Knobless Viruses and Other Capsid Proteins for Targeting

Structural conflicts emerging from knob modifications and the observation that fiber-deleted Ad vectors could be produced (66,67) provided the conceptual basis for replacing the native fiber with a chimeric knobless fiber, which would result in ablation of CAR binding. Simultaneous addition of a targeting ligand to the knobless fiber would result in a more specifically targeted Ad. The technical barrier to this approach is the innate trimerization function of the knob, required for proper fiber function and capsid assembly. A solution was devised by using the T4 bacteriophage fibritin protein for trimerization, while proof-of-concept targeting was achieved with a six-his-tidine (6-His) moiety and cells expressing an artificial 6-His receptor (68). A similar strategy was employed whereby the staphylococcal protein A allowed trimerization and subsequent utilization of an integrin-binding RGD motif or proof-of-concept sFv-mediated targeting (69,70). These approaches could be extremely valuable if they become fully applicable to versatile targeting moeties, such as sFvs, in vivo.

An exciting new approach to Ad targeting is incorporation of targeting proteins into other capsid proteins such as pIII and pIX, which have an accessory function in capsid assembly but have access to the surface of the virion and may thus be usable for incorporation of targeting moieties (71). Also, given that adapter molecules can change the biodistribution of Ad, perhaps alteration of length of the fiber could achieve the same. This hypothesis was tested with promising results, although packaging was compromised with longer shaft extensions (72,73).

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