Abstract
Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, αvβ5- and αvβ3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was ‘trans-complementation’ of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.
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
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Bergelson JM et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5 Science 1997 275: 1320–1323
Habib NA, Hodgson HJ, Lemoine N, Pignatelli M . A phase I/II study of hepatic artery infusion with wtp53-CMV-Ad in metastatic malignant liver tumours Hum Gene Ther 1999 10: 2019–2034
Diaz RM et al. Rapid adenoviral transduction of freshly resected tumour explants with therapeutically useful genes provides a rationale for genetic immunotherapy for colorectal cancer Gene Therapy 1998 5: 869–879
Harris MP et al. Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein Cancer Gene Ther 1996 3: 121–130
van der Eb MM et al. Severe hepatic dysfunction after adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration Gene Therapy 1998 5: 451–458
Ohwada A, Hirschowitz EA, Crystal RG . Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver Hum Gene Ther 1996 7: 1567–1576
Tomko RP, Xu R, Philipson L . HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses Proc Natl Acad Sci USA 1997 94: 3352–3356
Hong SS et al. Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells EMBO J 1997 16: 2294–2306
McDonald D et al. Coxsackie and adenovirus receptor (CAR)-dependent and major histocompatibility complex (MHC) class I-independent uptake of recombinant adenoviruses into human tumour cells Gene Therapy 1999 6: 1512–1519
Wickham TJ, Mathias P, Cheresh DA, Nemerow GR . Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment Cell 1993 73: 309–319
Wickham TJ, Filardo EJ, Cheresh DA, Nemerow GR . Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization J Cell Biol 1994 127: 257–264
Leon RP et al. Adenoviral-mediated gene transfer in lymphocytes Proc Natl Acad Sci USA 1998 95: 13159–13164
Kaner RJ et al. Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the Coxsackie/adenovirus receptor Am J Respir Cell Mol Biol 1999 20: 361–370
Li Y et al. Loss of adenoviral receptor expression in humanbladder cancer cells: a potential impact on the efficacy of genetherapy Cancer Res 1999 59: 325–330
Miller CR et al. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer Cancer Res 1998 58: 5738–5748
Hemmi S et al. The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures Hum Gene Ther 1998 9: 2363–2373
Dmitriev I et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism J Virol 1998 72: 9706–9713
Roelvink PW et al. Identification of a conserved receptor-binding site on the fiber proteins of a CAR-recognizing adenoviridae Science 1999 286: 1568–1571
Kasono K et al. Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector Clin Cancer Res 1999 5: 2571–2579
Pearson AS et al. Factors limiting adenovirus-mediated gene transfer into human lung and pancreatic cancer cell lines Clin Cancer Res 1999 5: 4208–4213
Lanuti M et al. Use of protamine to augment adenovirus-mediated cancer gene therapy Gene Therapy 1999 6: 1600–1610
Hidaka C et al. CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts J Clin Invest 1999 103: 579–587
Li D, Duan L, Freimuth P, O'Malley BWJ . Variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer Clin Cancer Res 1999 5: 4175–4181
Marienfeld U et al. ‘Autoreplication’ of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes Gene Therapy 1999 6: 1101–1113
Dion LD et al. E1A RNA transcripts amplify adenovirus-mediated tumor reduction Gene Therapy 1996 3: 1021–1025
Dion LD, Goldsmith KT, Garver RIJ . Quantitative and in vivo activity of adenoviral-producing cells made by cotransduction of a replication-defective adenovirus and a replication-enabling plasmid Cancer Gene Ther 1996 3: 230–237
Goldsmith KT, Dion LD, Curiel DT, Garver RIJ . Trans E1 component requirements for maximal replication of E1-defective recombinant adenovirus Virology 1998 248: 406–419
Goldsmith KT, Curiel DT, Engler JA, Garver RIJ . Trans complementation of an E1A-deleted adenovirus with codelivered E1A sequences to make recombinant adenoviral producer cells Hum Gene Ther 1994 5: 1341–1348
Bischoff JR et al. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells Science 1996 274: 373–376
Heise C et al. ONYX-015, an EIB gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents Nature Med 1997 3: 639–645
Kirn D, Hermiston T, McCormick F . ONYX-015: clinical data are encouraging Nature Med 1998 4: 1341–1342
Motoi F et al. Effective gene therapy for pancreatic cancer by cytokines mediated by restricted replication-competent adenovirus Hum Gene Ther 2000 11: 223–235
Freytag SO et al. A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy Hum Gene Ther 1998 9: 1323–1333
Lochmuller H et al. Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 + delta E3) during multiple passages in 293 cells Hum Gene Ther 1994 5: 1485–1491
Fallaux FJ, van der Eb AJ, Hoeben RC . Who's afraid of replication-competent adenoviruses Gene Therapy 1999 6: 709–712
Horwitz MS, Brayton C, Baum SG . Synthesis of type 2 adenovirus DNA in the presence of cycloheximide J Virol 1973 11: 544–551
Spitz FR et al. In vivo adenovirus-mediated p53 tumor suppressor gene therapy for colorectal cancer Anticancer Res 1996 16: 3415–3422
Spitz FR et al. Adenoviral-mediated wild-type p53 gene expression sensitizes colorectal cancer cells to ionizing radiation Clin Cancer Res 1996 2: 1665–1671
Anderson SC et al. p53 gene therapy in a rat model of hepatocellular carcinoma: intra-arterial delivery of a recombinantadenovirus Clin Cancer Res 1998 4: 1649–1659
Wills KN et al. Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene Cancer Gene Ther 1995 2: 191–197
Christenson SD et al. Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue in mice Stroke 1998 29: 1411–1415; Discussion 1416
Lusky M et al. Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3 J Virol 1999 73: 8308–8319
Loser P, Jennings GS, Strauss M, Sandig V . Reactivation of the previously silenced cytomegalovirus major immediate–early promoter in the mouse liver: involvement of NFkappaB J Virol 1998 72: 180–190
Brough DE et al. Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo J Virol 1997 71: 9206–9213
Goldman MJ, Wilson JM . Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway J Virol 1995 69: 5951–5958
Chiu CY, Mathias P, Nemerow GR, Stewart PL . Structure of adenovirus complexed with its internalization receptor, alpha v beta5 integrin J Virol 1999 73: 6759–6768
Bewley MC et al. Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR Science 1999 286: 1579–1583
Gorman CM, Gies D, McCray G, Huang M . The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins Virology 1989 171: 377–385
Gambaryan S et al. Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells Proc Natl Acad Sci USA 1998 95: 9003–9008
Poller W et al. Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host Gene Therapy 1996 3: 521–530
Fechner H et al. Expression of Coxsackie adenovirus receptor and alpha v-integrin does not correlate with adenovector targeting in vivo indicating anatomical vector barriers Gene Therapy 1999 6: 1520–1535
Fechner H et al. Alpha-and delta-tocopherol induce expression of hepatic alpha tocopherol transfer protein mRNA Biochem J 1998 331: 577–581
Acknowledgements
We thank Kerstin Hinze for assistance with the cell culture experiments. KH was supported by a grant from the Freie Universität Berlin and will incorporate part of the data presented here in her doctoral thesis. This work was supported by Deutsche Forschungsgemeinschaft through a Heisenberg Fellowship to WP (WP378/2–1 and 378/2–2), by a grant from the Centeon Pharmaceutical Company, and by the Cardiovascular Research Center at the University Hospital Benjamin Franklin, Freie Universität Berlin. This work was also supported by grants from the NIH (AI35667 and HL54734), and by an Established Investigator Award from the American Heart Association to JB.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Fechner, H., Wang, X., Wang, H. et al. Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells. Gene Ther 7, 1954–1968 (2000). https://doi.org/10.1038/sj.gt.3301321
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3301321
Keywords
This article is cited by
-
Expression of the coxsackie adenovirus receptor in neuroendocrine lung cancers and its implications for oncolytic adenoviral infection
Cancer Gene Therapy (2013)
-
The roles of cell surface attachment molecules and coagulation Factor X in adenovirus 5-mediated gene transfer in pancreatic cancer cells
Cancer Gene Therapy (2011)
-
Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro
Virology Journal (2010)
-
Coxsackievirus and adenovirus receptor expression in human endometrial adenocarcinoma: possible clinical implications
World Journal of Surgical Oncology (2008)
-
Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban
Gene Therapy (2007)