Peptide targeting of adenoviral vectors to augment tumor gene transfer.

Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild-type adenovirus in those cells. Nevertheless, the incorporation of relatively low affinity peptide ligands into the fiber knob, while effective in vitro, has only minimal targeting efficacy in vivo and highlights the importance of high affinity ligand:receptor interactions to achieve tumor targeting.

MicroRNA silencing improves the tumor specificity of adenoviral transgene expression.

Adenoviral technology has been thoroughly evaluated for delivering genetic material to tumor tissue and the surrounding microenvironment. Almost any gene can be cloned into an adenovirus (Ad) vector, which when combined with strong, constitutively active promoters permit up to a million-fold amplification of the transgene in a single adenoviral particle, thus facilitating their use in cancer therapy and imaging. However, widespread infection of the liver and other non-targeted tissues by Ad vectors is a substantial problem that often results in significant liver inflammation and hepatotoxicity at doses required to achieve efficient tumor transduction. miR-122 is a highly expressed liver-specific microRNA (miRNA) that provides a unique opportunity for downregulating adenoviral transgene expression in liver tissue. The binding of endogenous miRNAs to complementary miRNA-targeting elements (miRTs) incorporated into the 3' untranslated region of adenoviral transgenes interferes with message stability and/or protein translation, and miRT elements against miR-122 (miRT-122) can selectively reduce adenoviral transgene expression in the liver. Previous studies using miR-122-based regulation, with and without other types of transcriptional targeting, have yielded promising preliminary results. However, investigations to date evaluating miRT-122 elements for improving tumor specificity have used either non-tumor-bearing animals or direct intratumoral injection as the mode of delivery. In the present study, we confirmed the ability of miRT-122 sequences to selectively downregulate adenoviral luciferase expression in the liver in vitro and in vivo, and show that this strategy can improve tumor-specific transgene expression in a HT1080 human fibrosarcoma model. Rapid growth and the inefficient flow of blood through tumor neovasculature often results in profound hypoxia, which provides additional opportunities for targeting solid tumors and their microenvironment using vectors incorporating hypoxia-responsive promoters to drive transgene expression. We therefore used a combinatorial approach using miRT-122 elements with hypoxia-responsive transcriptional targeting to further improve the tumor-specific expression of an adenoviral reporter gene. Results from this investigation reveal that miRT122 elements alone decrease off-target liver expression and improve tumor specificity of adenoviral vectors. Furthermore, increased tumor specificity can be achieved by combining miRT-122 elements with hypoxia-responsive promoters.

Retargeting adenoviral vectors to improve gene transfer into tumors.

Gene targeting to tumors using adenoviral (Ad) vectors holds great potential for cancer imaging and therapy, but the limited efficacy of current methods used to improve delivery to target tissues and reduce unwanted interactions remain substantial barriers to further development. Progress in characterizing the set of molecular interactions used by Ad vectors to infect particular tissues has aided the development of novel strategies for retargeting vectors to tumor cells. One method is chemical retargeting of adenovirus using bispecific antibodies (bsAbs) against both viral capsid proteins and tumor-specific cell surface molecules. This approach can be combined either with competitive inhibitors designed to reduce viral tropism in undesired tissues, or with traditional therapeutics to increase the expression of surface molecules for improved tumor targeting. Ablating liver cell-specific interactions through mutation of capsid proteins or chemical means are promising strategies for reducing adenovirus-induced liver toxicity. The nature of tumor neovasculature also influences Ad delivery, and the use of vascular disrupting agents (VDAs) such as combretastatin can help elucidate these contributions. In this investigation, we evaluate a variety of these methods for retargeting Ad vectors to tumor cells in vitro and in vivo, and assess the contributions of specific molecular and tissue interactions that affect Ad transgene delivery.

Adenoviral targeting of gene expression to tumors.

Using biochemical, imaging and histological methods, we employed transcriptional targeting to increase the specificity of tumor gene expression in vivo for intravenously administered recombinant adenovirus vectors. Surprisingly, the relative specificity of tumor expression in comparison with other tissues was increased for a constitutively expressing recombinant adenovirus, AdCMVLuc, by simply reducing the viral dose. Even at lower doses, however, the high frequency of viral infection and transgene expression in the liver using constitutive promoters still represents a substantial problem. To further augment tumor specificity, we constructed a series of adenoviruses expressing luciferase from several other promoters and tested their ability to selectively transcribe genes in tumor cells, both in vitro and in vivo. Constitutively active viral promoters (RSV, SRalpha) varied widely in their tumor selectivity, but hypoxia-responsive promoters (carbonic anhydrase 9, PAI-1, SOD2 and several chimeric constructs) showed the most tumor-selective expression. Our results show that tumor targeting to HT1080 fibrosarcomas was readily achieved using transcriptional targeting mechanisms. We attribute the relatively high level of gene transfer and expression in HT1080 tumors in vivo to increased viral access to the tumor, presumably due to discontinuities in tumor vasculature and augmented expression from stress-responsive promoters in the hypoxic and inflammatory tumor microenvironment.