Cellular and Molecular Life Sciences

, Volume 69, Issue 24, pp 4191–4204 | Cite as

Replacement of huntingtin exon 1 by trans-splicing

  • Hansjörg Rindt
  • Pei-Fen Yen
  • Christina N. Thebeau
  • Troy S. Peterson
  • Gary A. Weisman
  • Christian L. Lorson
Research Article

Abstract

Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a “gain-of-function” mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.

Keywords

Neurodegeneration Huntington’s disease RNA-based therapeutics Spliceosome-mediated trans-splicing 

Supplementary material

18_2012_1083_MOESM1_ESM.pptx (96 kb)
Supplementary Fig. 1 Sequence of the trans-splicing product. The F2-R4 product shown in Fig. 3 was cloned and sequenced to confirm correct splicing. Exon junctions are indicated by the thick vertical bars. (PPTX 95 kb)

References

  1. 1.
    Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP Jr (1985) Neuropathological classification of Huntington’s disease. J Neuropathol Exp Neurol 44:559–577PubMedCrossRefGoogle Scholar
  2. 2.
    Nasir J, Floresco SB, O’Kusky JR, Diewert VM, Richman JM, Zeisler J, Borowski A, Marth JD, Phillips AG, Hayden MR (1995) Targeted disruption of the Huntington’s disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81:811–823PubMedCrossRefGoogle Scholar
  3. 3.
    Zeitlin S, Liu JP, Chapman DL, Papaioannou VE, Efstratiadis A (1995) Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington’s disease gene homologue. Nat Genet 11:155–163PubMedCrossRefGoogle Scholar
  4. 4.
    Duyao MP, Auerbach AB, Ryan A, Persichetti F, Barnes GT, McNeil SM, Ge P, Vonsattel JP, Gusella JF, Joyner AL et al (1995) Inactivation of the mouse Huntington’s disease gene homolog Hdh. Science 269:407–410PubMedCrossRefGoogle Scholar
  5. 5.
    MacDonald ME (2003) Huntingtin: alive and well and working in middle management. Sci STKE 207:pe48Google Scholar
  6. 6.
    Li SH, Li XJ (2004) Huntingtin-protein interactions and the pathogenesis of Huntington’s disease. Trends Genet 20:146–154PubMedCrossRefGoogle Scholar
  7. 7.
    Ferrante RJ, Gutekunst CA, Persichetti F, McNeil SM, Kowall NW, Gusella JF, MacDonald ME, Beal MF, Hersch SM (1997) Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum. J Neurosci 17:3052–3063PubMedGoogle Scholar
  8. 8.
    Fusco FR, Chen Q, Lamoreaux WJ, Figueredo-Cardenas G, Jiao Y, Coffman JA, Surmeier DJ, Honig MG, Carlock LR, Reiner A (1999) Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington’s disease. J Neurosci 19:1189–1202PubMedGoogle Scholar
  9. 9.
    DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, Young C, Martin E, Vonsattel JP, Carraway R, Reeves SA et al (1995) Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron 14:1075–1081PubMedCrossRefGoogle Scholar
  10. 10.
    Velier J, Kim M, Schwarz C, Kim TW, Sapp E, Chase K, Aronin N, DiFiglia M (1998) Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways. Exp Neurol 152:34–40PubMedCrossRefGoogle Scholar
  11. 11.
    Kegel KB, Meloni AR, Yi Y, Kim YJ, Doyle E, Cuiffo BG, Sapp E, Wang Y, Qin ZH, Chen JD, Nevins JR, Aronin N, DiFiglia M (2002) Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription. J Biol Chem 277:7466–7476PubMedCrossRefGoogle Scholar
  12. 12.
    Wexler NS, Lorimer J, Porter J, Gomez F, Moskowitz C, Shackell E, Marder K, Penchaszadeh G, Roberts SA, Gayan J, Brocklebank D, Cherny SS, Cardon LR, Gray J, Dlouhy SR, Wiktorski S, Hodes ME, Conneally PM, Penney JB, Gusella J, Cha JH, Irizarry M, Rosas D, Hersch S, Hollingsworth Z, MacDonald M, Young AB, Andresen JM, Housman DE, De Young MM, Bonilla E, Stillings T, Negrette A, Snodgrass SR, Martinez-Jaurrieta MD, Ramos-Arroyo MA, Bickham J, Ramos JS, Marshall F, Shoulson I, Rey GJ, Feigin A, Arnheim N, Acevedo-Cruz A, Acosta L, Alvir J, Fischbeck K, Thompson LM, Young A, Dure L, O’Brien CJ, Paulsen J, Brickman A, Krch D, Peery S, Hogarth P, Higgins DS Jr, Landwehrmeyer B (2004) Venezuelan kindreds reveal that genetic and environmental factors modulate Huntington’s disease age of onset. Proc Natl Acad Sci USA 101:3498–3503PubMedCrossRefGoogle Scholar
  13. 13.
    Walker FO (2007) Huntington’s disease. Lancet 369:218–228PubMedCrossRefGoogle Scholar
  14. 14.
    Cha JH (2000) Transcriptional dysregulation in Huntington’s disease. Trends Neurosci 23:387–392PubMedCrossRefGoogle Scholar
  15. 15.
    Bates G, Benn C (2002) The polyglutamine diseases. In: Bates G, Harper P and Jones L (eds) Huntington’s disease, Oxford University Press, London, p 429–474Google Scholar
  16. 16.
    Hickey MA, Chesselet MF (2003) Apoptosis in Huntington’s disease. Prog Neuropsychopharmacol Biol Psychiatry 27:255–265PubMedCrossRefGoogle Scholar
  17. 17.
    DiProspero NA, Chen EY, Charles V, Plomann M, Kordower JH, Tagle DA (2004) Early changes in Huntington’s disease patient brains involve alterations in cytoskeletal and synaptic elements. J Neurocytol 33:517–533PubMedCrossRefGoogle Scholar
  18. 18.
    Leoni V, Mariotti C, Tabrizi SJ, Valenza M, Wild EJ, Henley SM, Hobbs NZ, Mandelli ML, Grisoli M, Bjorkhem I, Cattaneo E, Di Donato S (2008) Plasma 24S-hydroxycholesterol and caudate MRI in pre-manifest and early Huntington’s disease. Brain 131:2851–2859PubMedCrossRefGoogle Scholar
  19. 19.
    Duennwald ML, Lindquist S (2008) Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity. Genes Dev 22:3308–3319PubMedCrossRefGoogle Scholar
  20. 20.
    Dragatsis I, Levine MS, Zeitlin S (2000) Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice. Nat Genet 26:300–306PubMedCrossRefGoogle Scholar
  21. 21.
    Zuccato C, Ciammola A, Rigamonti D, Leavitt BR, Goffredo D, Conti L, MacDonald ME, Friedlander RM, Silani V, Hayden MR, Timmusk T, Sipione S, Cattaneo E (2001) Loss of huntingtin-mediated BDNF gene transcription in Huntington’s disease. Science 293:493–498PubMedCrossRefGoogle Scholar
  22. 22.
    Puttaraju M, Jamison SF, Mansfield SG, Garcia-Blanco MA, Mitchell LG (1999) Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat Biotechnol 17:246–252PubMedCrossRefGoogle Scholar
  23. 23.
    Mansfield SG, Kole J, Puttaraju M, Yang CC, Garcia-Blanco MA, Cohn JA, Mitchell LG (2000) Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing. Gene Ther 7:1885–1895PubMedCrossRefGoogle Scholar
  24. 24.
    Kikumori T, Cote GJ, Gagel RF (2001) Promiscuity of pre-mRNA spliceosome-mediated trans splicing: a problem for gene therapy? Hum Gene Ther 12:1429–1441PubMedCrossRefGoogle Scholar
  25. 25.
    Puttaraju M, DiPasquale J, Baker CC, Mitchell LG, Garcia-Blanco MA (2001) Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing. Mol Ther 4:105–114PubMedCrossRefGoogle Scholar
  26. 26.
    Labrador M, Corces VG (2003) Extensive exon reshuffling over evolutionary time coupled to trans-splicing in Drosophila. Genome Res 13:2220–2228PubMedCrossRefGoogle Scholar
  27. 27.
    Flouriot G, Brand H, Seraphin B, Gannon F (2002) Natural trans-spliced mRNAs are generated from the human estrogen receptor-alpha (hER alpha) gene. J Biol Chem 277:26244–26251PubMedCrossRefGoogle Scholar
  28. 28.
    Dorn R, Krauss V (2003) The modifier of mdg4 locus in Drosophila: functional complexity is resolved by trans splicing. Genetica 117:165–177PubMedCrossRefGoogle Scholar
  29. 29.
    Finta C, Zaphiropoulos PG (2002) Intergenic mRNA molecules resulting from trans-splicing. J Biol Chem 277:5882–5890PubMedCrossRefGoogle Scholar
  30. 30.
    Caudevilla C, Serra D, Miliar A, Codony C, Asins G, Bach M, Hegardt FG (1998) Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver. Proc Natl Acad Sci USA 95:12185–12190PubMedCrossRefGoogle Scholar
  31. 31.
    Bruzik JP, Maniatis T (1992) Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells. Nature 360:692–695PubMedCrossRefGoogle Scholar
  32. 32.
    Li H, Wang J, Mor G, Sklar J (2008) A neoplastic gene fusion mimics trans-splicing of RNAs in normal human cells. Science 321:1357–1361PubMedCrossRefGoogle Scholar
  33. 33.
    Rickman DS, Pflueger D, Moss B, VanDoren VE, Chen CX, de la Taille A, Kuefer R, Tewari AK, Setlur SR, Demichelis F, Rubin MA (2009) SLC45A3-ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer. Cancer Res 69:2734–2738PubMedCrossRefGoogle Scholar
  34. 34.
    Coady TH, Baughan TD, Shababi M, Passini MA, Lorson CL (2008) Development of a single vector system that enhances trans-splicing of SMN2 transcripts. PLoS One 3:e3468PubMedCrossRefGoogle Scholar
  35. 35.
    Gropp M, Itsykson P, Singer O, Ben-Hur T, Reinhartz E, Galun E, Reubinoff BE (2003) Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther 7:281–287PubMedCrossRefGoogle Scholar
  36. 36.
    DiFiglia M, Sena-Esteves M, Chase K, Sapp E, Pfister E, Sass M, Yoder J, Reeves P, Pandey RK, Rajeev KG, Manoharan M, Sah DW, Zamore PD, Aronin N (2007) Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc Natl Acad Sci USA 104:17204–17209PubMedCrossRefGoogle Scholar
  37. 37.
    McBride JL, Boudreau RL, Harper SQ, Staber PD, Monteys AM, Martins I, Gilmore BL, Burstein H, Peluso RW, Polisky B, Carter BJ, Davidson BL (2008) Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci USA 105:5868–5873PubMedCrossRefGoogle Scholar
  38. 38.
    Drouet V, Perrin V, Hassig R, Dufour N, Auregan G, Alves S, Bonvento G, Brouillet E, Luthi-Carter R, Hantraye P, Deglon N (2009) Sustained effects of nonallele-specific Huntingtin silencing. Ann Neurol 65:276–285PubMedCrossRefGoogle Scholar
  39. 39.
    Mochizuki H, Yasuda T, Mouradian MM (2008) Advances in gene therapy for movement disorders. Neurotherapeutics 5:260–269PubMedCrossRefGoogle Scholar
  40. 40.
    Danos O (2008) AAV vectors for RNA-based modulation of gene expression. Gene Ther 15:864–869PubMedCrossRefGoogle Scholar
  41. 41.
    Harper SQ (2009) Progress and challenges in RNA interference therapy for Huntington disease. Arch Neurol 66:933–938PubMedCrossRefGoogle Scholar
  42. 42.
    Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, Deng Y, Oh R, Bissada N, Hossain SM, Yang YZ, Li XJ, Simpson EM, Gutekunst CA, Leavitt BR, Hayden MR (2003) Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Hum Mol Genet 12:1555–1567PubMedCrossRefGoogle Scholar
  43. 43.
    Wood M, Yin H, McClorey G (2007) Modulating the expression of disease genes with RNA-based therapy. PLoS Genet 3:e109PubMedCrossRefGoogle Scholar
  44. 44.
    Boado RJ, Kazantsev A, Apostol BL, Thompson LM, Pardridge WM (2000) Antisense-mediated down-regulation of the human huntingtin gene. J Pharmacol Exp Ther 295:239–243PubMedGoogle Scholar
  45. 45.
    Nellemann C, Abell K, Norremolle A, Lokkegaard T, Naver B, Ropke C, Rygaard J, Sorensen SA, Hasholt L (2000) Inhibition of Huntington synthesis by antisense oligodeoxynucleotides. Mol Cell Neurosci 16:313–323PubMedCrossRefGoogle Scholar
  46. 46.
    Hu J, Matsui M, Gagnon KT, Schwartz JC, Gabillet S, Arar K, Wu J, Bezprozvanny I, Corey DR (2009) Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs. Nat Biotechnol 27:478–484PubMedCrossRefGoogle Scholar
  47. 47.
    Boudreau RL, McBride JL, Martins I, Shen S, Xing Y, Carter BJ, Davidson BL (2009) Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington’s disease mice. Mol Ther 17:1053–1063PubMedCrossRefGoogle Scholar
  48. 48.
    White JK, Auerbach W, Duyao MP, Vonsattel JP, Gusella JF, Joyner AL, MacDonald ME (1997) Huntingtin is required for neurogenesis and is not impaired by the Huntington’s disease CAG expansion. Nat Genet 17:404–410PubMedCrossRefGoogle Scholar
  49. 49.
    Dietrich P, Shanmugasundaram R, Shuyu E, Dragatsis I (2009) Congenital hydrocephalus associated with abnormal subcommissural organ in mice lacking huntingtin in Wnt1 cell lineages. Hum Mol Genet 18:142–150PubMedCrossRefGoogle Scholar
  50. 50.
    van Bilsen PH, Jaspers L, Lombardi MS, Odekerken JC, Burright EN, Kaemmerer WF (2008) Identification and allele-specific silencing of the mutant huntingtin allele in Huntington’s disease patient-derived fibroblasts. Hum Gene Ther 19:710–719PubMedCrossRefGoogle Scholar
  51. 51.
    Schwarz DS, Ding H, Kennington L, Moore JT, Schelter J, Burchard J, Linsley PS, Aronin N, Xu Z, Zamore PD (2006) Designing siRNA that distinguish between genes that differ by a single nucleotide. PLoS Genet 2:e140PubMedCrossRefGoogle Scholar
  52. 52.
    Zhang Y, Engelman J, Friedlander RM (2009) Allele-specific silencing of mutant Huntington’s disease gene. J Neurochem 108:82–90PubMedCrossRefGoogle Scholar
  53. 53.
    Pfister EL, Kennington L, Straubhaar J, Wagh S, Liu W, DiFiglia M, Landwehrmeyer B, Vonsattel JP, Zamore PD, Aronin N (2009) Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr Biol 19:774–778PubMedCrossRefGoogle Scholar
  54. 54.
    Warby SC, Doty CN, Graham RK, Shively J, Singaraja RR, Hayden MR (2009) Phosphorylation of huntingtin reduces the accumulation of its nuclear fragments. Mol Cell Neurosci 40:121–127PubMedCrossRefGoogle Scholar
  55. 55.
    Carroll JB, Warby SC, Southwell AL, Doty CN, Greenlee S, Skotte N, Hung G, Bennett CF, Freier SM, Hayden MR (2011) Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/allele-specific silencing of mutant huntingtin. Mol Ther 19:2178–2185PubMedCrossRefGoogle Scholar
  56. 56.
    McBride JL, Pitzer MR, Boudreau RL, Dufour B, Hobbs T, Ojeda SR, Davidson BL (2011) Preclinical safety of RNAi-mediated HTT suppression in the rhesus macaque as a potential therapy for Huntington’s disease. Mol Ther 19:2152–2162PubMedCrossRefGoogle Scholar
  57. 57.
    Lee SJ, Lim HS, Masliah E, Lee HJ (2011) Protein aggregate spreading in neurodegenerative diseases: problems and perspectives. Neurosci Res 70:339–348PubMedCrossRefGoogle Scholar
  58. 58.
    Jucker M, Walker LC (2011) Pathogenic protein seeding in Alzheimer disease and other neurodegenerative disorders. Ann Neurol 70:532–540PubMedCrossRefGoogle Scholar
  59. 59.
    Ren PH, Lauckner JE, Kachirskaia I, Heuser JE, Melki R, Kopito RR (2009) Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. Nat Cell Biol 11:219–225PubMedCrossRefGoogle Scholar
  60. 60.
    Mitchell LG, McGarrity GJ (2005) Gene therapy progress and prospects: reprogramming gene expression by trans-splicing. Gene Ther 12:1477–1485PubMedCrossRefGoogle Scholar
  61. 61.
    Sassone J, Colciago C, Cislaghi G, Silani V, Ciammola A (2009) Huntington’s disease: the current state of research with peripheral tissues. Exp Neurol 219:385–397PubMedCrossRefGoogle Scholar
  62. 62.
    Morris DP, Greenleaf AL (2000) The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II. J Biol Chem 275:39935–39943PubMedCrossRefGoogle Scholar
  63. 63.
    Goldstrohm AC, Albrecht TR, Sune C, Bedford MT, Garcia-Blanco MA (2001) The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1. Mol Cell Biol 21:7617–7628PubMedCrossRefGoogle Scholar
  64. 64.
    Liu X, Jiang Q, Mansfield SG, Puttaraju M, Zhang Y, Zhou W, Cohn JA, Garcia-Blanco MA, Mitchell LG, Engelhardt JF (2002) Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat Biotechnol 20:47–52PubMedGoogle Scholar
  65. 65.
    Liu X, Luo M, Zhang LN, Yan Z, Zak R, Ding W, Mansfield SG, Mitchell LG, Engelhardt JF (2005) Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator function to polarized human cystic fibrosis airway epithelial cells. Hum Gene Ther 16:1116–1123PubMedCrossRefGoogle Scholar
  66. 66.
    Chao H, Mansfield SG, Bartel RC, Hiriyanna S, Mitchell LG, Garcia-Blanco MA, Walsh CE (2003) Phenotype correction of hemophilia A mice by spliceosome-mediated RNA trans-splicing. Nat Med 9:1015–1019PubMedCrossRefGoogle Scholar
  67. 67.
    Nakayama K, Pergolizzi RG, Crystal RG (2005) Gene transfer-mediated pre-mRNA segmental trans-splicing as a strategy to deliver intracellular toxins for cancer therapy. Cancer Res 65:254–263PubMedGoogle Scholar
  68. 68.
    Pergolizzi RG, Ropper AE, Dragos R, Reid AC, Nakayama K, Tan Y, Ehteshami JR, Coleman SH, Silver RB, Hackett NR, Menez A, Crystal RG (2003) In vivo trans-splicing of 5′ and 3′ segments of pre-mRNA directed by corresponding DNA sequences delivered by gene transfer. Mol Ther 8:999–1008PubMedCrossRefGoogle Scholar
  69. 69.
    Tahara M, Pergolizzi RG, Kobayashi H, Krause A, Luettich K, Lesser ML, Crystal RG (2004) Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency. Nat Med 10:835–841PubMedCrossRefGoogle Scholar
  70. 70.
    Wally V, Klausegger A, Koller U, Lochmuller H, Krause S, Wiche G, Mitchell LG, Hintner H, Bauer JW (2008) 5′ trans-splicing repair of the PLEC1 gene. J Invest Dermatol 128:568–574PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2012

Authors and Affiliations

  • Hansjörg Rindt
    • 1
  • Pei-Fen Yen
    • 1
  • Christina N. Thebeau
    • 2
  • Troy S. Peterson
    • 3
  • Gary A. Weisman
    • 2
    • 3
  • Christian L. Lorson
    • 1
  1. 1.Department of Veterinary Pathobiology, Life Sciences CenterUniversity of MissouriColumbiaUSA
  2. 2.Department of BiochemistryUniversity of MissouriColumbiaUSA
  3. 3.Interdisciplinary Neuroscience ProgramUniversity of MissouriColumbiaUSA

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