Abstract
Developing cell-targeting vectors is an important goal in gene therapy. Metabolic biotinylation of first-generation adenoviral vectors for cell targeting has already been shown. However, there are several advantages in using helper-dependent adenoviral vectors (HD-Ad) instead of first-generation vectors; HD-Ad contain only the noncoding termini of the viral genome, can deliver large DNA fragments of up to 36 Kb into target cells, elicit reduced toxicity and generate prolonged transgene expression in vivo. To enhance the potential of HD-Ad to transduce different cell types, we constructed a novel metablically biotinylated Helper Virus (Fib2102) to package HD-Ad vectors. Thus, co-infection of a helper-dependent packaging cell line with the fiber-modified helper virus and various HD-Ad constructs would allow the production of fiber-modified HD-Ad expressing different transgenes, obviating the requirement to fiber-modify each individual transgene-expressing HD-Ad. To this end, we modified the c-terminus of the Ad5 fiber by addition of a 70 amino acid biotin acceptor peptide (BAP) in our helper virus backbone by homologous recombination. Adenovirus particles bearing the BAP were metabolically biotinylated during vector production by the endogenous biotin ligase to produce covalently biotinylated virions. The resulting biotinylated vectors can subsequently be used to transduce different cell type receptors by conjugation to specific biotinylated antibodies. In particular, we tested whether a biotinylated HD-Ad generated with this system expressing the LacZ transgene could transduce chondrocytes in vitro. We found that a fiber-modified HD-Ad coupled to the chondrocyte specific α10 integrin antibody was more efficient at transducing a chondrocyte cell line than vectors bearing wild type fiber. In summary, we show the novel construction of a fiber-modified helper adenovirus which can be used to propagate high titers of fiber-modified HD-Ad, which, when coupled to cell-specific antibodies, results in improved transgene expression in vitro. This study demonstrates progress in retargeting strategies for helper-dependent vectors.