Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

Abstract

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

“…Besides its role as a structural protein, it is a transcriptional activator (Rosa-Calatrava et al, 2001;Sargent et al, 2004a) and reorganizes promyelocytic leukaemia (PML) nuclear bodies (Rosa-Calatrava et al, 2003) (reviewed by Parks, 2005). Protein IX has been extensively studied as a platform to express foreign peptides on the surface of the capsid (Dmitriev et al, 2002;Le et al, 2004;Li et al, 2005;Meulenbroek et al, 2004;Vellinga et al, 2004Vellinga et al, , 2007. Since protein IX has been so well characterized, we selected it as a fusion partner to test whether 2A sequences could be used to express foreign genes in adenoviruses.…”

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice

“…This can be avoided by genetic approaches that incorporate targeting peptides into viral capsid components, including the fiber, hexon and pIX proteins. 31,32,37,[70][71][72][73][74][75][76] Targeting ligands fused to hexon and pIX proteins are incorporated near the faces of the capsid rather than on the protruding fiber, which may reduce their ability to interact with cell surface receptors. However, Poulin et al 77 recently constructed a genetic fusion of pIX with a single-domain antibody against CD66c, which enhanced vector targeting to CD66c-positive cells, demonstrating that pIX may be a good target for large genetic modifications.…”

Selective induction of tumor-associated antigens in murine pulmonary vasculature using double-targeted adenoviral vectors

“…As a first proof of principle, we demonstrated succesful retargeting of Ad vectors to CEA in the pulmonary vasculature utilizing a bifunctional adapter molecule, comprised of the ectodomain of CAR and a single-chain antibody recognizing CEA (sCAR-MFE). Other more advanced retargeting paradigms comprise genetic engineering of capsid proteins such as fiber, 17,18 hexon, 19 or pIX, 20 which can now be compared for their targeting capabilities in an in vivo system. Additionally, this animal model may accurately parallel situations where targeted anticancer agents recognize tumor markers expressed on neoplastic endothelium, an area that is under active investigation.…”