A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the Hl loop of their fiber knob

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However the method of constructing an Ad vector containing a foreign liaand in the HI loop of the fiber knob has proved difficult. In this study, eye developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp451 and/or Clal site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the A;a-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)containing peptide motif (as a model) and containing a Csp451 and/or;or Clal recognition site, were ligated into the Csp451 and/or Clal-digested plasmid. The foreign transgene expression cassette was inserted into the Ef deletion site of the vector plasmid and the fiber-mutant ad vector was produced by transfection of the Pacl-digested plasmid into 293 cells, The virus containing the RGB or NC;R peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated C,CAR-independent cell entry pathway: The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.