Protocol

Construction of Fusion Proteins for Transductional Targeting

Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

Therapeutic 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 nontarget 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. One strategy for accomplishing such retargeting does so indirectly, by using bifunctional adapter molecules. One element of the bispecific adapter binds to the Ad fiber knob, blocking its interaction with CAR and, thus, its native tropism. The second component of the adapter introduces specificity for the target cells and is chemically or genetically conjugated to the knob-binding portion. Recombinant fusion proteins offer a number of technological advantages over chemical conjugates, including simplified production and purification. In this protocol, a recombinant fusion protein is generated that ablates the vector’s native tropism by blocking the CAR receptor and retargets the vector to cells expressing epidermal growth factor (EGF).