title: Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12).
abstract: IL-12 is a heterodimeric cytokine that is known to induce tumor regression and long-term antitumor immunity. Recombinant adeno-associated virus (rAAV) vectors are advantageous for gene therapy in that they lack pathogenicity in humans, infect dividing as well as nondividing cells, and show a broad range of infectivity. We constructed an rAAV vector expressing interleukin-12 (IL-12) for cancer immunotherapy studies in a mouse model by inserting murine IL-12 (mIL-12) p35 and p40 cDNAs into the plasmid pRep4 and inserting the encephalomyocarditis virus internal ribosomal entry site between the p35 and p40 cDNAs. The mIL-12 expression cassette containing the Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was subcloned into the AAV plasmid p008Sub/NeoR, which contains two AAV inverted terminal repeat sequences and the NeoR gene driven by the thymidine kinase promoter. rAAV virions (10(4) infectious particles/ml) were generated by cotransfection of rAAV-mIL-12 and a helper plasmid (pAAV/Ad) into 293 cells previously infected with adenovirus 5. After infection of D6 fibroblasts with rAAV-mIL-12, G418-resistant clones were isolated. Each of the 1D D6 clones isolated produced up to 5.2 ng/10(6) cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-gamma, enhanced lymphocyte proliferation, and cytotoxicity assays confirmed biologically functional IL-12 production by the vector. This is the first report indicating that an rAAV vector expresses mIL-12, which can be used to model the effects of mIL-12 alone and/or in combination with other antitumor agents.
authors.affiliation: Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: D
authors.initials: D
authors.lastname: Paul
authors.forename: M H
authors.initials: MH
authors.lastname: Qazilbash
authors.forename: K
authors.initials: K
authors.lastname: Song
authors.forename: H
authors.initials: H
authors.lastname: Xu
authors.forename: B K
authors.initials: BK
authors.lastname: Sinha
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: K H
authors.initials: KH
authors.lastname: Cowan
mesh_terms: D000818:Animals; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D018664:Interleukin-12 / Q000096:biosynthesis*; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D010835:Phytohemagglutinins / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D004274:DNA, Recombinant; D004279:DNA, Viral; D010835:Phytohemagglutinins; D018664:Interleukin-12; D007371:Interferon-gamma