Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12)

Devchand Paul, Muzaffar H. Qazilbash, Kaimei Song, Hui Xu, Birandra K. Sinha, Johnson Liu, Kenneth H. Cowan

Research output: Contribution to journalArticle

11 Citations (Scopus)

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 (104 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/106 cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-γ, 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.

Original languageEnglish (US)
Pages (from-to)308-315
Number of pages8
JournalCancer Gene Therapy
Volume7
Issue number2
DOIs
StatePublished - Jan 1 2000

Fingerprint

Dependovirus
Interleukin-12
Plasmids
Terminal Repeat Sequences
Interleukin-12 Subunit p35
Complementary DNA
Clone Cells
Inverted Repeat Sequences
Encephalomyocarditis virus
Rous sarcoma virus
Polyadenylation
Simian virus 40
Thymidine Kinase
Adenoviridae
Genetic Therapy
Virion
Antineoplastic Agents
Immunotherapy
Interferons
Virulence

Keywords

  • Adeno-associated virus
  • Gene transfer
  • Interleukin-12 vector

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Cancer Research

Cite this

Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12). / Paul, Devchand; Qazilbash, Muzaffar H.; Song, Kaimei; Xu, Hui; Sinha, Birandra K.; Liu, Johnson; Cowan, Kenneth H.

In: Cancer Gene Therapy, Vol. 7, No. 2, 01.01.2000, p. 308-315.

Research output: Contribution to journalArticle

Paul, Devchand ; Qazilbash, Muzaffar H. ; Song, Kaimei ; Xu, Hui ; Sinha, Birandra K. ; Liu, Johnson ; Cowan, Kenneth H. / Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12). In: Cancer Gene Therapy. 2000 ; Vol. 7, No. 2. pp. 308-315.
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AB - 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 (104 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/106 cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-γ, 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.

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