Cells were labeled with mouse anti-CAR monoclonal antibody, mouse anti-human integrin v3 (LM609) antibody or mouse anti-human integrin v5 (P1F6) antibody. reduce liver tropism and increase gene transfer in low-CAR or CAR-deficient AKR1C3-IN-1 cancer cells following intravascular delivery. However , anti-tumor effects of the fiber-mutated Ad5 expressing HSV-TK under control of the hTERT promoter was not found when compared with an unmodified Ad5 vector in cancer lines expressing different levels of CAR, likely due to the activity of the hTERT promoter being lower than that of the CMV promoter. Nevertheless, this study describes an enhanced Ad5 vector for intravascular gene delivery, and further modifications such as changes in the promoter may facilitate the development of this vector for cancer treatment. Keywords: Adenovirus, gene therapy, coxsackie-adenovirus receptor, liver tropism == Introduction == Human adenoviruses (Ad) are currently the most widely used viral vectors in gene therapy clinical trials due to their well-characterized biology, large transgene capacity, ability to transfer genes to a wide variety of cell types, amplification to high titers and relatively safe profile for use in humans [1]. Currently, 57 human Ad serotypes are defined and distributed into seven species, A to G, of which the most commonly used for cancer gene therapy is species C adenovirus, HAdV-C5 (Ad5) [2]. Systemic or intravenous (IV) administration is the ideal mode of delivery for Ad5-based cancer therapy. However , effective IV administration of Ad5-based vectors has been hindered by the innate liver tropism of Ad5 and concerns of hepatotoxicity. Liver tropism causes the Ad5 vector to be diverted away from the desired tumor targets and to be rapidly cleared from the body, thereby necessitating higher doses of administered vector and resulting in poor antitumor efficacy [3]. The mechanisms of systemic Ad supervision that leads to efficient liver tropism and transduction remain controversial. Infectivity of Ad5, at leastin vitro, is known to be mediated via a primary cell tethering interaction between the Ad5 fiber knob domain and the coxsackie and adenovirus receptor (CAR) [4], followed by a secondary, endocytosis-stimulating interaction between Arg-Gly-Asp (RGD) tri-peptide motifs in the Ad penton-base protein and v3 and v5 cellular integrins [5]. In adult humans, CAR is highly expressed in different tissues, including the liver. Its distribution is comparable to that in adult mice, AKR1C3-IN-1 except that higher levels are expressed in the mouse liver. However , the introduction of point mutations in the Ad5 fiber knob domain to abrogate CAR binding was shown to have a profound impact on transduction efficiencyin vitro, but it did not necessarily have an apparent effect on levels of liver transduction when introduced systemicallyin vivo[6, 7]. Ablating Ad5 liver tropism only partially contributes to improving the tumor targeting of vectors. In this context, Ad5-based vectors selectively transduce target cells via binding of the Ad fiber protein to CAR. However , CAR is poorly expressed on many tumor targets, thereby limiting transduction [8]. Prior Rabbit polyclonal to AACS studies showed that RGD-integrin interactions could be used as an alternative infection pathway and could dramatically improve the ability of the Ad vector to transduce several types of cells [9]. In a previous study, we constructed a fiber-modified Ad5 containing an RGD motif in the HI-loop and demonstrated that it could enhance anti-tumor effectsin vitroandin festn[10]. In the current study, a modified vector was made using this developed system for rapid construction of recombinant fiber-modified Ad5 vectors, which can be generated by in vitro ligation and easily monitored by a reporter gene in vitro and in vivo. This novel Ad5 vector that contains two amino acid mutations in the AB loop of the fiber-modified Ad5 fiber knob with the aim of reducing liver tropism and increasing the anti-tumor efficacy of the vector in CAR-deficient cancers. == Materials and methods == == Cell culture == Human embryonic kidney HEK293 cells AKR1C3-IN-1 were purchased from the American Type Culture Collection (Manassas, VA, USA). HL7702 (human normal liver), A549 (human lung adenocarcinoma), Caco-2 (human colorectal adenocarcinoma) and Skov3 (human ovarian cancer) cells.