Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Abstract

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues

“…Even so, only sbAb enhanced virus infection of cells expressing the targeted receptor. Thus, proving that the nature of the ligand can significantly affect vector targeting as already had been observed with fiber and hexo (Poulin et al, 2010).…”

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

“…hdAd⌬28E4 is the parental vector from which hdAd⌬28lacZ was derived and lacks a transgene. hdAd-PGKmSEAP contains the murine secreted alkaline phosphatase cDNA (12,36) under regulation by the murine phosphoglycerate kinase promoter (41,56). hdAd-PGK-mSEAP, hdAd-lacZ, and hdAd⌬28E4 were amplified with the helper virus AdNG163 (46) or AdJR46 using 116 cells and standard techniques (46,49).…”

A reduction in the human adenovirus virion size through use of a shortened fibre protein does not enhance muscle transduction following systemic or localised delivery in mice

“…Dmitry Shayakhmetov and Andre Lieber (University of Washington) (Shayakhmetov and Lieber, 2000). The modified fibre gene was subcloned into an E1/E3-deleted Ad genomic plasmid, pRP2014 (Christou and Parks, 2011;Poulin et al, 2010), and a cytomegalovirus immediate early enhancer/promoter-lacZ expression cassette (pCA38, (Addison et al, 1997)) was introduced to replace the E1-deletion, using RecA-mediated recombination (Chartier et al, 1996), as detailed previously (Willemsen, 2009), and designated Ad5SlacZ. To introduce the poly-lysine motif into the H-I loop of the knob domain of the short fibre, a BglII/PstI fragment containing part of the knob domain was first subcloned into pSP72 (Promega).…”