Gene therapy holds great promise for a variety of diagnostic and therapeutic applications, and adenoviral technology has emerged as one of the primary approaches for the targeted delivery of genes to mammalian cells. Replication-deficient adenoviral (Ad) vectors based on serotypes 5 show excellent gene transfer efficiency, have high stability in vivo
, can be grown to high titers, and techniques for their construction, propagation and purification are also well established.(1
) Ad vectors have been used in the majority of all clinical trials evaluating gene therapy for cancer, and only rarely result in any serious complications. Results from trials using nontargeted adenovirus, however, have been generally disappointing.(2
) This failure has mainly been attributed to the broad native tropism of adenoviral vectors which occurs primarily through high-affinity binding of the adenoviral fiber knob domain to its primary cellular receptor, the widely expressed coxsackie and adenovirus receptor (CAR).(3
) Other interactions are also involved in native adenoviral infection, including those between the RGD ligand in the penton base and αV
integrins, interactions between the KKTK sequence in the adenoviral fiber shaft and heparin sulfate glycosaminoglycans (HSGs),(4
) and binding of the adenoviral hexon protein to blood coagulation factors (VII and X) which serve as a bridge for HSPG-mediated hepatocyte transduction.(6
, the liver is a primary site of adenoviral infection due to hepatocyte transduction and Kuppfer cell uptake, leading to greater than 95% of hepatocytes being transduced following intravenous administration.(11
) This high level of liver tropism poses numerous problems for the development of adenoviral technology as a therapeutic or imaging modality directed to other tissues, including lower viral efficacy in target tissues and virus-induced liver inflammation and toxicity.(2
) A primary goal of adenoviral cancer therapy is therefore to simultaneously reduce liver transduction (detargeting) and improve the specificity of transgene expression in target tissues such as tumors and their surrounding microenvironment (targeting). Previous studies indicate that CAR and integrin-mediated interactions are unlikely to be the primary mechanisms of liver infection, since adenoviral vectors with modifications designed to ablate CAR and integrin-mediated binding have shown high residual levels of liver transduction in vivo
) Recent data, however, indicate that changing the HSG-binding site in the fiber shaft can enhance cancer cell infectivity while reducing liver transduction.(13
) A more effective strategy might involve blocking the coagulation factor-hexon interactions using anticoagulant therapy such as warfarin or nematode anticoagulant protein c2 (NapC2), (7
) or through mutation of the adenoviral hexon hypervariable regions.(8
Numerous approaches have been evaluated for detargeting and targeting adenoviral vectors. Ad vectors are typically detargeted using genetic modification of one or more of the three capsid proteins, hexon, penton and fiber, although chemical methods for transductional control incorporating bispecific adaptor molecules have also been used to effectively detarget adenovirus from their native tropism and retarget the virus to specific tissues. Bispecific fusion proteins or antibodies containing a high affinity knob-binding component that ablates CAR binding are attached either covalently or through a chemical linker to another component with high specificity for a surface molecule on the target cell. Successful retargeting of vectors has been achieved using anti-knob antibodies or truncated CAR constructs chemically or genetically linked either to a variety of targeted ligands or antibodies against cell surface receptors.(12
) Results from studies using adaptor molecules have shown a 10-20 fold increase in transgene expression in target tissues in vivo
) Studies have also established correlations between augmented gene transfer and cell surface receptor density, and demonstrated a substantial reduction of reporter gene expression in the liver compared to untargeted vector.(12
) Preliminary evidence also indicates high stability and good in vivo
performance for complexes involving adenoviral particles and adaptor molecules.(18
) These early results are promising, but additional studies need to be performed to achieve optimal retargeting of adenoviral vectors for cancer therapy or imaging.
Angiogenesis is an important process in the progression of solid tumors. The relatively disorganized tumor neovasculature might provide increased adenoviral access in the tumor microenvironment,(28
) and the utility of this effect could also be enhanced by the addition of genetic and/or physical detargeting/retargeting approaches. Tumor-specific endothelial markers of angiogenesis (TEMs) have been extensively characterized, and are rapidly being incorporated into targeted therapy strategies. Phosphatidylserine (PS) and other negatively charged phospholipids are usually restricted to the cytosolic side of cell membranes in most cells.(30
) However, anionic phospholipids become exposed on both tumor cells and tumor vascular endothelium, but not on normal vascular endothelium in mice, making PS one of the most specific tumor markers discovered to date.(30
) Bavituximab is a chimeric antibody that targets exposed PS by stabilizing a complex of two β2-glycoprotein I (β2GP1) molecules that are attached to PS at the cell surface.(30
) Mouse versions of this antibody (3G4 and 2aG4) have been shown to inhibit tumor growth in multiple animal models, and a recent study shows that clear images of subcutaneous prostate tumors can be obtained in rats following tail vein injection of isotopically-labeled bavituximab.(36
) Preliminary evidence indicates that antibodies against PS are rational candidates for incorporation into bispecific adaptor molecules designed to retarget adenoviral vectors to tumor tissue. The anti-microtubule chemotherapeutic docetaxel also induces the externalization of PS on several cell types, and has been shown to significantly improve antitumor activity in mice when combined with 3G4 in MDA-MB-435 tumor models.(34
) The use of docetaxel in combination with PS-retargeted vectors could theoretically improve the delivery of adenoviral vectors for imaging or therapeutic purposes.
Vascular disrupting agents (VDAs) are a relatively new group of compounds that show selective disruption of tumor vasculature, primarily through the depolymerization of microtubules in the endothelial cytoskeleton.(37
) Several VDAs such as combretastatin are currently in clinical development, and early results show that these agents have great potential for normalizing neovasculature, thereby reducing blood flow specifically in tumors. Although the exact mechanism of this specificity has not been firmly established, the predominant theory is that the immature and fragile nature of tumor vasculature makes it particularly susceptible to this type of therapy.(38
) However, these strategies can also have undesirable effects, by potentially selecting for more malignant cells, inducing cellular adaptations that promote tumor invasion, or restricting the access of therapeutic agents to the tumor.(39
) The clinical use of antiangiogenics and VDAs therefore needs to be carefully considered in the context of other therapeutic decisions, and many researchers have suggested that the timing of antiangiogenic therapy with other agents could be an important consideration.(40
) Characteristics of tumor vasculature could also affect adenoviral gene delivery for therapy or imaging purposes. Previous results in our lab have suggested that the disorganized and leaky nature of tumor blood vessels might improve adenoviral access to tumors in mice ,(28
) so VDAs could be valuable for resolving the contributions of irregular tumor vasculature on adenoviral transduction efficiency.
In the present study, we sought to explore strategies for the detargeting and retargeting of adenoviral vectors to develop a platform of tumor imaging agents using luciferase as an optical reporter. Various approaches for transductional retargeting with or without the administration of ancillary agents were evaluated to improve the tumor specificity of adenoviral-mediated luciferase delivery and expression in vitro and in vivo.