Construction of Adenovirus Vectors with RGD-Modified Fiber for Transductional Targeting

Abstract

INTRODUCTIONTherapeutic gene transfer strategies aim to maximize gene transfer and expression in target cells. However, gene delivery systems often fail to preferentially transduce target cells in mixed cell populations. In addition, limitations in vector specificity can lead to transduction of non-target cells, resulting in untoward toxicity, even with compartmental dosing. Thus, vector optimization is critical for the development of efficient genetic experiments. The life cycle and biology of adenovirus (Ad) infection and transgene expression in cells have been thoroughly characterized. Ad infection is initiated by recognition of the native Ad5 receptor, coxsackievirus-adenovirus receptor (CAR), on target cells by the carboxy-terminal portion (i.e., knob) of the fiber protein. The development of genetically modified Ad vectors with transductional specificity for a single cell type requires “retargeting”: the ablation of endogenous tropism and the introduction of novel tropism determinants for target cells. This protocol describes the construction of chimeric fibers. Homologous recombination between the pAdEasy-1 and the fiber shuttle plasmid (pFiber-dE3-RGD) is used to generate a rescue plasmid with a mutant fiber that inserts Arg-Gly-Asp (RGD) at the carboxyl terminus of the fiber knob region. The resultant fiber-modified rescue plasmid (pAdEasy-RGD) is isogenic to AdEasy-1 (except the fiber region) and can therefore be used for another round of homologous recombination with pShuttle-promoter-Luc to make a final Ad vector construct that expresses the luciferase gene and contains an RGD-modified fiber knob. This construct can then be tested for targeting efficacy against cells expressing αv integrin cell surface receptors.