Capsomer-specific fluorescent labeling of adenoviral vector particles allows for detailed analysis of intracellular particle trafficking and the performance of bioresponsive bonds for vector capsid modifications.

Adenoviral (Ad) vectors are widely used for gene therapy approaches. Because of the high abundance of the natural adenoviral receptors (coxsackievirus-adenovirus receptor and integrins) on a wide variety of cells, numerous methods have been developed to redirect the virions to specific receptors on target cell surfaces. Importantly, an increasing number of publications have shown that the success of targeting not only depends on receptor binding and cellular uptake, but also on intracellular trafficking processes. Therefore, improved knowledge about the intracellular fate of targeted Ad vector particles is mandatory for a rational design of targeted Ad vectors. However, the technologies currently available for fluorescent labeling of Ad vectors have significant limitations: (1) at present capsids are labeled all over the particle surface, and this imposes the risk of interference with particle infectivity; (2) capsomer-specific labeling requires extensive genetic modifications and has been demonstrated only at protein IX; and (3) two-color labeling approaches are not available. Here we present a novel, robust, and straightforward labeling procedure that overcomes these limitations. It allows for specific labeling of the capsomer's fiber, protein IX, or hexon and permits two-color labeling. We demonstrate the potential of this labeling technology by analyzing two different bioresponsive bonds that can be used for the attachment of shielding or targeting moieties to the capsids: disulfide and hydrazone bonds. We demonstrate that in contrast to disulfide bonds, hydrazone bonds are quickly hydrolyzed after uptake of the virions and are thus favorable for the generation of bioresponsive vectors.