Imaging Chemically Modified Adenovirus for Targeting Tumors Expressing Integrin α_vβ₃ in Living Mice with Mutant Herpes Simplex Virus Type 1 Thymidine Kinase PET Reporter Gene

Abstract

The aim of this study was to change adenovirus tropism by chemical modification of the fiber knobs with PEGylated RGD peptide for targeting integrin α_vβ₃ that is uniquely or highly expressed in tumor cells and neovasculature of tumors of various origins. Methods: The first generation Ad (Ad) vector, which expresses the herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) gene under the control of cytomegalovirus (CMV) promoter was conjugated with poly(ethylene glycol) (PEG) or RGD-PEG. The transduction efficiency of Ads (Adtk, PEG-Adtk, and RGD-PEG-Adtk) into different types of cells (293T, MCF7, MDA-MB-435, and U87MG) was analyzed and quantified by thymidine kinase (TK) assay using 8-³H-penciclovir (8-³H-PCV) as substrate. The in vivo infectivity of the Ad vectors after intravenous administration into integrin α_vβ₃–positive U87MG and MDA-MB-435 tumor-bearing athymic nude mice was measured by both noninvasive microPET using 9-[4-¹⁸F-fluoro-3-(hydroxymethyl)butyl]guanine (¹⁸F-FHBG) as a reporter probe and ex vivo TK assay of the tumor and tissue homogenates. Results: PEGylation completely abrogated coxsackievirus and adenovirus receptor (CAR)–knob interaction and the infectivity of PEG-Adtk is significantly lower than that of unmodified Adtk in CAR-positive cells. RGD-PEG–modified virus (RGD-PEG-Adtk) had significantly higher infectivity than PEG-Adtk and the extent of increase is related to both CAR and integrin α_vβ₃ expression levels. ¹⁸F-FHBG had minimal nonspecific uptake in the liver and tumors that are void of sr39tk. Mice preinjected intravenously with unmodified Adtk resulted in high hepatic uptake and moderate tumor accumulation of the tracer. In contrast, RGD-PEG-Adtk administration resulted in significantly lower liver uptake without compromising the tumor accumulation of ¹⁸F-FHBG. Expression of TK in the liver and tumor homogenates corroborated with the magnitude of ¹⁸F-FHBG uptake quantified by noninvasive microPET. Analysis of liver and tumor tissue integrin level confirmed that RGD–integrin interaction is responsible for the enhanced tumor infectivity of RGD-PEG-Adtk. Conclusion: The results of this study suggest that RGD-PEG conjugation is an effective way to modify Ad vector tropism for improved systemic gene delivery. Noninvasive PET and ¹⁸F-FHBG are able to monitor in vivo transfectivity of both Adtk and RGD-PEG-Adtk vectors in the liver and tumors after intravenous injection.