Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

Document Type : Original Article


1 Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran


Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases.
Materials and Methods
In brief, IL-4 coding sequence was amplified by PCR and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-' plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein.
Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell.
This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.


1.Kozarsky KF, Wilson JM. Gene therapy: adenovirus vectors. Curr Opin Genet Dev 1993; 3:499-503.
2.Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol 1999; 10:440¬447.
3. Yokota T, Otsuka T, Mosmann T, Banchereau J, DeFrance T, Blanchard D, et al. Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities. Proc Natl Acad Sci USA 1986; 83:894-898.
4.Whitehead RP, Lew D, Flanigan RC, Weiss GR, Roy V, Glode ML, et al. Phase II trial of recombinant human interleukin-4 in patients with advanced renal cell carcinoma: a southwest oncology group study. J Immunother 2002; 25:352-358.
5. Sutherland GR, Baker E, Callen DF, et al. Interleukin 4 is at 5q31 and interleukin 6 is at 7p15. Hum Genet 1988; 79:335-337.
6.Cook WJ, Ealick SE, Reichert P, Hammond GS, Le HV, Nagabhushan TL, et al. Crystallization and preliminary X-ray investigation of recombinant human interleukin-4. J Mol Biol 1991; 218:675-678.
7. Keane-Myers A, Maliszewski CR, Finkelman FD, Nickell SP. Recombinant IL-4 treatment augments resistance to Borrelia burgdorferi infections in both normal susceptible and antibody-deficient susceptible mice. J Immunol 1996; 156:2488-2494.
8.Chen YX, Wang LX, Tang LF, Zhang SK, Zhang J, Zeng XF, et al. Boost effect of recombinant IL-4 on protection of Schistosoma japonicum cathepsin B DNA vaccine in mice against the parasite. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 2005; 23:65-68.
9.Joshi BH, Leland P, Asher A, Prayson RA, Varricchio F, Puri RK . In situ expression of interleukin-4 (IL-4) receptors in human brain tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary glioblastoma cell cultures. Cancer Res 2001; 61:8058-8061.
10.Kay NE, Bone ND, Lee YK, Lee YK, Jelinek DF, Leland P, et al. A recombinant IL-4-Pseudomonas exotoxin inhibits protein synthesis and overcomes apoptosis resistance in human CLL B cells. Leuk Res 2005; 29:1009-1018.
11.Rand RW, Kreitman RJ, Patronas N, Varricchio F, Pastan I, Puri RK.. Intratumoral administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma. Clin Cancer Res 2000; 6:2157-2165.
12.Puri R, Hoon D, Leland P, Snoy P, Rand RW, Pastan, et al. Preclinical development of a recombinant toxin containing circularly permuted interleukin 4 and truncated Pseudomonas exotoxin for therapy of malignant astrocytoma. Cancer Res 1996; 56:5631-6537.
13.Casolaro V, Keane-Myers AM, Swendeman SL, Steindler C, Zhong F, Sheffery M, et al. Identification and characterization of a critical CP2-binding element in the human interleukin-4 promoter. J Biol Chem 2000; 275:36605-36611.
14.Lukacs NW, Addison CL, Gauldie J, Graham F, Simpson K, Strieter RM, et al. Transgene-induced production of IL-4 alters the development and collagen expression of T helper cell 1-type pulmonary granulomas. J Immunol 1997; 158:4478-4484.
15.Ritter T, Vogt K, Rieck P, Schilling-Schon A, Kolls J, Hartmann C, et al. Adenovirus-mediated gene transfer of interleukin-4 to corneal endothelial cells and organ cultured corneas leads to high IL-4 expression. Exp Eye Res 1999; 69:563-568.
16.Mobasheri MB, Modarressi MH, Shabani M, Asgarian H, Sharifian RA, Vossough P. Expression of the testis- specific gene, TSGA10, in Iranian patients with acute lymphoblastic leukemia (ALL). Leuk Res 2006; 30:883-889.
17.He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998; 95:2509-2514.
18.Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. New York: Cold spring harbour press; 2001.
19.Heiman M. webcutter. 1997 [cited 2007 April 19]; Available from:
20.Stoppacciaro A, Paglia P, Lombardi L, Parmiani G, Baroni C, Colombo MP. Genetic modification of a carcinoma with the IL-4 gene increases the influx of dendritic cells relative to other cytokines. Eur J Immunol 1997; 27:2375-2382.
21.Okada H, Lieberman FS, Edington HD, Witham TF, Wargo MJ, Cai Q, et al. Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of recurrent glioblastoma: preliminary observations in a patient with a favorable response to therapy. J Neuro-oncol 2003; 64:13-20.
22.Okada H, Villa L, Attanucci J, Witham TF, Wargo MJ, Cai Q, et al. Cytokine gene therapy of gliomas: effective induction of therapeutic immunity to intracranial tumors by peripheral immunization with interleukin-4 transduced glioma cells. Gene Ther 2001; 8:1157-1166. 
23.Sacco M, Benedetti S, Cato EM, Caniatti M, Ceruti R, Scanziani E, et al. Retrovirus-mediated IL-4 gene therapy in spontaneous adenocarcinomas from MMTV-neu transgenic mice. Gene Ther 1999; 6:1893-1897.
24.Okada H, Giezeman-Smits KM, Tahara H, Attanucci J, Fellows WK, Lotze MT, et al. Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSVtk tumor vaccine. Gene Ther 1999; 6:219-226.
25.Kluth DC, Ainslie CV, Pearce WP, Finlay S, Clarke D, Anegon I, et al. Macrophages transfected with adenovirus to express IL-4 reduce inflammation in experimental glomerulonephritis. J Immunol 2001; 166:4728¬4736.
26.Woods JM ,Katschke KJ, Volin MV. IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis. J Immunol 2001; 166:1214-1222.
27.Pousset F, Cremona S, Dantzer R. IL-10 and IL-4 regulate type-I and type-II IL-1 receptors expression on IL-1 beta-activated mouse primary astrocytes. J Neurochem 2001; 79:726-736.