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Extension of replicative lifespan by synthetic engineered telomerase RNA in patient induced pluripotent stem cells

Nature Biomedical Engineering volume 9, pages 2083–2097 (2025)Cite this article

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Abstract

RNA engineering has yielded a new class of medicines but faces limitations depending on RNA size and function. Here we demonstrate the synthesis and enzymatic stabilization of telomerase RNA component (TERC), a therapeutically relevant long non-coding RNA (lncRNA) that extends telomere length and replicative lifespan in human stem cells. Compared with therapeutic mRNAs, engineered TERC RNA (eTERC) depends on avoiding nucleoside base modifications and incorporates a distinct trimethylguanosine 5′ cap during in vitro transcription. We show that the non-canonical polymerase TENT4B can be repurposed to enzymatically stabilize synthetic RNAs of any size by catalysing self-limited 2′-O-methyladenosine tailing, which is critical for optimal eTERC function in cells. A single transient exposure to eTERC forestalls telomere-induced senescence in telomerase-deficient human cell lines and lengthens telomeres in induced pluripotent stem cells from nine patients carrying different mutations in telomere-maintenance genes, as well as primary CD34+ blood stem/progenitor cells. Our results provide methods and proof of functional reconstitution for a stabilized, synthetic human lncRNA. eTERC may have therapeutic potential to safely extend replicative capacity in human stem cells.

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Fig. 1: 3′ end protection via TENT4B-mediated 2′OMeA tailing increases stability and function of eGFP mRNA in 293T cells.
Fig. 2: Engineered synthetic TERC RNA (eTERC RNA) with a 5′-tmg cap and 3′ end protection via TENT4B-mediated 2′OMeA tailing rescues telomere length and TERC RNA levels in TERC-null 293T cells.
Fig. 3: eTERC RNA rescues cellular replicative capacity and senescence-induced DNA damage in TERC-null 293T cells.
Fig. 4: eTERC RNA rescues telomere length in iPSCs derived from a patient with TBD with mutations in the PARN gene.
Fig. 5: eTERC RNA rescues telomere length in iPSCs derived from patients with mutations in TBD-associated genes.
Fig. 6: eTERC RNA boosts telomere repeat synthesis in iPSCs and primary human HSPCs from healthy donors.

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Data availability

The data that support the findings of this study are available within the paper and associated extended data files. Unprocessed data will be provided on request. There are no restrictions on data availability from this study. Source data are provided with this paper.

References

  1. Zhu, Y., Zhu, L., Wang, X. & Jin, H. RNA-based therapeutics: an overview and prospectus. Cell Death Dis. 13, 644 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Sparmann, A. & Vogel, J. RNA-based medicine: from molecular mechanisms to therapy. EMBO J. 42, e114760 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Belgrad, J., Fakih, H. H. & Khvorova, A. Nucleic acid therapeutics: successes, milestones, and upcoming innovation. Nucleic Acid Ther. 34, 52–72 (2024).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Tang, Q. & Khvorova, A. RNAi-based drug design: considerations and future directions. Nat. Rev. Drug Discov. 23, 341–364 (2024).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Parhiz, H., Atochina-Vasserman, E. N. & Weissman, D. mRNA-based therapeutics: looking beyond COVID-19 vaccines. Lancet 403, 1192–1204 (2024).

    Article  PubMed  CAS  Google Scholar 

  6. Uszczynska-Ratajczak, B., Lagarde, J., Frankish, A., Guigo, R. & Johnson, R. Towards a complete map of the human long non-coding RNA transcriptome. Nat. Rev. Genet. 19, 535–548 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Mattick, J. S. et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nat. Rev. Mol. Cell Biol. 24, 430–447 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).

    Article  PubMed  CAS  Google Scholar 

  9. Yan, J. et al. Improving prime editing with an endogenous small RNA-binding protein. Nature 628, 639–647 (2024).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Winkle, M., El-Daly, S. M., Fabbri, M. & Calin, G. A. Noncoding RNA therapeutics—challenges and potential solutions. Nat. Rev. Drug Discov. 20, 629–651 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Nagpal, N. & Agarwal, S. Telomerase RNA processing: implications for human health and disease. Stem Cells 38, 1532–1543 (2020).

  12. Rossiello, F., Jurk, D., Passos, J. F. & d’Adda di Fagagna, F. Telomere dysfunction in ageing and age-related diseases. Nat. Cell Biol. 24, 135–147 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Cech, T. R. Beginning to understand the end of the chromosome. Cell 116, 273–279 (2004).

    Article  PubMed  CAS  Google Scholar 

  14. Codd, V. et al. Polygenic basis and biomedical consequences of telomere length variation. Nat. Genet. 53, 1425–1433 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Bodnar, A. G. et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352 (1998).

    Article  PubMed  CAS  Google Scholar 

  16. Vaziri, H. & Benchimol, S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr. Biol. 8, 279–282 (1998).

    Article  PubMed  CAS  Google Scholar 

  17. Revy, P., Kannengiesser, C. & Bertuch, A. A. Genetics of human telomere biology disorders. Nat. Rev. Genet. 24, 86–108 (2023).

    Article  PubMed  CAS  Google Scholar 

  18. Huang, F. W. et al. TERT promoter mutations and monoallelic activation of TERT in cancer. Oncogenesis 4, e176 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Jaskelioff, M. et al. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. Nature 469, 102–106 (2011).

    Article  PubMed  CAS  Google Scholar 

  20. Soerensen, M. et al. Genetic variation in TERT and TERC and human leukocyte telomere length and longevity: a cross-sectional and longitudinal analysis. Aging Cell 11, 223–227 (2012).

    Article  PubMed  CAS  Google Scholar 

  21. Codd, V. et al. Common variants near TERC are associated with mean telomere length. Nat. Genet. 42, 197–199 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Vulliamy, T. J. et al. Differences in disease severity but similar telomere lengths in genetic subgroups of patients with telomerase and shelterin mutations. PLoS ONE 6, e24383 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Nagpal, N. et al. Small-molecule PAPD5 inhibitors restore telomerase activity in patient stem cells. Cell Stem Cell 26, 896–909 e898 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Kirwan, M. et al. Exogenous TERC alone can enhance proliferative potential, telomerase activity and telomere length in lymphocytes from dyskeratosis congenita patients. Br. J. Haematol. 144, 771–781 (2009).

    Article  PubMed  CAS  Google Scholar 

  25. Wong, J. M. & Collins, K. Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes Dev. 20, 2848–2858 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Shukla, S., Jeong, H. C., Sturgeon, C. M., Parker, R. & Batista, L. F. Z. Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita. Blood Adv. 4, 2717–2722 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Bopst, M. et al. Unexpected neurotoxicity in chronic toxicity studies with a HBV viral expression inhibitor. Regul. Toxicol. Pharmacol. 141, 105407 (2023).

    Article  PubMed  CAS  Google Scholar 

  28. Lake, A. D. et al. Neurotoxicity of an hepatitis B virus (HBV) transcript inhibitor in 13-week rat and monkey studies. Toxicol. Sci. 186, 298–308 (2022).

    Article  PubMed  CAS  Google Scholar 

  29. Lam, A. M. et al. Preclinical antiviral and safety profiling of the HBV RNA destabilizer AB-161. Viruses 16, 323 (2024).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Buemi, V. et al. TGS1 mediates 2,2,7-trimethyl guanosine capping of the human telomerase RNA to direct telomerase dependent telomere maintenance. Nat. Commun. 13, 2302 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Jady, B. E., Bertrand, E. & Kiss, T. Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal. J. Cell Biol. 164, 647–652 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Mitchell, J. R., Cheng, J. & Collins, K. A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3′ end. Mol. Cell. Biol. 19, 567–576 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Mitchell, J. R. & Collins, K. Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. Mol. Cell 6, 361–371 (2000).

    Article  PubMed  CAS  Google Scholar 

  34. Fu, D. & Collins, K. Distinct biogenesis pathways for human telomerase RNA and H/ACA small nucleolar RNAs. Mol. Cell 11, 1361–1372 (2003).

    Article  PubMed  CAS  Google Scholar 

  35. Nguyen, D. et al. A polyadenylation-dependent 3′ end maturation pathway is required for the synthesis of the human telomerase RNA. Cell Rep. 13, 2244–2257 (2015).

    Article  PubMed  CAS  Google Scholar 

  36. Shukla, S., Schmidt, J. C., Goldfarb, K. C., Cech, T. R. & Parker, R. Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects. Nat. Struct. Mol. Biol. 23, 286–292 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Tseng, C. K. et al. Human telomerase RNA processing and quality control. Cell Rep. 13, 2232–2243 (2015).

    Article  PubMed  CAS  Google Scholar 

  38. Boyraz, B. et al. Posttranscriptional manipulation of TERC reverses molecular hallmarks of telomere disease. J. Clin. Invest. 126, 3377–3382 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Moon, D. H. et al. Poly(A)-specific ribonuclease (PARN) mediates 3′-end maturation of the telomerase RNA component. Nat. Genet. 47, 1482–1488 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Schwartz, S. et al. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159, 148–162 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Mannherz, W. & Agarwal, S. Thymidine nucleotide metabolism controls human telomere length. Nat. Genet. 55, 568–580 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Cristofari, G. & Lingner, J. Telomere length homeostasis requires that telomerase levels are limiting. EMBO J. 25, 565–574 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Beattie, T. L., Zhou, W., Robinson, M. O. & Harrington, L. Reconstitution of human telomerase activity in vitro. Curr. Biol. 8, 177–180 (1998).

    Article  PubMed  CAS  Google Scholar 

  44. Tseng, C. K., Wang, H. F., Schroeder, M. R. & Baumann, P. The H/ACA complex disrupts triplex in hTR precursor to permit processing by RRP6 and PARN. Nat. Commun. 9, 5430 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Roake, C. M. et al. Disruption of telomerase RNA maturation kinetics precipitates disease. Mol. Cell 74, 688–700 e683 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Lim, J. et al. Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation. Science 361, 701–704 (2018).

    Article  PubMed  CAS  Google Scholar 

  47. Cummins, L. L. et al. Characterization of fully 2′-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res. 23, 2019–2024 (1995).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Wahlestedt, C. et al. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl Acad. Sci. USA 97, 5633–5638 (2000).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Rammelt, C., Bilen, B., Zavolan, M. & Keller, W. PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif. RNA 17, 1737–1746 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Kariko, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

    Article  PubMed  CAS  Google Scholar 

  51. Kariko, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008).

    Article  PubMed  CAS  Google Scholar 

  52. Gao, M., Zhang, Q., Feng, X. H. & Liu, J. Synthetic modified messenger RNA for therapeutic applications. Acta Biomater. 131, 1–15 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Allsopp, R. C. et al. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl Acad. Sci. USA 89, 10114–10118 (1992).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Chiba, K. et al. Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism. Science 357, 1416–1420 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Han, X. et al. Discovery of RG7834: the first-in-class selective and orally available small molecule hepatitis B virus expression inhibitor with novel mechanism of action. J. Med. Chem. 61, 10619–10634 (2018).

    Article  PubMed  CAS  Google Scholar 

  56. Gazzaniga, F. S. & Blackburn, E. H. An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity. Blood 124, 3675–3684 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Tummala, H. et al. Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita. Am. J. Hum. Genet. 109, 1472–1483 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Nagpal, N., Tai, A. K., Nandakumar, J. & Agarwal, S. Domain specific mutations in dyskerin disrupt 3′ end processing of scaRNA13. Nucleic Acids Res. 50, 9413–9425 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Agarwal, S. et al. Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature 464, 292–296 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Uringa, E. J. et al. RTEL1 contributes to DNA replication and repair and telomere maintenance. Mol. Biol. Cell 23, 2782–2792 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Dokal, I. Dyskeratosis congenita in all its forms. Br. J. Haematol. 110, 768–779 (2000).

    Article  PubMed  CAS  Google Scholar 

  62. Aubert, G., Baerlocher, G. M., Vulto, I., Poon, S. S. & Lansdorp, P. M. Collapse of telomere homeostasis in hematopoietic cells caused by heterozygous mutations in telomerase genes. PLoS Genet. 8, e1002696 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Ramunas, J. et al. Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells. FASEB J. 29, 1930–1939 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Ferrari, S. et al. Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy. Cell Stem Cell 30, 549–570 (2023).

    Article  PubMed  CAS  Google Scholar 

  65. Warlich, E. et al. Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming. Mol. Ther. 19, 782–789 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Casirati, G. et al. Epitope editing enables targeted immunotherapy of acute myeloid leukaemia. Nature 621, 404–414 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Mender, I. & Shay, J. W. Telomere dysfunction induced foci (TIF) analysis. Bio Protoc. 5, e1656 (2015).

    Article  PubMed  Google Scholar 

  68. Maryati, M. et al. A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. Nucleic Acids Res. 42, e30 (2014).

    Article  PubMed  CAS  Google Scholar 

  69. Lycka, M. et al. WALTER: an easy way to online evaluate telomere lengths from terminal restriction fragment analysis. BMC Bioinform. 22, 145 (2021).

    Article  Google Scholar 

  70. Lai, T. P., Wright, W. E. & Shay, J. W. Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity. BioTechniques 60, 306–309 (2016).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank patients and families for their participation in research. We thank A. Shimamura, M. Fleming and the BCH Bone Marrow Failure/Myelodysplastic Syndrome Registry (NIH grant number 1RC2DK122533). We thank Y. Fong for rPARN and rTENT4B protein. We thank H. Cramer and C.-H. Chien from the BCH Cellular Imaging Core. N.N. discloses support for the research described in this study from BCH Manton Center for Orphan Disease Research, Uplifting Athletes Young Investigator Draft, Team Telomere. S.A. discloses support for the research described in this study from the NIH (grant number R01DK107716), BCH Translational Research Program, Team Telomere, philanthropic gifts (Agudelo, Brizio and Martin families). The BCH Cellular Imaging Core is supported by a grant from the NIH (S10OD030322). This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Division of Hematology/Oncology, Stem Cell Program, and Manton Center for Orphan Disease Research, Boston Children’s Hospital, Boston, MA, USA

    Neha Nagpal & Suneet Agarwal

  2. Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Neha Nagpal & Suneet Agarwal

  3. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Neha Nagpal & Suneet Agarwal

  4. Harvard Initiative for RNA Medicine, Boston, MA, USA

    Neha Nagpal & Suneet Agarwal

  5. Harvard Stem Cell Institute, Cambridge, MA, USA

    Neha Nagpal & Suneet Agarwal

Authors
  1. Neha Nagpal
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  2. Suneet Agarwal
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Contributions

N.N. and S.A. conceived this study and designed the experiments. N.N. performed in vitro and cell-based studies; synthesis, engineering and delivery of TERC RNA; generation of induced pluripotent stem cells; and molecular, cellular and biochemical analyses. N.N. and S.A. wrote the paper.

Corresponding authors

Correspondence to Neha Nagpal or Suneet Agarwal.

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Competing interests

N.N. and S.A. are listed as co-inventors on patent applications 63/642,292 and 63/728,464 that include engineering of TERC RNA described in this paper.

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Nature Biomedical Engineering thanks Lin Liu 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 IVT, 5′ capped synthetic TERC RNA does not increase telomere length in TERC-null 293T cells by TRF analysis.

A, Top: Full length (451 nt) m7g-capped (Cap-0) synthetic IVT TERC RNA evaluated by agarose gel electrophoresis. n = 3 biological replicates, one representative shown. Bottom: Representation of IVT TERC with 5’Cap-0 (orange). B, TERC RNA by northern blot in TERC-null 293T cells compared to normal (WT) 293T cells, using 18S rRNA loading control. n = 3 biological replicates, one representative shown. C, Analysis of telomerase activity by TRAP assay in TERC-null 293T cells compared to normal (WT) 293T cells. n = 3 biological replicates, one representative shown. D, TRF analysis in TERC-null 293T cells compared to normal (WT) 293T cells. n = 3 biological replicates, one representative shown. E, TRF analysis in TERC-null 293T cells 96 h post-transfection of m7g-capped (Cap-0) synthetic TERC RNA and human TERT expressing plasmid vector. n = 3 biological replicates, one representative shown, compared to normal (WT) 293T cells. n = 3, one representative shown.

Extended Data Fig. 2 Recombinant TENT4B (rTENT4B) can add 2′OMeATP at the 3’end of RNA oligonucleotide to generate an exonuclease-resistant block.

A, Extension of a 20-mer RNA oligonucleotide (Oligo 1) using rTENT4B and ATP analogs demonstrating differential incorporation. (ADP: Adenosine-5’-diphosphate; ATP: Adenosine-5’-triphosphate; Azido-ATP: 3’-O-azidomethyl-ATP; AlphaS-ATP: Adenosine-5’-(α-thio)-triphosphate; GammaS-ATP: Adenosine-5’-(γ-thio)-triphosphate; ApCpp: Adenosine-5’-[(α,β)-methyleno]triphosphate; AppCp: Adenosine-5’-[(β,γ)-methyleno]triphosphate; AppNhp: Adenosine-5’-[(β,γ)-imido]triphosphate; 2’OMe-ATP: 2’-O-Methyladenosine-5’-triphosphate; Cordycepin-TP: Cordycepin-5’-triphosphate; dd-ATP: dideoxyadenosine-5’-triphosphate; LNA-ATP: Locked nucleic acid-adenosine-5’-triphosphate). n = 3 replicates, one representative shown. B, Upper: Structure of 2’-O-methyladenosine-5’-triphosphate. Bottom: Structure of Locked nucleic acid-adenosine-5’-triphosphate. C, 3’exonuclease activity detection via fluorescence-based assay using exonuclease T (ExoT) and recombinant PARN (PARN) for Oligo 3 alone or Oligo 3 treated with 2’OMeATP or LNA-ATP using rTENT4B. Fluorescently-labeled Oligo 3 is quenched by the addition of complementary anti-sense probe at the end of exonuclease reaction. Degradation of the Oligo 3 prevents hybridization and quenching by the anti-sense oligonucleotide, resulting in higher fluorescence. Degradation-resistant Oligo 3 will have a lower fluorescence. n = 3 replicates, mean ± S.D., p values calculated by 2way-ANOVA. n.s. not significant D, Self-limiting extension of a 20-mer RNA oligonucleotide (Oligo 1) using rTENT4B and 2’OMeATP. n = 3, one representative shown. E,F, RNA oligonucleotide degradation assay using rPARN (E) or exonuclease T (F) for Oligo 2 variants (Oligo 2, 2-mA, 2-mA2, 2-mA3) which have no (0), one, two or three 2’OMeA terminal residues at the 3’ ends, +/- extension with rTENT4B and 2’OMeATP. n = 3, one representative shown. G, RNA oligonucleotide degradation assay using exonuclease T (ExoT) and recombinant PARN (PARN) for Oligo 2 alone or Oligo 2 treated with 2’OMeATP using rTENT4B versus E. Coli poly(A)-polymerase (EPAP). n = 3 replicates, one representative shown.

Extended Data Fig. 3 rTENT4B-mediated but not EPAP-mediated treatment of eGFP mRNA using 2’OMeATP increases its expression when transfected in 293T cells.

A, Integrity of full length IVT eGFP mRNA by agarose gel electrophoresis, +/- treatment with rTENT4B or EPAP and 2’OMeATP. n = 3 replicates, one representative shown. B, Flow-cytometry of percent GFP positive 293T cells over the course of six days post-transfection with synthetic eGFP mRNA +/- treatment with rTENT4B and 2’OMeATP. n = 3, mean ± S.D., p values calculated by 2way-ANOVA. n.s. not significant C, Mean fluorescence intensity of GFP by flow-cytometry in 293T cells, 1-3 days post-transfection of synthetic eGFP-mRNA with or without treatment with EPAP and 2’OMeATP. n = 3, mean ± S.D., p values calculated by 2way-ANOVA. n.s. not significant D, Flow-cytometry of percent GFP positive 293T cells over the course of six days post-transfection with synthetic eGFP mRNA +/- treatment with EPAP and 2’OMeATP. n = 3, mean ± S.D., p values calculated by 2way-ANOVA. n.s. not significant.

Extended Data Fig. 4 2’OMeA-blocked m7g-capped TERC RNA increases telomerase activity but not telomere length in TERC-null 293T cells.

A, Top: Integrity of full length synthetic m7g-capped (Cap-0) TERC RNA +/- rTENT4B-mediated 3’-2’OMeA-block, by agarose gel electrophoresis. n = 3, one representative shown. Bottom: Representation of synthetic TERC RNAs with 5’Cap-0 (orange) and 5’Cap-0 (orange) plus rTENT4B-incorporated 3’-2’OMeA-block (red). B, Analysis of telomerase activity by TRAP assay, 72 h post-transfection, in TERC-null 293T cells transfected with synthetic m7g-capped (Cap-0), 3’-2’OMeA-blocked-TERC, compared to m7g-capped (Cap-0) TERC. n = 3 biological replicates, mean ± S.D., p values calculated by 2way-ANOVA. a.u., arbitrary units. C, TRF analysis 72 h post-transfection in TERC-null 293T cells transfected with synthetic m7g-capped (Cap-0)-TERC RNA + /- 3’-2’OMeA-block compared to normal (WT) 293T cells. n = 3 biological replicates, one representative shown. D, TRF analysis 72 h post-transfection in TERC-null 293T cells transfected with increasing concentrations of synthetic m7g-capped (Cap-0) TERC + /- 3’-2’OMeA-block and human TERT expressing plasmid vector, compared to normal (WT) 293T cells. n = 3 biological replicates, one representative shown.

Extended Data Fig. 5 eTERC RNA increases telomerase activity and telomere length in TERC-null 293T cells.

A, Top: Integrity of full length IVT synthetic tmg-capped-TERC RNA +/- rTENT4B-mediated 3’-2’OMeA-block by agarose gel electrophoresis. n = 3 replicates, one representative shown. Bottom: Representations of synthetic engineered TERC RNAs with 5’-tmg-cap (green) or 5’-tmg-cap (green) plus rTENT4B-incorporated 3’-2’OMeA-block (red). B, TRF analysis 72 h post-transfection in TERC-null 293T cells transfected with 4.5 µg of synthetic tmg-capped-TERC + /- 3’-2’OMeA-block compared to normal (WT) 293T cells. n = 3 biological replicates, one representative shown. C, TRF analysis 72 h post-transfection TERC-null 293T cells transfected with 4.5 µg of synthetic tmg-capped-TERC + /- 3’-2’OMeA-block and human TERT expressing plasmid vector, compared to normal (WT) 293T cells. n = 3 biological replicates shown. D, TRF analysis followed by quantitation of mean telomere length 72 h post-transfection in TERC-null 293T cells transfected with 4.5 µg of synthetic tmg-capped TERC + /- 3’-2’OMeA-block and synthetic TERT mRNA. n = 3, p values calculated by two-tailed unpaired t test. E, Analysis of telomerase activity by TRAP assay 72 h post-transfection in TERC-null 293T cells transfected with synthetic tmg-capped, 3’-2’OMeA-blocked TERC, compared to tmg-capped TERC. n = 3 biological replicates shown. F, Telomerase activity as in (E) quantified. n = 3, mean ± S.D., p values calculated by 2way-ANOVA. a.u., arbitrary units.

Extended Data Fig. 6 rTENT4B-mediated incorporation of 2’OMeA-block to the 3’ end of tmg-capped TERC RNA increases its stability and rescues telomere length and replicative capacity in TERC-null 293T cells.

A, TERC RNA levels by northern blot in TERC-null 293T cells, 24-96 h post-transfection of synthetic tmg-capped TERC + /- 3’-2’OMeA-block, compared to untransfected and WT 293T cells. 18S rRNA loading control. n = 2 biological replicates, one representative shown. B,C same blot as in (A) with increased autoradiographic exposures. D, Top: Agarose gel electrophoresis of full length eTERC RNA +/- nucleobase modifications (substituting ATP with N6-methyladenosine-5’-triphosphate (A), UTP with N1-methylpseudouridine-5’-triphosphate (U), or CTP with 5-methylcytidine-5’-triphosphate (C), or all three substitutions (ACU)). Bottom: Structures of modified nucleosides. n = 3 different modified nucleobases, one biological replicate as shown. E, TRF telomere length analysis in late passage, near-senescent TERC-null 293T cells 3 days after transfection with eTERC plus synthetic TERT mRNA, with synthetic eGFP mRNA as a control, and compared to WT and unmanipulated TERC-null 293T cells. n = 2 biological replicates, one representative shown. F, Morphology of TERC-null cells 293T 23 days post-RNA transfection as in (E), compared to WT 293T cells. Phase contrast; 100X magnification, scale bar 100 µm.

Extended Data Fig. 7 rTENT4B-mediated incorporation of 2’OMeA-block to the 3’ end of tmg-capped TERC RNA increases its stability and telomere length without triggering cellular inflammatory responses in TBD patient iPSCs.

A, TRF analysis 72 h post-transfection of PARN-mutant patient iPSCs transfected with synthetic tmg-capped-TERC + /- 3’-2’OMeA-block and human TERT expressing plasmid vector, compared to normal (WT) iPSCs. n = 3 biological replicates shown. B, TRF analysis 72 h post-electroporation. PARN-mutant patient iPSCs were transfected with increasing concentrations of eTERC RNA, and human TERT expressing plasmid vector, and compared to normal (WT) iPSCs. n = 3 biological replicates, one representative shown. C, Comparison of mean telomere length in PARN-mutant patient iPSCs transfected versus those electroporated with synthetic tmg-capped-TERC + /- 3’-2’OMeA-block and human TERT expressing plasmid vector for 72 h. n = 2 biological replicates shown. D, TRF analysis in PARN-mutant patient iPSCs 8 days post-electroporation of synthetic TERC RNA with 5’-tmg-cap +/- 3’-2’OMeA-block, and human TERT expressing plasmid vector, and compared to unmanipulated patient iPSCs and normal (WT) iPSCs. n = 3 biological replicates, one representative shown. E, Top: Integrity of full length, IVT tmg-capped TERC RNA, +/- rTENT4B-mediated 3’-2’OMeA-block, by agarose gel electrophoresis. Bottom: Representations of synthetic engineered TERC RNAs. F, Integrity of RNAs as in (E) by northern blot. G, TERC RNA by northern blot in PARN-mutant patient iPSCs, 24, 48 and 72 h post-electroporation of synthetic tmg-capped TERC + /- 3’-2’OMeA-block, compared to patient and normal (WT) iPSCs. 18S rRNA as loading control. n = 2 biological replicates, one representative shown. See also same blot with lighter exposure in Fig. 3b. H, Quantification of TERC RNA levels as in (G). Normalized to 18S rRNA levels, and to the normalized value of unmanipulated patient iPSCs. n = 2 biological replicates shown. I-K, IFNB1 (I), ISG15 (J) and RANTES (K) mRNA levels by quantitative RT-PCR in PARN-mutant patient iPSCs, 24 h post-electroporation of 4 µg of poly(I):(C) or eTERC RNA. Normalized to 18S rRNA levels, and relative to the normalized value of untreated patient iPSCs. n = 3 biological replicates shown, mean ± S.D., p values calculated by one-way ANOVA. n.s. not significant.

Extended Data Fig. 8 eTERC RNA stability and function is independent of the TENT4B pathway, and does not affect apoptosis in patient iPSCs.

A, TERC RNA levels by northern blot in PARN-mutant patient iPSCs, 24-72 h post-electroporation of eTERC RNA ± TENT4B inhibitor (RG7834; 1 µM), compared to unmanipulated patient and normal (WT) iPSCs. 18S rRNA loading control. n = 2 biological replicates, one representative shown. B,C same blot as in (A) with increased autoradiographic exposures. D, Quantification of intact TERC RNA levels at 72 h, normalized to 18S rRNA levels, and relative to the normalized value of unmanipulated patient iPSCs. n = 2 biological replicates shown. E, TRF analysis of PARN-mutant patient iPSCs, 72 h post-electroporation of eTERC RNA ± RG7834 (1 µM), and compared to unmanipulated patient iPSCs and normal (WT) iPSCs. n = 2 biological replicates, one representative shown. F, TRF analysis in PARN-mutant patient iPSCs 28 days post-treatment with RG7834 compared to vehicle (DMSO). n = 3 biological replicates, one representative shown. G, Cell viability by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 24 h after etoposide (0.1 µM) treatment of PARN-mutant patient iPSCs, 72 h post-electroporation of eTERC RNA. n = 2 biological replicates with 3 technical replicates each, mean ± S.D., p values calculated by one-way ANOVA. n.s. not significant. H, Flow cytometry-based apoptosis analysis using Annexin V and propidium iodide (PI) staining 24 h after etoposide (0.1 µM) treatment of PARN-mutant patient iPSCs, 72 h post-electroporation of eTERC RNA. n = 3 biological replicates shown, mean ± S.D., p values calculated by 2way-ANOVA.

Extended Data Fig. 9 eTERC RNA increases telomere length in iPSCs derived from patients with various genetic forms of TBDs.

TRF analyses in iPSCs from patients with compound heterozygous mutations in RTEL1 (p.E615D/p.R998X), heterozygous mutation in TERT (p.A716V) and heterozygous mutations in TINF2 (p.R282H) genes after electroporation with synthetic tmg-capped TERC + /- 3’-2’OMeA-block, compared to normal (WT) iPSCs. n = 3 biological replicates for each genotype, one representative of each shown.

Extended Data Fig. 10 eTERC RNA boosts telomerase activity and hematopoietic output in PARN-deficient primary human HSPCs.

A, Telomerase activity by TRAP assay in primary human CD34+ HSPCs 72 h after CRISPR/Cas9-mediated disruption of the PARN gene (93% indels), followed by eTERC or eGFP mRNA electroporation or RG7834 treatment for 48 h, compared to unmanipulated HSPCs (WT). n = 2 biological replicates, one representative is shown. B, Quantification of TRAP assay data at lysate concentration 0.5 µg as in (A), n = 2 biological replicates as shown. a.u. arbitrary units.

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Nagpal, N., Agarwal, S. Extension of replicative lifespan by synthetic engineered telomerase RNA in patient induced pluripotent stem cells. Nat. Biomed. Eng 9, 2083–2097 (2025). https://doi.org/10.1038/s41551-025-01429-1

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