24. Context-Specific Peptide-Presenting Phage Libraries for Adenoviral Vector Targeting

Abstract

Top of pageAbstract Adenoviruses have developed as attractive method for gene delivery due to their ability to transduce a wide variety of dividing and non-dividing cells. However, their ability to successfully target a given cell type is limited by its evolution to infect a multitude of cell types. As one approach to develop more specific vectors, peptide-presenting phage libraries have been utilized to identify cell-binding peptides to be engineered into gene therapy vectors for retargeting. However, incorporation of selected peptide ligands from the structural context of phage into the differing structural context of a viral capsid protein can ablate the function of the ligand for targeting or disrupt viral assembly and function. In order to circumvent this ligand compatibility problem, we have engineered a |[ldquo]|context-specific|[rdquo]| peptide-presenting phage library. In this library, we have displayed the H and I sheets from the fiber protein of adenovirus type 5 (Ad5) on the pIII protein of fd bacteriophage. A twelve amino acid (12-mer) random peptide library was inserted between the H and I sheets on phage. This 10e9 member library was used for peptide selection against C2C12 mouse skeletal muscle cells. Five rounds of selection combined with four rounds of clearing on non-target cells selected one primary peptide designated 12.51, which bound target C2C12 cells approximately 100-fold better than the control RGD peptide. Translation of 12.51 back into the fiber protein produced a ligand-modified adenoviral vector that mediated 17-fold better transduction of target C2C12 cells. Preliminary data also suggest that 12.51 may exhibit tropism towards human skeletal muscle cells. In summary, we demonstrate proof of principle for the use of relatively large repertoire |[ldquo]|context-specific|[rdquo]| peptide-presenting phage libraries as a potential approach to generate and identify ligands that are compatible when incorporated back into the viral capsid for retargeting.