MODIFICATION OF THE ADENOVIRUS FIBER TO ALTER THE TARGET RECEPTOR SPECIFICITY

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Current use of adenovirus as a gene therapy vector for treatment of cystic fibrosis (CF) is based on its high affinity binding and subsequent efficient uptake into airway epithelial cells. Adenovirus binding is mediated by an undefined cell surface receptor (Adv-R) which is believed to bind the C terminal portion of the adenovirus fiber polypeptide. A combination of fiber gene organization and fiber protein structural elements make genetic manipulation of the fiber polypeptide a particularly interesting and potentially very useful scientific endeavor for the purpose of gene therapy. Using a mutagenesis protocol which is directed towards the fiber gene, we are going to i). identify the general receptor ligand binding domain of fiber and ii). Target adenovirus in a cell specific manner through a modified fiber ligand which binds via a designated cell surface receptor. The strategy we will employ begins with insertional mutagenesis of fiber, using an inframe insertion of a hexapeptide cassette throughout the gene. Insertion of the cassette will give some information about functional domains, but it's primary purpose is not to disrupt fiber function. The peptide cassette contains a protease cleavage site recognized by thrombin. Using constructs generated in this manner, we will be able to study the effects of the mutations after we have generated the intact virus. This strategy will allow us to focus on mutations which affect the virus domains responsible for attachment to the receptor. The system as it is developed will allow us to use the specified protease to displace the ligand binding domain , resulting in a virus which is null for cell attachment. Using the fiber defective virus, we will develop suitable chemistry for the covalent coupling of distinct ligand moieties to the defective virus creating a virus which will bind through an altered ligand receptor interaction. For certain well defined receptor ligands, we will modify the fiber polypeptide by genetic engineering and create a chimeric fiber with altered receptor affinity. Carry out appropriate biological characterization of the chimeric fiber viruses. At the most basic level, these studies will expand our knowledge of the fiberligand binding domain and it's attachment to the cell receptor Adv-R. The proposed studies should also result in the production of a safer and more effective adenovirus vector to be used in cystic fibrosis somatic cell gene therapy and finally but possibly of greatest importance, these studies should create a new adenovirus vector system which can be used for tissue specific targeting of genes with the intention of correcting tissue specific pathology.