Biomedical subjects

George T Mercier

Publications and source records attributed to George T Mercier.

3 recordsLinked to original sources

PubMed2004

A chimeric adenovirus vector encoding reovirus attachment protein sigma1 targets cells expressing junctional adhesion molecule 1.

The utility of adenovirus (Ad) vectors for gene transduction can be limited by receptor specificity. We developed a gene-delivery vehicle in which the potent Ad5 vector was genetically reengineered to display the mucosal-targeting sigma1 protein of reovirus type 3 Dearing (T3D). A sigma1 construct containing all but a small virion-anchoring domain was fused to the N-terminal 44 aa of Ad5 fiber. This chimeric attachment protein Fibtail-T3Dsigma1 forms trimers and assembles onto Ad virions. Fibtail-T3Dsigma1 was recombined into the Ad5 genome, replacing sequences encoding wild-type fiber. The resulting vector, Ad5-T3Dsigma1, expresses Fibtail-T3Dsigma1 and infects Chinese hamster ovary cells transfected with human or mouse homologs of the reovirus receptor, junctional adhesion molecule 1 (JAM1), but not the coxsackievirus and Ad receptor. Treatment of Caco-2 intestinal epithelial cells with either JAM1-specific antibody or neuraminidase reduced transduction by Ad5-T3Dsigma1, and their combined effect decreased transduction by 95%. Ad5-T3Dsigma1 transduces primary cultures of human dendritic cells substantially more efficiently than does Ad5, and this transduction depends on expression of JAM1. These data provide strong evidence that Ad5-T3Dsigma1 can be redirected to cells expressing JAM1 and sialic acid for application as a vaccine vector.

Adenoviridae↗

PubMed2003

Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification.

Development of cell-targeting vectors is an important focus for gene therapy. While some ligands can be genetically inserted into virus capsid proteins for cell targeting, for many ligands, this approach can disrupt either ligand function or vector function. To address this problem for adenovirus type 5 vectors, the fiber capsid protein was genetically fused to a biotin acceptor peptide (BAP). Adenovirus particles bearing this BAP were metabolically biotinylated during vector production by the endogenous biotin ligase in 293 cells to produce covalently biotinylated virions. The resulting biotinylated vector could be retargeted to new receptors by conjugation to biotinylated antibodies using tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). Finally, this vector was used as a ligand screening platform for dendritic cells in which a variety of structurally diverse protein, carbohydrate, and nucleic acid ligands were easily added to the vector using the biotin-avidin interaction. This work demonstrates the utility of metabolically biotinylated viruses for ligand screening, vector targeting, and virus purification applications.

Adenoviridae↗

PubMed2003

Biotinylated gene therapy vectors.

The avidin-biotin system is a fundamental technology in biomedicine for immunolocalisation, imaging, nucleic acid blotting and protein labelling. This technology has recently been adapted for use in gene therapy vector applications to add proteins or cell-targeting ligands to non-viral and viral vectors. Two biotinylation technologies are being used in these applications: chemical biotinylation and metabolic biotinylation. In chemical biotinylation, reactive alkylating agents couple biotin to proteins by random covalent attachment to amino acid side chains. In metabolic biotinylation, proteins are genetically engineered with a biotin acceptor peptide (BAP), such that they are covalently biotinylated by cellular biotin ligases during viral vector production. Both technologies show promise for cell-targeting in vitro and in vivo, and for ligand screening applications. Metabolic biotinylation has the added feature of allowing viruses, vectors and vaccines to be produced from cells already biotinylated, thereby allowing them to purified by affinity chromatography on monomeric avidin columns.

Animals↗