Abstract
Recombinant adenovirus vectors have provided a major advance in gene delivery systems for post-mitotic neurons. however, the use of these first generation vectors has been limited due to the onset of virally mediated effects on cellular function and viability. in the present study we have used primary cultures of cerebellar granule neurons to examine the efficacy and cytotoxic effects of a helper-dependent adenovirus vector (hdad) in comparison with a first generation vector. our results demonstrate that the hdad system provides equally efficient infectivity with significantly reduced toxicity in comparison to first generation vectors. neurons transduced with a high titre of a first generation vector exhibited a time-dependent shut down in global protein synthesis and impaired physiological function as demonstrated by a loss of glutamate receptor responsiveness. this was followed by an increase in the fraction of tunel-positive cells and a loss of neuronal survival. in contrast, hdads could be used at titres that transduce >85% of neurons with little cytotoxic effect: cellular glutamate receptor responses and rates of protein synthesis were indistinguishable from uninfected controls. Furthermore, cell viability was not significantly affected for at least 7 days after infection. At excessive viral titres, however, infection with hdAd did cause moderate but significant changes in cell function and viability in primary neuronal cultures. Thus, while these vectors are remarkably improved over first generation vectors, these also have limitations with respect to viral effects on cellular function and viability.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Karpati G et al. The principles of gene therapy for the nervous system Trends Neurosci 1996 19: 49–54
Slack RS, Miller FD . Viral vectors for modulating gene expression in neurons Curr Opin Neurobiol 1996 6: 576–583
Hitt M, Addison C, Graham FL . Human adenovirus vectors for gene transfer into mammalian cells. In: August JT (ed) Advances in Pharmacology – Gene Therapy Academic Press: San Diego 1997 137–206
La Salle GLG et al. An adenovirus vector for gene transfer into neurons and glia in the brain Science 1993 259: 988–990
Akli S et al. Transfer of a foreign gene into the brain using adenovirus vectors Nat Genet 1993 3: 224–228
Davidson BL et al. A model system for in vivo gene transfer into the central nervous system using an adenoviral vector Nat Genet 1993 3: 219–223
Slack RS et al. Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons J Cell Biol 1996 135: 1085–1096
Xiang H et al. Evidence for p53-mediated modulation of neuronal viability J Neurosci 1996 16: 6753–6765
Jordan J et al. p53 expression induces apoptosis in hippocampal pyramidal neuron cultures J Neurosci 1997 17: 1397–1405
Xu DG et al. Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus Nat Med 1997 3: 997–1004
Acsadi G et al. Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer Hum Gene Ther 1996 7: 129–140
Franklin RJM, Quick MM, Haase G . Adenoviral vectors for in vivo gene delivery to oligodendrocytes: transgene expression and cytopathic consequences Gene Therapy 1999 6: 1360–1367
Lawrence MS et al. Inflammatory responses and their impact on β-galactosidase transgene expression following adenovirus vector delivery to primate caudate nucleus Gene Therapy 1999 6: 1368–1379
Yang Y et al. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy Proc Natl Acad Sci USA 1994 91: 4407–4411
Easton RM, Johnson EM, Creedon MJ . Analysis of events leading to neuronal death after infection with E1-deficient adenoviral vectors Mol Cell Neurosci 1998 11: 334–347
Yang Y, Wilson JM . Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo J Immunol 1995 5: 2564–2570
Yang Y et al. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses J Virol 1995 69: 2004–2015
Morral N et al. Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: comparison of E2a wild type and E2a deleted vectors Hum Gene Ther 1997 8: 1275–1286
Morral N et al. High doses of a helper-dependent adnoviral vector yield supraphysiological levels of alpha1-antitrypsin with negligible toxicity Hum Gene Ther 1998 9: 2709–2716
Shenk T, Flint J . Transcriptional and transforming activities of the adenovirus E1A proteins Adv Cancer Res 1991 57: 47–85
Cress WD, Nevins JR . Use of the E2F transcription factor by DNA tumor virus regulatory proteins Curr Top Microbiol Immunol 1996 208: 63–78
Armentano D, Sookdeo CC . Characterization of an adenovirus gene transfer vector containing an E4 deletion Hum Gene Ther 1995 6: 1343–1353
Dedieu JF, Vigne E . Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses J Virol 1997 71: 4626–4637
Engelhardt JF, Ye X . Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver Proc Natl Acad Sci USA 1994 91: 6196–6200
Fang B, Wang H . Lack of persistence of E1-recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs Gene Therapy 1996 3: 217–222
Gao GP, Yang Y, Wilson JM . Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy J Virol 1996 70: 8934–8943
Goldman MJ et al. Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study Hum Gene Ther 1995 7: 839–851
Gorziglia MI et al. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy J Virol 1996 70: 4173–4178
Yang Y et al. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis Nat Genet 1994 7: 362–369
Parks RJ et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal Proc Natl Acad Sci USA 1996 93: 13565–13570
Chen L, Anton M, Graham FL . Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre Somat Cell Mol Genet 1996 22: 477–488
Hardy S et al. Construction of adenovirus vectors through Cre lox recombination J Virol 1997 71: 1842–1849
Schiedner G et al. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity Nat Genet 1998 2: 180–183
Bello LJ, Ginsberg HS . Inhibition of host protein synthesis in type 5 adenovirus-infected cells J Virol 1967 1: 843–850
Beltz GA, Flint SJ . Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus J Mol Biol 1979 131: 353–373
Durham HD et al. Toxicity of replication-defective recombinants in dissociated cultures of nervous tissue Exp Neurol 1996 140: 14–20
Kafri T et al. Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy Proc Natl Acad Sci USA 1998 95: 11377–11382
Amalfitano A et al. Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted J Virol 1998 72: 926–933
Mitani K, Graham FL, Caskey CT, Kochanek S . Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector Proc Natl Acad Sci USA 1995 92: 3854–3858
Fisher KJ et al. Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis Virology 1996 217: 11–22
Haecker SE et al. In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes Hum Gene Ther 1996 7: 1907–1914
Kumar-Singh R, Chamberlain JS . Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells Hum Mol Genet 1996 5: 913–921
Kochanek S et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase Proc Natl Acad Sci USA 1996 93: 5731–5736
Morral N et al. High doses of a helper-dependent adenoviral vector yield supraphysiological levels of alpha 1-antitrypsin with negligible toxicity Hum Gene Ther 1998 9: 2709–2716
Morral N et al. Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons Proc Natl Acad Sci USA 1999 96: 12816–12821
Parks RJ et al. Effects of stuffer DNA on transgene expression from helper-dependent adenoviral vectors J Virol 1999 73: 8027–8034
Cowdery JS et al. Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides J Immunol 1996 12: 4570–4575
Krieg AM et al. CpG motifs in bacterial DNA trigger direct B-cell activation Nature 1995 374: 546–549
Stacey KJ, Sweet MJ, Hume DA . Macrophages ingest and are activated by bacterial DNA J Immunol 1996 157: 2116–2122
Addison CL et al. Comparison of the human versus murine cytomegalovirus immediate early gene promoters for transgene expression by adenoviral vectors J Gen Virol 1997 78: 1653–1661
Graham FL, Prevec L . Manipulation of adenovirus vectors Methods in Molecular Biology Humana Press: Clifton, NJ 1991 pp 109–128
Levi G et al. Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures Brain Res 1984 290: 77–86
Miller TM, Johnson EM . Metabolic and genetic analyses of apoptosis in potassium/serum-deprived rat cerebellar granule cells J Neurosci 1996 23: 7487–7495
Morley P et al. Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells J Cereb Blood Flow Metab 1998 18: 396–406
Deckwerth TL, Johnson EM . Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor J Cell Biol 1993 123: 1207–1222
Acknowledgements
This work was supported by grants from the Heart and Stroke Foundation of Ontario (HSFO) and the Medical Research Council of Canada (MRC) to RSS, and an MRC, NCI, NIH and a Merck grant to FLG, and a HSFO grant to PM. SPC is supported by a fellowship from the HSFO, OSCAR group grant. RSS is an MRC scholar.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Cregan, S., MacLaurin, J., Gendron, T. et al. Helper-dependent adenovirus vectors: their use as a gene delivery system to neurons. Gene Ther 7, 1200–1209 (2000). https://doi.org/10.1038/sj.gt.3301208
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3301208
Keywords
This article is cited by
-
Nuclear receptor NR5A2 controls neural stem cell fate decisions during development
Nature Communications (2016)
-
Safe, Long-term Hepatic Expression of Anti-HCV shRNA in a Nonhuman Primate Model
Molecular Therapy (2012)
-
Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)
Cell Biology and Toxicology (2010)
-
A rapid protocol for construction and production of high-capacity adenoviral vectors
Nature Protocols (2009)
-
Transcriptional activation of caspase-6 and -7 genes by cisplatin-induced p53 and its functional significance in cisplatin nephrotoxicity
Cell Death & Differentiation (2008)