Cellular and Molecular Neurobiology

, Volume 13, Issue 5, pp 503–515 | Cite as

Herpesvirus-mediated gene delivery into the rat brain: specificity and efficiency of the neuron-specific enolase promoter

  • Julie K. Andersen
  • David M. Frim
  • Ole Isacson
  • Xandra O. Breakefield


  1. 1.

    Herpesvirus infection with genetically engineered vectors is a way to deliver foreign gene products to various cell populations in culture andin vivo. Selective neuronal gene expression can be achieved using the neuron-specific enolase (NSE) promoter regulating expression of a transgene placed in and delivered by a herpesvirus vector.

  2. 2.

    We sought to determine the anatomical specificity and efficiency of herpesvirus-mediated gene transfer into the rat brain following placement of virus particles carrying a transgene (lacZ) under control of the NSE promoter. The virus utilized was thymidine kinase (TK) deficient and therefore replication deficient in the brain.

  3. 3.

    Infusion of 106 plaque-forming units of virus into the striatum caused a limited number of striatal neurons to express thelacZ transgene mRNA and protein product 7 days postinfection. In addition, small numbers of neurons expressing the transgene mRNA and protein were found ipsilateral to the viral injection in the frontal cortex, substantia nigra pars compacta, and thalamus. Neurons at these anatomic loci project directly to the striatal injection site. No other cells within the brains of injected animals expressed thelacZ gene.

  4. 4.

    While this herpesvirus NSE vector was capable of introducing novel functional genetic information into postmitotic neurons within defined neuroanatomic constraints, the numbers of neurons expressing detectable levels ofβ-galactosidase was minimal. The calculated efficiency of delivery and transgene expression at 7 days postinfection was 1 transgenic neuron per 104 virus particles infused.

  5. 5.

    We conclude that NSE probably is not an optimal promoter for use in gene delivery to CNS neurons in herpesvirus vectors and that the efficacy of gene delivery using other neuron-specific promoters placed at various sites in the herpes viral genome needs to be explored.


Key words

herpesvirus vector gene transfer neuron-specific enolase (NSE) promoter lacZ, stereotactic delivery rat CNS 


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  1. Andersen, J. K., Garber, D., Meaney, C. A., and Breakefield, X. O. (1992). Gene transfer into mammalian central nervous system using herpes virus vectors: Extended expression of bacterial LacZ in neurons using the neuron-specific enolase promoter.Hum. Gene Ther. 3487–499.Google Scholar
  2. Breakefield, X. O. (1989). Combining CNS transplantation and gene transfer.Neurobiol. Aging 10647–648.Google Scholar
  3. Breakefield, X. O., and Deluca, N. A. (1991). Herpes simplex virus for gene delivery to neurons.New Biol. 3203–218.Google Scholar
  4. Breakefield, X. O., Chin, Q., Andersen, J. K., Kramer, M. F., Bebrin, W. R., Davar, G., Vos, B., GArber, D. A., Difiglia, M., and Coen, D. M. (1992). Gene transfer into the nervous system using recombinant herpes virus vectors. InGene Transfer and Therapy in the Nervous System (C. Gage, Ed.), Springer-Verlag, Heidelberg, Vol. 16, pp. 45–48.Google Scholar
  5. Cai, W., and Schaffer, P. A. (1989). Herpes simplex type 1 ICPO plays a critical role in the de novo synthesis of infections virus following transfection of viral DNA.J. Virol. 634579–4589.Google Scholar
  6. Carpenter, M. B. (1982). Anatomy of the corpus striatum and brain stem integrating systems. InHandbook of Physiology:The Nervous System II. (J. M. Brookhart and V. B. Mountcastle, Eds.); City Publisher, New York, part 2, pp. 947–995.Google Scholar
  7. Cepko, C. (1989). Retrovirus vectors and their applications in neurobiology.Neuron 1345–353.Google Scholar
  8. Chang, P. L., Capone, J. P., and Brown, G. M. (1990). Autologous fibroblast implantation: Feasibility and potential problems in gene replacement.Mol. Biol. Med. 7461–470.Google Scholar
  9. Chiocca, E. A., Choi, B. B., Cai, W., Deluca, N. A., Schaffer, P. A., Difiglia, M., Breakefield, X. O., and Martuza, R. L. (1990). Transfer and expressions of the lacZ gene in rat brain neurons mediated by herpes simplex virus insertion mutants.New Biol. 2739–746.Google Scholar
  10. Chou, J., Kern, E. R., Whitley, R. J., and Roizman, B. (1991). Mapping of herpes simplex virus-1 neurovirulence to g134.5, a gene nonessential for growth in culture.Science 2501262–1266.Google Scholar
  11. Coen, D. M., Kosz-Vnenchak, M., Jacobson, J. G., Leib, D. A., Board, C. L., Schaffer, P. A., Taylor, K. L., and Knipe, D. M. (1989). Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate.Proc. Natl. Acad. Sci. USA 8647836–47840.Google Scholar
  12. Comb, M., Mermod, N., Hyman, S. E., Pearlberg, J., Ross, M. E., and Goodman, H. M. (1988). Proteins bound to adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcriptions.EMBO J. 173793–3805.Google Scholar
  13. Cunningham, L. A., Hansen, J. P., Short, M. P., and Bohn, M. C. (1991). The use of genetically altered astrocytes to provide nerve growth factor (NGF) to adrenal chromaffin cells grafted into the striatum.Brain Res. 61192–202.Google Scholar
  14. Davidson, B. L., Allen, E. D., Kozarsky, K. F., Wilson, J. M., and Roessler, B. L. (1993). A model system for in vivo gene transfer into the central nervous system using an adenovirus vector.Nature Gen. 3 219–223.Google Scholar
  15. Dobson, A. T., Sederati, F., Devi-Rao, G., Flanagan, W. M., Farrell, M. J., Stevens, J. G., Wagner, E. K., and Feldman, L. T. (1989). Identification of the latency-associated transcript promoter by expression of rabbitβ-galactosidase mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus.J. Virol. 633844–3851.Google Scholar
  16. Dobson, A. T., Margolis, T. P., Sederati, F., Stevens, J. G., and Feldman, I. T. (1990). A latent, nonpathogenic HSV-1-derived vector stably expressesβ-galactosidase in mouse neurons.Neuron 5353–360.Google Scholar
  17. Efstathiou, S., Kamp, S., Darby, G., and Minson, A. C. (1989). The role of herpes simplex type 1 thymidine kinase in pathogenesis.J. Gen. Virol. 70869–879.Google Scholar
  18. Emson, P. C., Kamp, S., Darby, G., and Minson, A. C. (1990). The use of a retroviral vector to identify foetal striatal neurones transplanted into adult striatum.Exp. Brain Res. 7927–430.Google Scholar
  19. Federoff, H. J., Geschwind, M. D., Geller, A. I., and Kessler, J. A. (1992). Expression of nerve growth factor in vivo from a defective herpes simplex virus 1 vector prevents effects of axotomy on sympathetic ganglia.Proc. Natl. Acad. Sci. 891636–1640.Google Scholar
  20. Fenwick, M. L., and Everett, R. D. (1990). Transfer of UL41, the gene controlling virion-associated host cell shut-off, between different strains of herpes simplex virus.J. Gen. Virol. 71411–418.Google Scholar
  21. Fink, D. J., Sternberg, L. R., Weber, P. C., Mata, M., Goins, W. F., and Glorioso, J. C. (1992). In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer.Hum. Gene Ther. 111–19.Google Scholar
  22. Frim, D. M., Short, M. P., Rosenberg, W. S., Simpson, J., Breakefield, X. O., and Isacson, O. (1993). Nerve growth factor secreting fibroblasts protect only locally against excitotoxicity in the rat striatum.J. Neurosurg. 78267–273.Google Scholar
  23. Forss-Peter, S., Danielson, P. E., Catsicas, S., Battenberg, E., Price, J., Nurenberg, M., and Sutcliffe, J. G. (1990). Transgenic mice expressingβ-galactosidase in mature neurons under neuron specific enolase promoter control.Neuron 5187–197.Google Scholar
  24. Gage, F. H., Wolf, J. A., Rosenberg, M. B., Xu, L., Yee, J. K., Shults, C., and Friedmann, T. (1987). Grafting genetically modified cells to the brain: Possibilities for the future.Neuroscience 23795–807.Google Scholar
  25. Gage, F. H., Rosenberg, M. B., Tuszynski, M. H., Yoshida, K., Armstrong, D. M., Hayes, R. C., and Friedmann, T. (1990). Gene therapy in the CNS: Intracerebral grafting of genetically modified cells.Prog. Brain Res. 86205–217.Google Scholar
  26. Geller, A. I., During, M. J., and Neve, R. L. (1991). Molecular analysis of neuronal physiology by gene transfer into neurons with herpes simplex virus vectors.Trends Neurosci. 14428–432.Google Scholar
  27. Glorioso, J. C., Sternberg, L. R., Groins, W. F., and Fink, D. J. (1992). Development of herpes simplex virus as a gene transfer vector for the central nervous system. InGene Transfer and Therapy in the Nervous System. (F. Gage and Y. Christen, Eds.); Springer-Verlag, New York, pp. 133–145.Google Scholar
  28. Groenewegen, H. J., Berendse, H. W., Wolters, J. G., and Lohman, A. H. (1990). The anatomical relationship of the prefrontal cortex with the striatopallidal system, the thalamus, and the amygdala: Evidence for a parallel organization.Prog. Brain Res. 8595–118.Google Scholar
  29. Huang, Q., Vonsattel, J. P., Schaffer, P. A., Martuza, R. L., Breakefield, X. O., and Difiglia, M. (1992). Introduction of a large foreign gene (E. Coli LacZ) into rat neostriatal neurons using herpes simplex virus mutants: A light and electron microscope study.Exp. Neurol. 115303–316.Google Scholar
  30. Javier, R. T., Izumi, K. M., and Steven, J. G. (1988). Localization of a herpes simplex virus neurovirulence gene dissociated from high-titer virus replication in the brain.J. Viriol. 621381–1387.Google Scholar
  31. Kaplitt, M. G., Pfaus, J. G., Kleopoulos, S. P., Hanlon, B. A., Rabkin, S. D., and Pfaff, D. W. (1991). Expression of a functional foreign gene in adult mammalian brain following in vivo transfer via a herpes simplex virus type 1 defective viral vector.Mol. Cell. Neurosci. 2320–330.Google Scholar
  32. Kosz-Vnenchak, M., Coen, D. M., and Knipe, D. M. (1990). Restricted expression of herpes simplex virus lytic genes during establishment of latent infection by thymidine kinase negative viruses.J. Virol. 645396–5402.Google Scholar
  33. Kwong, A. D., and Frenkel, N. (1989). The herpes simplex virus virion host shutoff function.J. Virol. 634834–4839.Google Scholar
  34. Leist, T. P., Sandri-Goldin, R. M., and Stevens, J. G. (1989). Latent infections in spinal ganglia with thymidine kinase-deficient herpes simplex virus.J. Virol. 634976–4978.Google Scholar
  35. Martin, X., and Dolivo, M. (1983). Neuronal and transneuronal tracing in the trigeminal system of the rat using the herpes virus suis.Brain Res. 273253–276.Google Scholar
  36. Palella, T. D., Silverman, L. J., Schroll, C. T., Homa, F. L., Levine, M., and Kelley, W. N. (1988). Herpes simplex virus mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.Mol. Cell. Biol. 8457–468.Google Scholar
  37. Rosenberg, M. B., Friedman, T., Robertson, R. C., Tuszynski, M., Wolff, J. A., Breakefield, X. O., and Gage, F. H. (1988). Grafting genetically modified cells to the damaged brain: Restorative effects of NGF expression.Science 242575–1578.Google Scholar
  38. Rosenberg, W. S., Breakefield, X. O., DeAntonio, C., and Isacson, O. (1992). Authentic and artifactual detection of theE. coli lacZ gene product in the rat brain by histochemical techniques.Mol. Brain Res. 16311–315.Google Scholar
  39. Rouiller, E. M., Capt, M., Dolivo, M., and De Ribaupierre, F. (1989). Neuronal organization of the stapedious reflex pathways in the rat: A retrograde HRP and viral transneuronal tracing study.Brain Res. 47621–28.Google Scholar
  40. Sambrook, J., Fritsch, E. F., and Manatis, T. (1989). InMolecular Cloning (C. Nolan, Ed.), Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY.Google Scholar
  41. Schumacher, J. M., Short, M. P., Hyman, B. T., Breakefield, X. O., and Isacon, I. (1991). Intracerebral implantation of nerve growth factor-producing fibroblasts protects striatum against neurotoxic levels of excitatory amino acids.Neuroscience 45561–570.Google Scholar
  42. Shimohama, S., Rosenberg, M. B., Fagan, A. M., Wolff, J. A., Short, M. P., and Breakefield, X. O. (1989). Grafting genetically modified cells into the rat brain: Characteristics of E. coli galactosidase as a reporter gene.Mol. Brain Res. 5271–276.Google Scholar
  43. Stevens, J. G. (1989). Human herpesviruses: A consideration of the latent state.Microbiol. Rev. 53318–332.Google Scholar
  44. Turner, D. L., and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development.Nature 328131–136.Google Scholar
  45. Thompson, R. L., Rogers, S. K., and Zerhusen, M. A. (1989). Herpes simplex virus neurovirulence and productive infection of neural cells is associated with a function which maps between 0.82 and 0.832 map units on the HSV genome.Virology 172435–450.Google Scholar
  46. Ugolini, G., Kuypers, H. G., and Strick, P. L. (1989). Transneuronal transfer of herpes virus from peripheral nerves to cortex and brainstem.Science 24389–91.Google Scholar
  47. Wolfe, J. H., Deshmane, S. L., and Fraser, N. W. (1992). Herpesvirus vector gene transfer and expression ofβ-glucuronidase in the central nervous system of MPS VII mice.Nature Genet. 1379–384.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Julie K. Andersen
    • 1
    • 3
  • David M. Frim
    • 1
    • 2
    • 4
  • Ole Isacson
    • 3
    • 4
  • Xandra O. Breakefield
    • 1
    • 3
  1. 1.Molecular Neurogenetics UnitMassachusetts General Hospital, Harvard Medical SchoolBostonUSA
  2. 2.Neurosurgery ServiceMassachusetts General Hospital, Harvard Medical SchoolBostonUSA
  3. 3.Neurology ServiceMassachusetts General Hospital, Harvard Medical SchoolBostonUSA
  4. 4.Neuroregeneration LaboratoryMcLean HospitalBelmontUSA

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