Imaging chemically modified adenovirus for targeting tumors expressing integrin alphavbeta3 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 alpha(v)beta(3) 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-(3)H-penciclovir (8-(3)H-PCV) as substrate. The in vivo infectivity of the Ad vectors after intravenous administration into integrin alpha(v)beta(3)-positive U87MG and MDA-MB-435 tumor-bearing athymic nude mice was measured by both noninvasive microPET using 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-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 alpha(v)beta(3) expression levels. (18)F-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 (18)F-FHBG. Expression of TK in the liver and tumor homogenates corroborated with the magnitude of (18)F-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 (18)F-FHBG are able to monitor in vivo transfectivity of both Adtk and RGD-PEG-Adtk vectors in the liver and tumors after intravenous injection.

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; RGD is a head-to-tail cyclic pentapeptide carrying arginine-glycine-aspartic acid (RGD) sequence; PEG is poly(ethylene glycol) (molecular weight, 3,400 Da) with N-hydroxysuccinimide ester, which reacts with primary amino groups on the adenovirus, and maleimide group, which reacts with the thiolated RGD peptide, at each end of the PEG linker.

FIGURE 2

Flow cytometric analysis of expression levels of α_vβ₃ integrin (A) and CAR (B) in 293T, MCF-7, U87MG, and MDA-MB-435 cells. The median of α_vβ₃-positive and CAR-positive cells is shown in C and D, respectively.

FIGURE 3

Transduction of Adtk, PEG-Adtk, and RGD-PEG-Adtk in 293T (A), MCF-7 (B), U87MG (C), and MDA-MB-435 (D) cells. Results are expressed as the mean % conversion of ³H-PCV/min/μg protein of the cell lysates ± SD. Significantly higher transduction efficiency of RGD-PEG-Adtk than that of PEG-Adtk was found in all 4 cell types. PA = PEG-Adtk.

FIGURE 4

microPET of tumor-bearing mice (top: subcutaneous U87MG tumor on right shoulder; bottom: orthotopic MDA-MB-435 tumor on left mammary fat pad) 1 h after injection of ¹⁸F-FHBG (10-min static scan). Images (both trans-axial and coronal) were normalized to the same scale. The tumors are indicated by white arrows in all cases.

FIGURE 5

(A) ¹⁸F-FHBG retention in the form of %ID/g by measuring ROIs in organs of liver and in U87MG and MDA-MB-435 tumors after transfection of unmodified and modified Ad vectors. Data are presented as average ± SD (n = 3). Dose of virus was normalized to 20-g mouse body weight. (B) Percent conversion of ³H-PCV in tissue lysates from mice after 48 h of exposure to Adtk and RGD-PEG-Adtk. *P < 0.001.

FIGURE 6

Comparison of integrin expression in liver and in MDA-MB-435 and U87MG tumor tissue lysates by SDS-PAGE/autoradiography using ¹²⁵I-echistatin as α_vβ₃-specific radio-ligand. DLU = digital light unit.