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Retargeting of Helper-Dependent Adenovirus by Modification of the Fiber with Metabolic Biotinylation

技術優勢

1) Efficient in vitro and in vivo targeting of different cell types and tissues depending only availability of monoclonal antibody to cell type surface protein2) Ability to utilize HD-Ad system to delivery up to 36 kb of transgenic DNA3) Low immunogenicity of HD-Ad because of absence of Adenovirus proteins4) Detargeting from natural Adenovirus target tissues and cells that depend on Coxackie Adenovirus Receptor (CAR) uptake

詳細技術說明

None

*Abstract

In order to enhance the potential of helper-dependent or gutted adenoviral vectors (HD-Ad) to transduce different cell types, the developers constructed a novel metabolically biotinylated Helper Virus (Fib2102) to package HD-Ad vectors. 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. The developers modified the c-terminus of the Ad5 fiber by addition of a 70 amino acid biotin acceptor peptide (BAP) into the helper virus backbone by homologous recombination. Adenovirus particles bearing the BAP are metabolically biotinylated during vector production by the endogenous producer cell biotin ligase to produce covalently biotinylated virions. The resulting biotinylated vectors can be used to transduce different cell type receptors by conjugation to specific biotinylated antibodies or ligands using a multivalent neutravidin linker.In one example, biotinylated HD-Ad carrying the LacZ reporter gene was coupled to ╬▒10 integrin specific antibodies. Fiber-modified HD-Ad coupled to the chondrocyte specific ╬▒10 integrin antibody was more efficient at transducing chondrocytes in vitro than vectors bearing wild type fiber. Moreover intra-articular injection of this vector in C57BL/6 mice allows efficient transduction of articular chondrocytes. Furthermore, the ╬▒10 integrin-LacZ complexed vector was injected into the intervetebral disc of a pig and assayed for ╬▓-Gal expression in the disc.This 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 as well as in vivo. Any transgene can be easily expressed in any cell type by growing any HD-Ad vector with the modified helper virus. This system is suitable for both clinical and research purposes. In one exemplary application, the developers retargeted HD-Ad to transducer cartilage both in vitro and in vivo for the first time.

國家/地區

美國