Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Abstract

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse b-glucuronidase (mbG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-b-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional b-glucuronidase (bG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (bG) on CT26 tumors. The fluorescent intensity in bG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral bG vector into carcinoma xenografts. Importantly, mbG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membraneanchored form of human bG also allowed in vivo imaging, demonstrating that human bG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

Enhancement of CPT-11 antitumor activity by adenovirus-mediated expression of β–glucuronidase in tumors

“…Glucuronides, on the other hand, do not efficiently enter cells, resulting in poor activation of glucuronide prodrugs when bG is expressed in the cytosol of mammalian cells. 19,39 To overcome poor penetration of glucuronides into cells, we anchored bG on the plasma membrane to allow effective contact with SN-38G. High levels of bG could be expressed on all three tumor cells investigated by fusing an optimized transmembrane domain derived from the B7-1 antigen to the c-terminus of bG.…”

“…Systemic activation of glucuronides by endogenous bG, however, appears to be limited by the compartmentalization of bG in lysosomes, which effectively limits contact of extracellular glucuronides with endogenous bG in most tissues. 39 bG does accumulate in the interstitial space in tumors due to secretion by tumorinfiltrating monocytes and release from necrotic cancer cells. [42][43][44] This explains the effectiveness of cancer monotherapy with synthetic glucuronide prodrugs of Figure 4 Growth of tumors expressing membrane-bound b-glucuronidase (bG) was suppressed to a greater degree than parental tumors.…”

PET Imaging of β-Glucuronidase Activity by an Activity-Based 124I-Trapping Probe for the Personalized Glucuronide Prodrug Targeted Therapy

“…D-saccharic acid 1,4-lactone monohydrate (SAL) was purchased from Sigma-Aldrich. We previously generated CT26/mbG cells (2) All these cell lines were maintained in Dulbecco's minimal essential medium (DMEM; SigmaAldrich) supplemented with 10% heat-inactivated bovine calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 C in an atmosphere of 5% CO 2 . The cell lines were not authenticated by our laboratory.…”

“…It has been widely used as an attractive enzyme for reporter imaging (2)(3)(4) and cancer prodrug therapies (5)(6)(7)(8). Several glucuronide prodrugs have been used in antibody-directed enzyme prodrug therapy (ADEPT; refs.…”

“…However, the fluorescent product FDGlcU rapidly leaked from bG-expressing sites and exhibited poor penetrative properties, limiting imaging to subcutaneous tumors but not deeper tumors (2). To overcome these problems, we also developed fluorescent glucuronide trapping probes by conjugating a fluorescein isothiocyanate (FITC; lex ¼ 495 nm, lem ¼ 519 nm) and the near infrared dye IR-820 isothiocyanate (NIR; lex ¼ 710 nm, lem ¼ 820 nm) with the difluoromethylphenol betaglucuronide trapping moiety to form FITC-TrapG and NIR-TrapG probes, respectively.…”