Protocol

Construction of Adenovirus Vectors for Transcriptional 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

The balance between target and nontarget cell toxicity determines the efficacy of any therapeutic agent. 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. While genetically modified Ad vectors with transductional specificity for a single cell type can be generated by direct or indirect modifications of the carboxyl terminus of the vectors’ receptor recognition site, Ad vectors can also be targeted at the level of protein expression of the transgene in the targeted cells (i.e., transcription/translation). Target-cell-specific gene expression is accomplished using tissue-specific promoters (TSPs), DNA elements that restrict expression to specific cellular subsets. The major drawback of transcriptional retargeting is that pathological change in target tissues or organs, such as degeneration or malignant transformation, can ectopically activate TSPs. In this protocol, recombinant Ad vectors that express the luciferase gene are constructed through homologous recombination in Escherichia coli. TSPs are placed in front of the luciferase gene for selective expression.