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In utero delivery of rAAV2/9 induces neuronal expression of the transgene in the brain: towards new models of Parkinson’s disease

Abstract

Animal models are essential tools for basic pathophysiological research as well as validation of therapeutic strategies for curing human diseases. However, technical difficulties associated with classical transgenesis approaches in rodent species higher than Mus musculus have prevented this long-awaited development. The availability of viral-mediated gene delivery systems in the past few years has stimulated the production of viruses with unique characteristics. For example, the recombinant adeno-associated virus serotype 9 (rAAV2/9) crosses the blood–brain barrier, is capable of transducing developing cells and neurons after intravenous injection and mediates long-term transduction. Whilst post-natal delivery is technically straightforward, in utero delivery bears the potential of achieving gene transduction in neurons at embryonic stages during which the target area is undergoing development. To test this possibility, we injected rAAV2/9 carrying either A53T mutant human α-synuclein or green fluorescent protein, intracerebroventricularly in rats at embryonic day 16.5. We observed neuronal transgene expression in most regions of the brain at 1 and 3 months after birth. This proof-of-concept experiment introduces a new opportunity to model brain diseases in rats.

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References

  1. Dehay B, Bourdenx M, Gorry P, Przedborski S, Vila M, Hunot S et al. Targeting alpha-synuclein for treatment of Parkinson's disease: mechanistic and therapeutic considerations. Lancet Neurol 2015; 14: 855–866.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mattson MP, Magnus T . Ageing and neuronal vulnerability. Nat Rev Neurosci 2006; 7: 278–294.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bezard E, Yue Z, Kirik D, Spillantini MG . Animal models of Parkinson's disease: limits and relevance to neuroprotection studies. Movement Disorders: Official Journal of the Movement Disorder Society 2013; 28: 61–70.

    Article  CAS  Google Scholar 

  4. Blesa J, Przedborski S . Parkinson's disease: animal models and dopaminergic cell vulnerability. Front Neuroanat 2014; 8: 155.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Dehay B, Fernagut PO . Alpha-synuclein-based models of Parkinson's disease. Revue Neurol 2016; 172: 371–378.

    Article  CAS  Google Scholar 

  6. Bourdenx M, Dutheil N, Bezard E, Dehay B . Systemic gene delivery to the central nervous system using Adeno-associated virus. Front Mol Neurosci 2014; 7: 50.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Weinberg MS, Samulski RJ, McCown TJ . Adeno-associated virus (AAV) gene therapy for neurological disease. Neuropharmacology 2013; 69: 82–88.

    Article  CAS  PubMed  Google Scholar 

  8. Bourdenx M, Dovero S, Engeln M, Bido S, Bastide MF, Dutheil N et al. Lack of additive role of ageing in nigrostriatal neurodegeneration triggered by alpha-synuclein overexpression. Acta Neuropathologica Communications 2015; 3: 46.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Zhang H, Xie J, Xie Q, Wilson JM, Gao G . Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production. Hum Gene Ther 2009; 20: 922–929.

    Article  CAS  PubMed  Google Scholar 

  10. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE . Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 2008; 16: 1073–1080.

    Article  CAS  PubMed  Google Scholar 

  11. Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK . Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 2009; 27: 59–65.

    Article  CAS  PubMed  Google Scholar 

  12. Zhang H, Yang B, Mu X, Ahmed SS, Su Q, He R et al. Several rAAV vectors efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system. Mol Ther 2011; 19: 1440–1448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dehay B, Dalkara D, Dovero S, Li Q, Bezard E . Systemic scAAV9 variant mediates brain transduction in newborn rhesus macaques. Sci Rep 2012; 2: 253.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Duque S, Joussemet B, Riviere C, Marais T, Dubreil L, Douar AM et al. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther 2009; 17: 1187–1196.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Foust KD, Wang X, McGovern VL, Braun L, Bevan AK, Haidet AM et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol 2010; 28: 271–274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stott SR, Kirik D . Targeted in utero delivery of a retroviral vector for gene transfer in the rodent brain. Eur J Neurosci 2006; 24: 1897–1906.

    Article  PubMed  Google Scholar 

  17. Rahim AA, Wong AM, Howe SJ, Buckley SM, Acosta-Saltos AD, Elston KE et al. Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors. Gene Ther 2009; 16: 509–520.

    Article  CAS  PubMed  Google Scholar 

  18. Walsh C, Cepko CL . Widespread dispersion of neuronal clones across functional regions of the cerebral cortex. Science 1992; 255: 434–440.

    Article  CAS  PubMed  Google Scholar 

  19. Bowers WJ, Mastrangelo MA, Howard DF, Southerland HA, Maguire-Zeiss KA, Federoff HJ . Neuronal precursor-restricted transduction via in utero CNS gene delivery of a novel bipartite HSV amplicon/transposase hybrid vector. Mol Ther 2006; 13: 580–588.

    Article  CAS  PubMed  Google Scholar 

  20. Shen JS, Meng XL, Yokoo T, Sakurai K, Watabe K, Ohashi T et al. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease. J Gene Med 2005; 7: 540–551.

    Article  CAS  PubMed  Google Scholar 

  21. Shen JS, Meng XL, Maeda H, Ohashi T, Eto Y . Widespread gene transduction to the central nervous system by adenovirus in utero: implication for prenatal gene therapy to brain involvement of lysosomal storage disease. J Gene Med 2004; 6: 1206–1215.

    Article  CAS  PubMed  Google Scholar 

  22. Rahim AA, Wong AM, Ahmadi S, Hoefer K, Buckley SM, Hughes DA et al. In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression. Gene Ther 2012; 19: 936–946.

    Article  CAS  PubMed  Google Scholar 

  23. Haddad MR, Donsante A, Zerfas P, Kaler SG . Fetal brain-directed AAV gene therapy results in rapid, robust, and persistent transduction of mouse choroid plexus epithelia. Mol Ther Nucleic Acids 2013; 2: e101.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Tabata H, Nakajima K . Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience 2001; 103: 865–872.

    Article  CAS  PubMed  Google Scholar 

  25. Yoshida A, Yamaguchi Y, Nonomura K, Kawakami K, Takahashi Y, Miura M . Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system. Genes Cells 2010; 15: 501–512.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. dal Maschio M, Ghezzi D, Bony G, Alabastri A, Deidda G, Brondi M et al. High-performance and site-directed in utero electroporation by a triple-electrode probe. Nat Commun 2012; 3: 960.

    Article  PubMed  Google Scholar 

  27. Beach TG, White CL, Hamilton RL, Duda JE, Iwatsubo T, Dickson DW et al. Evaluation of alpha-synuclein immunohistochemical methods used by invited experts. Acta Neuropathol 2008; 116: 277–288.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Neumann M, Kahle PJ, Giasson BI, Ozmen L, Borroni E, Spooren W et al. Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies. J Clin Invest 2002; 110: 1429–1439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Saunders NR, Liddelow SA, Dziegielewska KM . Barrier mechanisms in the developing brain. Front Pharmacol 2012; 3: 46.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Glascock JJ, Osman EY, Wetz MJ, Krogman MM, Shababi M, Lorson CL . Decreasing disease severity in symptomatic, Smn(-/-);SMN2(+/+), spinal muscular atrophy mice following scAAV9-SMN delivery. Hum Gene Ther 2012; 23: 330–335.

    Article  CAS  PubMed  Google Scholar 

  31. Lipshutz GS, Gruber CA, Cao Y, Hardy J, Contag CH, Gaensler KM . In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression. Mol Ther 2001; 3: 284–292.

    Article  CAS  PubMed  Google Scholar 

  32. Almeida-Porada G, Atala A, Porada CD . In utero stem cell transplantation and gene therapy: rationale, history, and recent advances toward clinical application. Mol Ther 2016; 5: 16020.

    Google Scholar 

  33. Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet 2007; 369: 2097–2105.

    Article  CAS  PubMed  Google Scholar 

  34. Marks Jr WJ, Bartus RT, Siffert J, Davis CS, Lozano A, Boulis N et al. Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomised, controlled trial. Lancet Neurol 2010; 9: 1164–1172.

    Article  CAS  PubMed  Google Scholar 

  35. Bartus RT, Baumann TL, Brown L, Kruegel BR, Ostrove JM, Herzog CD . Advancing neurotrophic factors as treatments for age-related neurodegenerative diseases: developing and demonstrating 'clinical proof-of-concept' for AAV-neurturin (CERE-120) in Parkinson's disease. Neurobiol Aging 2013; 34: 35–61.

    Article  CAS  PubMed  Google Scholar 

  36. Engeln M, Fasano S, Ahmed SH, Cador M, Baekelandt V, Bezard E et al. Levodopa gains psychostimulant-like properties after nigral dopaminergic loss. Ann Neurol 2013; 74: 140–144.

    Article  CAS  PubMed  Google Scholar 

  37. Baker M . Statisticians issue warning over misuse of P values. Nature 2016; 531: 151.

    Article  CAS  PubMed  Google Scholar 

  38. Mattar CN, Waddington SN, Biswas A, Johana N, Ng XW, Fisk AS et al. Systemic delivery of scAAV9 in fetal macaques facilitates neuronal transduction of the central and peripheral nervous systems. Gene Ther 2013; 20: 69–83.

    Article  CAS  PubMed  Google Scholar 

  39. Wang DB, Dayton RD, Henning PP, Cain CD, Zhao LR, Schrott LM et al. Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed. Mol Ther 2010; 18: 2064–2074.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. McLean JR, Smith GA, Rocha EM, Hayes MA, Beagan JA, Hallett PJ et al. Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection. Neurosci Lett 2014; 576: 73–78.

    Article  CAS  PubMed  Google Scholar 

  41. Klein RL, Dayton RD, Tatom JB, Henderson KM, Henning PP . AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method. Mol Ther 2008; 16: 89–96.

    Article  CAS  PubMed  Google Scholar 

  42. Cearley CN, Vandenberghe LH, Parente MK, Carnish ER, Wilson JM, Wolfe JH . Expanded repertoire of AAV vector serotypes mediate unique patterns of transduction in mouse brain. Mol Ther 2008; 16: 1710–1718.

    Article  CAS  PubMed  Google Scholar 

  43. Hadaczek P, Eberling JL, Pivirotto P, Bringas J, Forsayeth J, Bankiewicz KS . Eight years of clinical improvement in MPTP-lesioned primates after gene therapy with AAV2-hAADC. Mol Ther 2010; 18: 1458–1461.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Martins-Branco D, Esteves AR, Santos D, Arduino DM, Swerdlow RH, Oliveira CR et al. Ubiquitin proteasome system in Parkinson's disease: a keeper or a witness? Exp Neurol 2012; 238: 89–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jackson MP, Hewitt EW . Cellular proteostasis: degradation of misfolded proteins by lysosomes. Essays Biochem 2016; 60: 173–180.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Goedert M, Spillantini MG, Del Tredici K, Braak H . 100 years of Lewy pathology. Nat Rev Neurol 2013; 9: 13–24.

    Article  CAS  PubMed  Google Scholar 

  47. Del Tredici K, Braak H . Review: Sporadic Parkinson's disease: development and distribution of alpha-synuclein pathology. Neuropathol Appl Neurobiol 2016; 42: 33–50.

    Article  CAS  PubMed  Google Scholar 

  48. Jellinger KA . Neuropathobiology of non-motor symptoms in Parkinson disease. J Neural Transm 2015; 122: 1429–1440.

    Article  CAS  PubMed  Google Scholar 

  49. Blandini F, Armentero MT, Martignoni E . The 6-hydroxydopamine model: news from the past. Parkinsonism Relat Disord 2008; 14 (Suppl 2): S124–S129.

    Article  PubMed  Google Scholar 

  50. Lo Bianco C, Ridet JL, Schneider BL, Deglon N, Aebischer P . Alpha -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease. Proc Natl Acad Sci USA 2002; 99: 10813–10818.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Decressac M, Mattsson B, Lundblad M, Weikop P, Bjorklund A . Progressive neurodegenerative and behavioural changes induced by AAV-mediated overexpression of alpha-synuclein in midbrain dopamine neurons. Neurobiol Dis 2012; 45: 939–953.

    Article  CAS  PubMed  Google Scholar 

  52. Mulcahy P, O'Doherty A, Paucard A, O'Brien T, Kirik D, Dowd E . The behavioural and neuropathological impact of intranigral AAV-alpha-synuclein is exacerbated by systemic infusion of the Parkinson's disease-associated pesticide, rotenone, in rats. Behav Brain Res 2013; 243: 6–15.

    Article  CAS  PubMed  Google Scholar 

  53. Caudal D, Alvarsson A, Bjorklund A, Svenningsson P . Depressive-like phenotype induced by AAV-mediated overexpression of human alpha-synuclein in midbrain dopaminergic neurons. Exp Neurol 2015; 273: 243–252.

    Article  CAS  PubMed  Google Scholar 

  54. Dehay B, Decressac M, Bourdenx M, Guadagnino I, Fernagut PO, Tamburrino A et al. Targeting alpha-synuclein: Therapeutic options. Movement Disord 2016; 31: 882–888.

    Article  CAS  PubMed  Google Scholar 

  55. Ysselstein D, Dehay B, Costantino IM, McCabe GP, Frosch MP, George JM et al. Endosulfine-alpha inhibits membrane-induced alpha-synuclein aggregation and protects against alpha-synuclein neurotoxicity. Acta Neuropathol Commun 2017; 5: 3.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Bourdenx M, Daniel J, Genin E, Soria FN, Blanchard-Desce M, Bezard E et al. Nanoparticles restore lysosomal acidification defects: implications for Parkinson and other lysosomal-related diseases. Autophagy 2016; 12: 472–483.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999; 6: 973–985.

    Article  CAS  PubMed  Google Scholar 

  58. Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods 2012; 23: 18–28.

    Article  CAS  PubMed  Google Scholar 

  59. Engeln M, Ahmed SH, Vouillac C, Tison F, Bezard E, Fernagut PO . Reinforcing properties of Pramipexole in normal and parkinsonian rats. Neurobiol Dis 2013; 49: 79–86.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

MB was the recipient of a MESR fellowship and the France Parkinson Foundation. Slide scanning was performed at the Bordeaux Imaging Center a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging. Accommodations and experiments were performed in the Service des Animaleries of the University of Bordeaux, supported by the Région Aquitaine. This work was supported by the LABEX BRAIN. The help of Romain Desgranges for Figure 1, as well as Florian Roguet and Laetitia Medan for animal care is acknowledged. We also thank A. Crossman for valuable comments on the manuscript.

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Correspondence to B Dehay.

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EB is Chief Scientific Officer of Motac neuroscience Ltd. The remaining authors declare no conflict of interest.

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Chansel-Debordeaux, L., Bourdenx, M., Dovero, S. et al. In utero delivery of rAAV2/9 induces neuronal expression of the transgene in the brain: towards new models of Parkinson’s disease. Gene Ther 24, 801–809 (2017). https://doi.org/10.1038/gt.2017.84

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