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Gene therapy
Mar, 2025;32 (2): 163-171.

A peptide conjugate enables systemic injection of the morpholino inducer and more durable induction of T3H38 ribozyme-controlled AAV transgene in mice.

Xiaojuan Tang, Haimin Wang, Yandong Yin, Guocai Zhong
Author Information
  1. Xiaojuan Tang: State Key Laboratory of Chemical Oncogenomics, Shenzhen Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, Guangdong, China. ORCID
  2. Haimin Wang: Department of Genetic & Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. ORCID
  3. Yandong Yin: State Key Laboratory of Chemical Oncogenomics, Shenzhen Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, Guangdong, China. yinyd@szbl.ac.cn. ORCID
  4. Guocai Zhong: Department of Genetic & Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. guocai.zhong@umassmed.edu. ORCID
PMID: 39939797 DOI: 10.1038/s41434-025-00519-8

Abstract

Genetic switches that allow for precise control over transgene expression timing or levels may improve the safety and expand the use of adeno-associated viral (AAV) vector-based gene therapy technologies. We previously engineered an efficient RNA switch system that comprises a novel self-cleaving ribozyme (T3H38) and an octaguanidine dendrimer-conjugated morpholino oligonucleotide (v-M8) complementary to the ribozyme. This switch system can be used to efficiently regulate AAV-delivered transgenes with an up to 200-fold regulatory range in mice. However, this switch system has a relatively short induction half-life and only works well when v-M8 was locally but not systemically administered, representing two key limitations of the system. To address these issues, here, we tested replacing the octa-guanidine dendrimer in the v-M8 morpholino oligo with a cell-penetrating peptide (CPP). Two CPP-conjugated morpholino oligos (B-M8 and B-MSP-M8) were synthesized and compared with v-M8 for the induction of T3H38-regulated AAV-luciferase in mice. One of the CPP-conjugated oligos (B-MSP-M8) not only showed significantly improved induction half-life over that of v-M8, but also enabled efficient induction of AAV transgene expression when the oligo was systemically administered. This study improves in vivo performance and broadens the utility of the T3H38 ribozyme-based RNA switch system in gene therapy applications.

References

  1. Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;359:eaan4672. [PMID: 29326244]
  2. Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18:358–78. [PMID: 30710128]
  3. Samulski RJ, Muzyczka N. AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol. 2014;1:427–51. [PMID: 26958729]
  4. Yla-Herttuala S. Endgame: glybera finally recommended for approval as the first gene therapy drug in the European Union. Mol Ther. 2012;20:1831–2. [PMID: 23023051]
  5. Russell S, Bennett J, Wellman JA, Chung DC, Yu ZF, Tillman A, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390:849–60. [PMID: 28712537]
  6. Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N. Engl J Med. 2017;377:1713–22. [PMID: 29091557]
  7. George LA, Sullivan SK, Giermasz A, Rasko JEJ, Samelson-Jones BJ, Ducore J, et al. Hemophilia B gene therapy with a high-specific-activity Factor IX variant. N. Engl J Med. 2017;377:2215–27. [PMID: 29211678]
  8. Rangarajan S, Walsh L, Lester W, Perry D, Madan B, Laffan M, et al. AAV5-Factor VIII gene transfer in severe Hemophilia A. N. Engl J Med. 2017;377:2519–30. [PMID: 29224506]
  9. Keam SJ. Eladocagene Exuparvovec: First approval. Drugs. 2022;82:1427–32. [PMID: 36103022]
  10. Hoy SM. Delandistrogene Moxeparvovec: First approval. Drugs. 2023;83:1323–9. [PMID: 37566211]
  11. Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol. 1996;70:8098–108. [PMID: 8892935]
  12. Zhou S, Murphy JE, Escobedo JA, Dwarki VJ. Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates. Gene Ther. 1998;5:665–70. [PMID: 9797871]
  13. Rivera VM, Ye X, Courage NL, Sachar J, Cerasoli F Jr., Wilson JM, et al. Long-term regulated expression of growth hormone in mice after intramuscular gene transfer. Proc Natl Acad Sci USA. 1999;96:8657–62. [PMID: 10411931]
  14. Ye X, Rivera VM, Zoltick P, Cerasoli F Jr., Schnell MA, Gao G, et al. Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer. Science. 1999;283:88–91. [PMID: 9872748]
  15. Rivera VM, Gao GP, Grant RL, Schnell MA, Zoltick PW, Rozamus LW, et al. Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer. Blood. 2005;105:1424–30. [PMID: 15507527]
  16. Bohl D, Salvetti A, Moullier P, Heard JM. Control of erythropoietin delivery by doxycycline in mice after intramuscular injection of adeno-associated vector. Blood. 1998;92:1512–7. [PMID: 9716577]
  17. Chenuaud P, Larcher T, Rabinowitz JE, Provost N, Cherel Y, Casadevall N, et al. Autoimmune anemia in macaques following erythropoietin gene therapy. Blood. 2004;103:3303–4. [PMID: 14739218]
  18. Zhong G, Wang H, Bailey CC, Gao G, Farzan M. Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells. Elife. 2016;5:e18858. [PMID: 27805569]
  19. Luo L, Jea JD, Wang Y, Chao PW, Yen L. Control of mammalian gene expression by modulation of polyA signal cleavage at 5’ UTR. Nat Biotechnol. 2024;42:1454–66.
  20. Monteys AM, Hundley AA, Ranum PT, Tecedor L, Muehlmatt A, Lim E, et al. Regulated control of gene therapies by drug-induced splicing. Nature. 2021;596:291–5. [PMID: 34321659]
  21. Zhong G, Wang H, He W, Li Y, Mou H, Tickner ZJ, et al. A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo. Nat Biotechnol. 2020;38:169–75. [PMID: 31873216]
  22. Fukunaga K, Dhamodharan V, Miyahira N, Nomura Y, Mustafina K, Oosumi Y, et al. Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals. J Am Chem Soc. 2023;145:7820–8.
  23. Theil D, Kuhle J, Brees D, Tritto E, Pognan F, Frieauff W, et al. Neurofilament light chain: a translational safety biomarker for drug-induced peripheral neurotoxicity. Toxicol Pathol. 2023;51:135–47. [PMID: 37439009]
  24. Hastie E, Samulski RJ. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success–a personal perspective. Hum Gene Ther. 2015;26:257–65. [PMID: 25807962]
  25. Favre D, Blouin V, Provost N, Spisek R, Porrot F, Bohl D, et al. Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus. J Virol. 2002;76:11605–11. [PMID: 12388721]
  26. Wang PR, Xu M, Toffanin S, Li Y, Llovet JM, Russell DW. Induction of hepatocellular carcinoma by in vivo gene targeting. Proc Natl Acad Sci USA. 2012;109:11264–9. [PMID: 22733778]
  27. David RM, Doherty AT. Viral vectors: the road to reducing genotoxicity. Toxicol Sci. 2017;155:315–25. [PMID: 27803388]
  28. Wilton-Clark H, Yokota T. Recent trends in Antisense therapies for Duchenne Muscular Dystrophy. Pharmaceutics. 2023;15:778. [PMID: 36986639]
  29. Woo S, Jusko WJ. Interspecies comparisons of pharmacokinetics and pharmacodynamics of recombinant human erythropoietin. Drug Metab Dispos. 2007;35:1672–8. [PMID: 17576810]
  30. Blum S, Shapir N, Miari R, Lerner B, Koren B, Doenyas-Barak K, et al. TARGT gene therapy platform for correction of anemia in end-stage renal disease. N. Engl J Med. 2017;376:189–91. [PMID: 28076704]
  31. Bunn HF. Erythropoietin. Cold Spring Harb Perspect Med. 2013;3:a011619. [PMID: 23457296]
  32. Fang J, Wang J, Wang Y, Liu X, Chen B, Zou W. Ribo-On and Ribo-Off tools using a self-cleaving ribozyme allow manipulation of endogenous gene expression in C. elegans. Commun Biol. 2023;6:816. [PMID: 37542105]
  33. Hughes AC, Pittman BG, Xu B, Gammons JW, Webb CM, Nolen HG, et al. A single-vector intersectional AAV strategy for interrogating cellular diversity and brain function. Nat Neurosci. 2024;27:1400–10.
  34. Ronzitti G. Let’s switch on AAV! Sci Transl Med. 2020;12:eaba9016.
  35. Morcos PA, Li Y, Jiang S. Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. Biotechniques. 2008;45:613–4. [PMID: 19238792]
  36. Moulton HM. In vivo delivery of morpholino oligos by cell-penetrating peptides. Curr Pharm Des. 2013;19:2963–9. [PMID: 23140456]
  37. Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020;19:673–94. [PMID: 32782413]
  38. Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE, Nelson MH, Iversen PL, et al. Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res. 2007;35:5182–91. [PMID: 17670797]
  39. Moulton HM, Moulton JD. Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy. Biochim Biophys Acta. 2010;1798:2296–303. [PMID: 20170628]
  40. Yin H, Moulton HM, Betts C, Seow Y, Boutilier J, Iverson PL, et al. A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice. Hum Mol Genet. 2009;18:4405–14. [PMID: 19692354]
  41. Yin H, Moulton HM, Betts C, Merritt T, Seow Y, Ashraf S, et al. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO. Mol Ther. 2010;18:1822–9. [PMID: 20700113]
  42. Gernoux G, Gruntman AM, Blackwood M, Zieger M, Flotte TR, Mueller C. Muscle-directed delivery of an AAV1 vector leads to Capsid-Specific T cell exhaustion in nonhuman primates and humans. Mol Ther. 2020;28:747–57. [PMID: 31982038]
  43. Mueller C, Gernoux G, Gruntman AM, Borel F, Reeves EP, Calcedo R, et al. 5 year expression and neutrophil defect repair after gene therapy in Alpha-1 Antitrypsin deficiency. Mol Ther. 2017;25:1387–94. [PMID: 28408179]
  44. Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M, et al. Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol. 2005;23:584–90. [PMID: 15834403]
  45. Deckers J, Anbergen T, Hokke AM, de Dreu A, Schrijver DP, de Bruin K, et al. Engineering cytokine therapeutics. Nat Rev Bioeng. 2023;1:286–303. [PMID: 37064653]
  46. Rosenstock J, Juneja R, Beals JM, Moyers JS, Ilag L, McCrimmon RJ. The basis for weekly insulin therapy: evolving evidence with Insulin Icodec and Insulin Efsitora Alfa. Endocr Rev. 2024;45:379–413. [PMID: 38224978]
  47. Bhoopalan SV, Huang LJ, Weiss MJ. Erythropoietin regulation of red blood cell production: from bench to bedside and back. F1000Res. 2020;9:1153.
  48. Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA. 2000;97:13003–8. [PMID: 11087855]
  49. Samoylova TI, Smith BF. Elucidation of muscle-binding peptides by phage display screening. Muscle Nerve. 1999;22:460–6. [PMID: 10204780]
  50. Yu CY, Yuan Z, Cao Z, Wang B, Qiao C, Li J, et al. A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery. Gene Ther. 2009;16:953–62. [PMID: 19474807]
  51. Yin H, Boisguerin P, Moulton HM, Betts C, Seow Y, Boutilier J, et al. Context dependent effects of chimeric peptide morpholino conjugates contribute to Dystrophin Exon-skipping Efficiency. Mol Ther Nucleic Acids. 2013;2:e124. [PMID: 24064708]
  52. Zhang H, Zhang Y, Zhang C, Yu H, Ma Y, Li Z, et al. Recent advances of cell-penetrating peptides and their application as vectors for delivery of peptide and protein-based cargo molecules. Pharmaceutics. 2023;15:2093. [PMID: 37631307]
  53. Mullard A. Antibody-oligonucleotide conjugates enter the clinic. Nat Rev Drug Discov. 2022;21:6–8. [PMID: 34903879]
  54. Smith CIE, Zain R. Therapeutic Oligonucleotides: state of the art. Annu Rev Pharmacol Toxicol. 2019;59:605–30. [PMID: 30285540]

MeSH Term

Animals
RNA, Catalytic
Mice
Morpholinos
Dependovirus
Transgenes
Genetic Vectors
Dendrimers
Cell-Penetrating Peptides
Genetic Therapy
Humans

Chemicals

RNA, Catalytic
Morpholinos
Dendrimers
Cell-Penetrating Peptides
Journal Article

Word Cloud

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