382. The Polysaccharide Mannan as a Novel Chemical Coupling Partner for Adenovirus Vectors with a Potential Use for Genetic Vaccination
Authors: Sigrid Espenlaub, Andreas Wortmann, Tatjana Dick, Stefan Kochanek, Florian Kreppel
Journal: Molecular Therapy (2006)

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Safe and successful genetic vaccination with adenovirus gene transfer vectors requires specific and efficient transduction of antigen- presenting cells (APCs) in vivo. Due to the high promiscuity of adenovirus and the presence of its natural receptor CAR on a wide variety of cells there is a strong demand for vectors which on the one hand are efficiently detargeted from their natural receptor and on the other hand possess specificity for endocytic receptors on antigen- presenting cells like dendritic cells. However, current genetic and chemical de- and retargeting strategies suffer from significant disadvantages. For example, genetically detargeted vectors are often difficult to produce and only a very limited number of peptidic ligands can be incorporated into the particles. Chemical detargeting strategies based on polymers like PEG and HPMA frequently fail to produce retargeted particles. Here we propose to use high molecular weight polysaccharide mannan as a polymer for chemical detargeting.

We coupled mannan from S. cerevisiae onto the Ad vector particle surface by reductive amination. Mannan was partially oxidized and the resulting carbonyl groups were coupled to the amine groups on the vector particle surface under formation of Schiff bases. Subsequently, the Schiff bases were reduced to amine bonds which covalently linked the polysaccaride moiety to the vector particle surface. We carefully analyzed each step of the amination process for interference with physical or biological vector particle integrity and we established a protocol that allows for reductive amination maintaining particle integrity. SDS-PAGE analysis of the mannan- modified vector particles suggested highly efficient mannanylation of the vector surface. Photon correlation spectroscopy revealed that no particle aggregates had formed during the amination process.

The mannanylated particles were tested for infectivity in vitro on the high and low CAR-expressing cell lines A549 and K562, respectively. Flow cytometric analysis of transgene expression (EGFP) after transduction with different MOIs revealed that, compared to unmodified controls, efficient detargeting of the mannanylated vectors was achieved. At the vector genome level this finding was supported by a DNA-based slot-blot assay. The detargeting efficiency was correlated with the extent of surface mannanylation. Using a fluorescamine-based assay we could show that modification of 25% of the amine groups on the vector particle surface was sufficient to detarget the vector particles 30-fold.

To analyze whether mannan-detargeted particles were still capable to induce immune responses against the transgene in vivo we subcutaneously injected 5E+08 particles of a mannanylated vector coding for EGFP in Balb/c mice. Four weeks after injection a surprisingly strong cellular immune response against the transgene was detected.

In summary, we could efficiently couple mannan to Ad vectors by reductive amination resulting in an efficient detargeting. Furthermore, the detargeted particles retained their potential to induce cytotoxic T cell responses against the transgene in vivo. This renders them potential candidates for vaccination.