 sense strand targeting, ASOs mitigate deficits in TDP-43 function in authentic C9orf72 ALS/FTD patient iPSNs. Collectively, our data suggest that the G2C4 antisense repeat RNA may be an important therapeutic target and provide insights into a possible explanation for the recent G4C2 ASO clinical trial failure.

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Data availability

All data and materials are detailed within the main text or the supplementary materials. Requests for materials should be addressed to Alyssa N. Coyne, PhD email: acoyne3@jhmi.edu. ASOs used in this study can be obtained from Ionis Pharmaceuticals (Frank Bennett, PhD email: fbennett@ionisph.com) under a standard research MTA.

References

  1. Aladesuyi Arogundade O, Stauffer JE, Saberi S, Diaz-Garcia S, Malik S, Basilim H et al (2019) Antisense RNA foci are associated with nucleoli and TDP-43 mislocalization in C9orf72-ALS/FTD: a quantitative study. Acta Neuropathol 137:527–530. https://doi.org/10.1007/s00401-018-01955-0

    Article  PubMed  Google Scholar 

  2. Baskerville V, Rapuri S, Mehlhop E, Coyne AN (2023) SUN1 facilitates CHMP7 nuclear influx and injury cascades in sporadic amyotrophic lateral sclerosis. Brain. https://doi.org/10.1093/brain/awad291

    Article  PubMed  Google Scholar 

  3. Baxi EG, Thompson T, Li J, Kaye JA, Lim RG, Wu J et al (2022) Answer ALS, a large-scale resource for sporadic and familial ALS combining clinical and multi-omics data from induced pluripotent cell lines. Nat Neurosci 25:226–237. https://doi.org/10.1038/s41593-021-01006-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Brown AL, Wilkins OG, Keuss MJ, Hill SE, Zanovello M, Lee WC et al (2022) TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature 603:131–137. https://doi.org/10.1038/s41586-022-04436-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chew J, Cook C, Gendron TF, Jansen-West K, Del Rosso G, Daughrity LM et al (2019) Aberrant deposition of stress granule-resident proteins linked to C9orf72-associated TDP-43 proteinopathy. Mol Neurodegener 14:9. https://doi.org/10.1186/s13024-019-0310-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Conlon EG, Lu L, Sharma A, Yamazaki T, Tang T, Shneider NA et al (2016) The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains. Elife. https://doi.org/10.7554/eLife.17820

    Article  PubMed  PubMed Central  Google Scholar 

  7. Cooper-Knock J, Higginbottom A, Stopford MJ, Highley JR, Ince PG, Wharton SB et al (2015) Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy. Acta Neuropathol 130:63–75. https://doi.org/10.1007/s00401-015-1429-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Coyne AN, Baskerville V, Zaepfel BL, Dickson DW, Rigo F, Bennett F et al (2021) Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS. Sci Transl Med. https://doi.org/10.1126/scitranslmed.abe1923

    Article  PubMed  PubMed Central  Google Scholar 

  9. Coyne AN, Zaepfel BL, Hayes L, Fitchman B, Salzberg Y, Luo EC et al (2020) G(4)C(2) Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD. Neuron. https://doi.org/10.1016/j.neuron.2020.06.027

    Article  PubMed  PubMed Central  Google Scholar 

  10. Davidson YS, Barker H, Robinson AC, Thompson JC, Harris J, Troakes C et al (2014) Brain distribution of dipeptide repeat proteins in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathol Commun 2:70. https://doi.org/10.1186/2051-5960-2-70

    Article  PubMed  PubMed Central  Google Scholar 

  11. DeJesus-Hernandez M, Finch NA, Wang X, Gendron TF, Bieniek KF, Heckman MG et al (2017) In-depth clinico-pathological examination of RNA foci in a large cohort of C9ORF72 expansion carriers. Acta Neuropathol 134:255–269. https://doi.org/10.1007/s00401-017-1725-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ et al (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256. https://doi.org/10.1016/j.neuron.2011.09.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. DeVos SL, Miller TM (2013) Antisense oligonucleotides: treating neurodegeneration at the level of RNA. Neurotherapeutics 10:486–497. https://doi.org/10.1007/s13311-013-0194-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Donnelly CJ, Zhang PW, Pham JT, Haeusler AR, Mistry NA, Vidensky S et al (2013) RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80:415–428. https://doi.org/10.1016/j.neuron.2013.10.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Freibaum BD, Taylor JP (2017) The Role of Dipeptide Repeats in C9ORF72-Related ALS-FTD. Front Mol Neurosci 10:35. https://doi.org/10.3389/fnmol.2017.00035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gendron TF, Bieniek KF, Zhang YJ, Jansen-West K, Ash PE, Caulfield T et al (2013) Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol 126:829–844. https://doi.org/10.1007/s00401-013-1192-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gendron TF, Chew J, Stankowski JN, Hayes LR, Zhang YJ, Prudencio M et al (2017) Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis. Sci Transl Med. https://doi.org/10.1126/scitranslmed.aai7866

    Article  PubMed  PubMed Central  Google Scholar 

  18. Gitler AD, Tsuiji H (2016) There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS. Brain Res 1647:19–29. https://doi.org/10.1016/j.brainres.2016.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gleixner AM, Verdone BM, Otte CG, Anderson EN, Ramesh N, Shapiro OR et al (2022) NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility. Nat Commun 13:3380. https://doi.org/10.1038/s41467-022-31098-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gupta R, Lan M, Mojsilovic-Petrovic J, Choi WH, Safren N, Barmada S et al (2017) The Proline/Arginine Dipeptide from Hexanucleotide Repeat Expanded C9ORF72 Inhibits the Proteasome. eNeuro. https://doi.org/10.1523/eneuro.0249-16.2017

    Article  PubMed  PubMed Central  Google Scholar 

  21. Irwin KE, Jasin P, Braunstein KE, Sinha I, Bowden KD, Moghekar A et al (2023) A fluid biomarker reveals loss of TDP-43 splicing repression in pre-symptomatic ALS. bioRxiv. https://doi.org/10.1101/2023.01.23.525202

    Article  PubMed  PubMed Central  Google Scholar 

  22. Jiang J, Zhu Q, Gendron TF, Saberi S, McAlonis-Downes M, Seelman A et al (2016) Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron 90:535–550. https://doi.org/10.1016/j.neuron.2016.04.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA et al (2019) ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci 22:167–179. https://doi.org/10.1038/s41593-018-0300-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lagier-Tourenne C, Baughn M, Rigo F, Sun S, Liu P, Li HR et al (2013) Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc Natl Acad Sci U S A 110:E4530-4539. https://doi.org/10.1073/pnas.1318835110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lee SM, Asress S, Hales CM, Gearing M, Vizcarra JC, Fournier CN et al (2019) TDP-43 cytoplasmic inclusion formation is disrupted in C9orf72-associated amyotrophic lateral sclerosis/frontotemporal lobar degeneration. Brain Commun. https://doi.org/10.1093/braincomms/fcz014

    Article  PubMed  PubMed Central  Google Scholar 

  26. Lee YB, Chen HJ, Peres JN, Gomez-Deza J, Attig J, Stalekar M et al (2013) Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep 5:1178–1186. https://doi.org/10.1016/j.celrep.2013.10.049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lin Y, Mori E, Kato M, Xiang S, Wu L, Kwon I et al (2016) Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell 167:789-802.e712. https://doi.org/10.1016/j.cell.2016.10.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ling JP, Pletnikova O, Troncoso JC, Wong PC (2015) TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science 349:650–655. https://doi.org/10.1126/science.aab0983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Liu Y, Pattamatta A, Zu T, Reid T, Bardhi O, Borchelt DR et al (2016) C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD. Neuron 90:521–534. https://doi.org/10.1016/j.neuron.2016.04.005

    Article  CAS  PubMed  Google Scholar 

  30. Ma XR, Prudencio M, Koike Y, Vatsavayai SC, Kim G, Harbinski F et al (2022) TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. Nature 603:124–130. https://doi.org/10.1038/s41586-022-04424-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mackenzie IR, Frick P, Neumann M (2014) The neuropathology associated with repeat expansions in the C9ORF72 gene. Acta Neuropathol 127:347–357. https://doi.org/10.1007/s00401-013-1232-4

    Article  CAS  PubMed  Google Scholar 

  32. Melamed Z, López-Erauskin J, Baughn MW, Zhang O, Drenner K, Sun Y et al (2019) Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci 22:180–190. https://doi.org/10.1038/s41593-018-0293-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mizielinska S, Gronke S, Niccoli T, Ridler CE, Clayton EL, Devoy A et al (2014) C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 345:1192–1194. https://doi.org/10.1126/science.1256800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mizielinska S, Lashley T, Norona FE, Clayton EL, Ridler CE, Fratta P et al (2013) C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci. Acta Neuropathol 126:845–857. https://doi.org/10.1007/s00401-013-1200-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Moens TG, Mizielinska S, Niccoli T, Mitchell JS, Thoeng A, Ridler CE et al (2018) Sense and antisense RNA are not toxic in Drosophila models of C9orf72-associated ALS/FTD. Acta Neuropathol 135:445–457. https://doi.org/10.1007/s00401-017-1798-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nana AL, Sidhu M, Gaus SE, Hwang JL, Li L, Park Y et al (2019) Neurons selectively targeted in frontotemporal dementia reveal early stage TDP-43 pathobiology. Acta Neuropathol 137:27–46. https://doi.org/10.1007/s00401-018-1942-8

    Article  PubMed  Google Scholar 

  37. Prudencio M, Humphrey J, Pickles S, Brown AL, Hill SE, Kachergus J et al (2020) Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia. J Clin Invest. https://doi.org/10.1172/jci139741

    Article  PubMed  PubMed Central  Google Scholar 

  38. Prudencio M, Jansen-West KR, Lee WC, Gendron TF, Zhang YJ, Xu YF et al (2012) Misregulation of human sortilin splicing leads to the generation of a nonfunctional progranulin receptor. Proc Natl Acad Sci U S A 109:21510–21515. https://doi.org/10.1073/pnas.1211577110

    Article  PubMed  PubMed Central  Google Scholar 

  39. Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–268. https://doi.org/10.1016/j.neuron.2011.09.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Roczniak-Ferguson A, Ferguson SM (2019) Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis. Life Sci Allian https://doi.org/10.26508/lsa.201900358

  41. Sareen D, O’Rourke JG, Meera P, Muhammad AK, Grant S, Simpkinson M et al (2013) Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci Transl Med. https://doi.org/10.1126/scitranslmed.3007529

    Article  PubMed  PubMed Central  Google Scholar 

  42. Schoch KM, Miller TM (2017) Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases. Neuron 94:1056–1070. https://doi.org/10.1016/j.neuron.2017.04.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Seddighi S, Qi YA, Brown A-L, Wilkins OG, Bereda C, Belair C et al (2023) Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD. bioRxiv. https://doi.org/10.1101/2023.01.23.525149

    Article  PubMed  PubMed Central  Google Scholar 

  44. Vatsavayai SC, Nana AL, Yokoyama JS, Seeley WW (2019) C9orf72-FTD/ALS pathogenesis: evidence from human neuropathological studies. Acta Neuropathol 137:1–26. https://doi.org/10.1007/s00401-018-1921-0

    Article  CAS  PubMed  Google Scholar 

  45. Vatsavayai SC, Yoon SJ, Gardner RC, Gendron TF, Vargas JN, Trujillo A et al (2016) Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia. Brain 139:3202–3216. https://doi.org/10.1093/brain/aww250

    Article  PubMed  PubMed Central  Google Scholar 

  46. Wen X, Tan W, Westergard T, Krishnamurthy K, Markandaiah SS, Shi Y et al (2014) Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84:1213–1225. https://doi.org/10.1016/j.neuron.2014.12.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yuva-Aydemir Y, Almeida S, Krishnan G, Gendron TF, Gao FB (2019) Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD. Nat Commun 10:5466. https://doi.org/10.1038/s41467-019-13477-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P et al (2015) The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525:56–61. https://doi.org/10.1038/nature14973

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhang YJ, Guo L, Gonzales PK, Gendron TF, Wu Y, Jansen-West K et al (2019) Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity. Science. https://doi.org/10.1126/science.aav2606

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the ALS patients and their families for essential contributions to this research. iPSC lines acquired through the Answer ALS program made this research possible. These and other ALS iPSC lines can be obtained from: https://csbiomfg.com/cellcollection. This work was supported by The Robert Packard Center for ALS Research (ANC), NIH NINDS/NIA R00 NS123242 (ANC), Target ALS IL-2023-C6-L4 (ANC, JDR, and PJN), along with funding from NIH-NINDS, NIH-NIA, Department of Defense, ALS Association, Muscular Dystrophy Association, F Prime, and the Chan Zuckerberg Initiative.

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Authors and Affiliations

  1. Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Jeffrey D. Rothstein, Victoria Baskerville, Sampath Rapuri, Emma Mehlhop & Alyssa N. Coyne

  2. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Jeffrey D. Rothstein, Victoria Baskerville, Sampath Rapuri, Emma Mehlhop & Alyssa N. Coyne

  3. Ionis Pharmaceuticals, Carlsbad, CA, 92010, USA

    Paymaan Jafar-Nejad, Frank Rigo & Frank Bennett

  4. UK Dementia Research Institute at King’s College London, London, UK

    Sarah Mizielinska

  5. Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, Maurice Wohl Clinical Neuroscience Institute, King’s College London, London, UK

    Sarah Mizielinska

  6. UK Dementia Research Institute at UCL, London, UK

    Adrian Isaacs

  7. Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK

    Adrian Isaacs

  8. UCL Queen Square Motor Neuron Disease Centre, UCL Queen Square Institute of Neurology, London, UK

    Adrian Isaacs

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  1. Jeffrey D. Rothstein
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Contributions

Conceived and designed the experiments: ANC and JDR. Performed the experiments: ANC, VB, SR, EM. Analyzed the data: ANC. Contributed reagents and materials: ANC, PHN, FR, FB, SM, AMI, and JDR. Wrote the manuscript: ANC and JDR with input from co-authors.

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Correspondence to Jeffrey D. Rothstein or Alyssa N. Coyne.

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Rothstein, J.D., Baskerville, V., Rapuri, S. et al. G2C4 targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in human C9orf72 ALS/FTD induced pluripotent stem cell derived neurons. Acta Neuropathol 147, 1 (2024). https://doi.org/10.1007/s00401-023-02652-3

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