30. AdenoLibrary for Construction and Selection of Targeted Adenoviruses

Top of pageAbstract Vector systems derived from the human adenovirus serotype 5 (Ad5) have shown promise as gene delivery vehicles for a variety of clinical applications. However, the paucity of the Ad5 primary receptor (coxsackie and adenovirus receptor, CAR) on target cells of interest has rendered many tissues refractory to Ad-mediated transduction. Additionally, this endogenous tropism may potentially lead to undesired transduction of tissues expressing CAR. In view of this, strategies have been proposed to modify Ad5 to achieve targeted gene delivery, by ablating native tropism while redirecting Ad to target cell markers of interest. Such strategies involve modification of the trimeric capsid fiber ligands tend to lose specificity and/or affect virion integrity. As an alternative, several so-called knob-less fiber platforms have been utilized. These protein, whose C-terminal knob domain has been shown to dictate the native tropism associated with Ad5. While approaches employing genetic fusion of simple targeting ligands to the fiber have shown promise in retargeting, in many cases, the incorporated modifications are based on the concept of replacing the native fiber with an alternative protein capable of providing the trimerization functions that the knob usually confers. Such a strategy would accrue the additional benefit of an expanded repertoire of incorporable ligands, based on removal of the structural constraints imposed by the fiber knob. Previously, our group has utilized the bacteriophage T4 fibritin protein for fiber replacement, with subsequent display of complex targeting ligands providing for expanded tropism. However, vectors employing these knob-less fiber platforms often exhibit poor capsid incorporation of the alternate fibers in comparison to that of wild-type Ad5 fiber, which consequently leads to limited gene transfer efficacy and difficulty in vector propagation. In view of these obstacles, we have hypothesized that direct analysis of modified adenoviruses during their rescue/propagation is the only straightforward approach that guarantees the selection of functional, targeted vectors with optimized structural integrity and targeting efficiency. Herein, we describe a new schema for Ad vector construction, which allows for the selection of vectors with the optimal modified capsid configuration at the level of viral rescue. Based on our first generation fiber-fibritin molecule, several new fibers employing variant amounts of fibritin and Ad5 fiber shaft were analyzed via this library selection schema. Our selected variant, composed of the entire Ad5 fiber shaft connected to 12th coiled-coil segment of fibritin, is capable of efficient fiber incorporation, ligand display, and CAR-independent transduction. Furthermore, we have concluded that various targeting ligands require individual optimization, which may be accomplished by utilizing this adenoviral library approach.