Imaging Chemically Modified Adenovirus for Targeting Tumors Expressing Integrin αvβ3 in Living Mice with Mutant Herpes Simplex Virus Type 1 Thymidine Kinase PET Reporter Gene

Recombinant adenovirus (rAd) has been widely used as an attractive gene delivery vehicle to mammalian cells because of its unparalleled efficacy in accomplishing gene transfer in vivo (1,2). There are, however, some limitations associated with Ad for gene delivery. One such disadvantage is related to the wide native tropism of Ad, which often results in high accumulation in nontargeting tissues (3–9). Another major disadvantage for using Ad vector in vivo is that administration of Ad to a host leads to the host immune response against Ad (10–12). Vector targeting to a specific tissue of cell type would enhance gene therapy efficacy and permit the delivery of lower doses, which would consequently result in reduced toxicity. Cell-specific gene delivery via Ad vectors has been achieved by both genetic (13–15) and chemical (16–22) approaches. Chemical modification is a nongenetic strategy to modify the surface of a viron by covalently attaching a polymer containing the targeting ligand with the lysine residues on the surface of Ad. Modification of Ad vectors with poly(ethylene glycol) (PEG) prolongs persistence in the blood and circumvents inflammatory and humoral immune responses (16–22). However, the PEGylation of Ad vectors also leads to loss of infectivity due to the steric hindrance induced by PEG chains. To overcome the decreased efficiency of infection of PEGylated Ad, vectors containing functional molecules on the tip of PEG restore target-specific infectivity. Lanciotti et al. reported targeted Ad vectors using heterofunctional PEG and FGF-2 (22). Ogawara et al. (17) reported PEGylated Ad vectors containing E-selectin–specific antibody or αv integrin–specific RGD peptide for targeting activated endothelial cells. Ad vectors coated with polymers other than PEG have also been developed. Fisher et al. used a multivalent hydrophilic polymer based on poly[N-(2-hydroxypropyl) methacrylamide] to modify Ad vectors (23). Although improved pharmacokinetic properties of polymer-coated Ad vectors without ligands have been reported, those of polymer-coated Ad vectors with ligands have not been reported in detail. Most studies performed in animal models to optimize vector targeting and functional gene delivery use protocols that require the animals to be sacrificed to monitor reporter gene expression. Recently, however, several noninvasive technologies for monitoring reporter gene expression in vivo have been described (24). The most commonly used PET reporter gene is herpes simplex virus 1 thymidine kinase (HSV1-tk). Many of the enzyme substrates have been labeled with 18F or 124I to image HSV1-tk gene expression. Two major substrates for HSV1-tk enzyme/PET include 9-[4-18F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) and 124I- or 18F-labeled 2′-fluoro-2′-deoxy-5′-iodo-1-β-D-arabinofuranosyluracil (124I-FIAU or 18F-FIAU) (25–27). It has also been reported that the mutant HSV1-sr39tk is more effective than the wild-type HSV1-tk at using acycloguanosines as substrates. We have demonstrated that the utility of HSV1-sr39tk and 18F-FHBG is able to monitor the reporter gene expression over a 3-mo period after somatic gene transfer (26,28). In this study, a replication-deficient Ad was modified with bifunctional PEG and then conjugated with cyclic RGD peptide (Fig. 1). We hypothesized that surface modification of Ad fiber knobs with PEGylated-RGD peptide will ablate the normal tropism and reduce transduction of nontarget tissues in vivo; incorporation of integrin αvβ3–specific RGD peptides will enhance the gene delivery to tumor neovasculature and integrin-positive tumor cells. To monitor the localization and expression of the chemically modified virus we packed the HSV1-sr39tk mutant as a reporter gene under the control of cytomegalovirus (CMV) promoter. The homing selectivity and transgene expression after intravenous administration of integrin-directed adenovirus were studied by microPET using 18F-FHBG as a reporter probe. FIGURE 1 Schematic representation of Ad vector modified with RGD-PEG. Synthesis of RGD-PEG-NHS (where NHS = N-hydroxysuccinimide) is shown on the top and coupling is shown on the bottom. Where Adtk is the first-generation Ad vector encoding the HSV1-sr39tk gene; ...