Construction and Characterization of Adenovirus Vectors Encoding Aspartate-β-Hydroxylase to Preliminary Application in Immunotherapy of Hepatocellular Carcinoma

Abstract

Dendritic cells (DCs) harboring tumor-associated antigen are supposed to be a potential immunotherapy for hepatocellular carcinoma (HCC). Aspartate-β-hydroxylase (AAH), an overexpressed tumor-associated cell surface protein, is considered as a promising biomarker and therapeutic target for HCC. In this study, we constructed adenovirus vector encoding AAH gene by gateway recombinant cloning technology and preliminarily explored the antitumor effects of DC vaccines harboring AAH. Firstly, the total AAH mRNA was extracted from human HCC tissues; the cDNA was amplified by RT-PCR, verified, and sequenced after TA cloning. Gateway technology was used and the obtained 18T-AAH was used as a substrate, to yield the final expression vector Ad-AAH-IRES2-EGFP. Secondly, bone marrow-derived DCs were infected by Ad-AAH-IRES2-EGFP to yield AAH-DC vaccines. Matured DCs were demonstrated by increased expression of CD11c, CD80, and MHC-II costimulatory molecules. A dramatically cell-killing effect of T lymphocytes coculturing with AAH-DCs on HepG2 HCC cell line was demonstrated by CCK-8 and FCM assays in vitro. More importantly, in an animal experiment, the lysis effect of cytotoxic T lymphocytes (CTLs) on HepG2 cells in the AAH-DC group was stronger than that in the control groups. In conclusion, the gateway recombinant cloning technology is a powerful method of constructing adenovirus vector, and the product Ad-AAH-IRES2-EGFP may present as a potential candidate for DC-based immunotherapy of HCC.

Construction and characterization of an adenoviral vector encoding human bone morphogenetic protein-2

“…We constructed a recombinant adenoviral vector encoding BMP-2 using Gateway TM technology with significantly reduced production time and increased efficiency. 10,11 Our work makes high level gene expression of BMP-2 possible and lays the foundation for future applications of BMP-2 in gene therapies for orthopedic conditions including bone defects 12,13 as well as in bone tissue engineering. 14,15 Material and methods…”

Notch ligand-expressing adenovirus infection enhances the efficacy of dendritic cell-based immunotherapy for allergic asthma in mice

“…Additionally, DCs has been shown to be permissive of adenoviral (Ad) vector infection in vitro, and Ad vectors are a promising gene delivery platform for a variety of therapeutic and vaccine applications. 5 In this regard, overexpressing immunoregulatory proteins in Ad-modified DCs would be an effective way to suppress Th2 cell immunity and establish an antiallergic response.…”