Genetic Targeting of Adenovirus Vectors Using a Reovirus σ1-Based Attachment Protein

Abstract

Targeting adenovirus vectors (AdV's) for selective transduction of specific cell types requires ablation of native adenovirus tropism and introduction of a unique target-binding moiety. To bring these requirements within reach, we developed a novel strategy to target AdV's genetically that relies on replacement of the entire adenovirus fiber protein with a fusion molecule comprising the virion-anchoring domain of fiber and the oligomerization domain of reovirus attachment protein sigma1. The chimeric molecule forms trimers, is transported to the nucleus, and assembles onto the adenovirus capsid. In contrast to previously reported genetically targeted vectors, the AdV presented herein propagates efficiently without a requirement for complementing fiber. Due to ablation of the native adenovirus tropism, the infectivity of this AdV was at least 35-fold reduced on 293 cells. Importantly, a His tag incorporated into the chimeric attachment protein conferred His-tag-dependent tropism to the AdV, which resulted in a 12- to 40-fold greater transduction efficiency on two different cell lines expressing a His-tag-binding receptor. In addition, the infection efficiency was strongly reduced by preincubation with a His-tag-specific Ab. Thus, this sigma1-based chimeric attachment molecule provides a promising new platform for the generation of truly targeted AdV's.

Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

“…The Ad fiber has also been replaced by the structurally similar reovirus σl protein [Chappell et al, 2002], resulting in a chimeric vector that could transduce cells through the reo virus receptors JAM1 and sialic acid [Mercier et al, 2004]. These fiber replacement strategies have proven useful as scaffolds for the display of various targeting ligands like small peptide ligands [Schagen et al, 2006], S. aureus protein A domains for subsequent display of immunoglobulins [Henning et al, 2005], human CD40L , and stabilized single chain antibodies, engineered to properly fold in the reducing environments of the cytoplasm and nucleus . Other reports of "knobless" vectors whereby the knobs are proteolylically removed after vector production, thereby exposing cell targeting domains have also been described [Hong et al, 2003].…”

Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24

“…In these approaches, the knob of the Ad5 fiber is deleted and trimerization of the knobless protein restored by fusing it with a trimerization moiety, such as the coiled-coil domain of the retrovirus envelope glycoprotein (53), the neck region peptide of human lung surfactant D (32,48), or reovirus sigma-1 protein (35,47,52).…”

Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

“…This position was chosen since, in one study, an hexahistidine tag was already reported to be functional at this position . Also, when a chimeric protein between the adenovirus fiber protein and σ1 is added to adenovirus virions, the presence of both hexahistidine and myc tag at its carboxyl-terminal end still allowed cell binding by the σ1 moiety (Mercier et al, 2004;Schagen et al, 2006;Tsuruta et al, 2005;. In the present study, the hexahistidine epitope (6H) was first used and, as a further proof that different epitopes can be accommodated at this position, the more complex HA epitope, YPYDVPDYA (Kolodziej and Young, 1991;Wilson et al, 1984), was examined in parallel (Fig.…”