 |
| Chemical name: | Ad5-(PSE-BC)-(GAL4-(VP16)2)-(GAL4)5-sr39tk | |
| Abbreviated name: | AdTSTA-sr39tk | |
| Synonym: | ||
| Agent Category: | Protein (virus) | |
| Target: | Androgen receptor | |
| Target Category: | Receptor | |
| Method of detection: | Positron emission tomography (PET) | |
| Source of signal/contrast: | 18FHBG | |
| Activation: | Yes | |
| Studies: |
| No structure is current available in PubChem. |
[PubMed]
Adenoviruses (Ads) comprise a non-enveloped icosahedral protein shell (capsid) 70–100 nm in diameter surrounding an inner vial genome core (1). Human Ads consist of 51 distinct serotypes that are classified into six subgroups (species) designated A–F (2). Some serotypes such as serotypes 2 (Ad2) and 5 (Ad5) of species C only induce a mild, non-oncogenic respiratory infection, making them suitable for development of Ad-based vaccines and gene delivery vehicles for systematic administrations (1). Their core genome, a linear, double-stranded DNA of ~36 kb, contains five early transcription units (E1A, E1B, E2, E3, and E4), four intermediate units (IVA2, IX, VAI, and VAII), and one later transcription unit (2). The functions in the early transcription regions have been well identified: E1A activates cell cycles and initiates DNA replication; E1B blocks apoptosis induced by E1A activity; E2 facilitates viral DNA replication; E3 modulates host immune responses; and E4 regulates DNA replication, mRNA transport, and apoptosis (3). As a gene delivery vehicle, the genome core in Ad2 or Ad5 is usually modified with recombinant transcription activators (RTAs), in which foreign DNA with a size of up to 7.5 kb can be inserted in the deleted E1 or E3 regions in the Ads (4). Such genetic modification can incorporate ligands that can recognize specific cellular receptors and/or block the adenoviral naïve receptor (coxsackie-adenovirus receptor (CAR)), so that their tissue specificity is enhanced significantly. For example, the gene for the androgen receptor (AR) regulates the growth of prostate epithelial cells, induces the expression of prostate-specific antigen (PSA), and controls the growth of prostate cancer in the early phase. The AR gene can be introduced to the adenoviral genome to target AR-responsive prostate cancer or metastasis (5). Furthermore, reporter genes such as firefly luciferase (Fluc) (6) can be included for in vivo optical imaging, or suicide genes such as herpes simplex virus type 1-thymidine kinase (HSV1-TK, also a reporter gene for positron emission tomography (PET)), can be used for therapeutic applications (7).
HSV1-TK is a homodimer composed of two 376-residue subunits, and it functions as a key enzyme in the pyrimidine-salvage pathway to catalyze the phosphorylation of thymidine (dT) to thymidine monophosphate (dTMP) in the presence of ATP and Mg2+ (7). Unlike mammalian thymidine kinases, HSV1-TK can efficiently phosphorylate substrates such as acycloguanosines and uracils (8). Thus, radiolabeled acycloguanosines such as 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG, [18F]FPCV) or uracils such as 2’-fluoro-2’-deoxy-1-β-D-arabino-furanosyl-5-iodo-uracil ([131I]FIAU) are used as HSV1-TK imaging probes for positron emission tomography (PET) or single-photon emission tomography (SPECT). As a mutant of HSV1-TK, HSV1-sr39tk has five nonpolar amino acids being replaced at its active site: L159I, I160F, F161L, A162F, and L163M (9). Such remodeling in the active site leads to a substantial increase in the binding affinity to acycloguanosines; i.e., the 50% inhibition concentration of ganciclovir reduces ~300-fold. This also increases the imaging detection sensitivity; i.e., a more than two-fold enhancement has been observed in detection of [18F]FHBG with HSV1-sr39tk compared to that with HSV1-TK, which provides an efficient gene-reporter probe combination (HSV1-sr39tk-[18F]FHBG) for PET imaging (8).
The transcriptional activity of adenoviral genes can be amplified several orders of magnitude with the use of the GAL4-VP2 fusion protein, where GAL4 is a DNA-binding domain (147 amino acids) and VP2 is a two-tandem repeat of the herpes simplex virus VP16 acidic activation domain (78 amino acids) (10). The two-step transcriptional amplification (TSTA) system is a novel recombinant transcription activation approach for designing a PSA promoter/enhancer, which can provide as high as ~800-fold enhancement in transcription activity compared with its conventional analog (11, 12). The TSTA system is composed of two parts in general; i.e., an activator PBC-VP2 that contains a PSA promoter with a duplicated ARE4 enhancer core (each core has four binding sites for AR) to control the expression of the chimeric protein GAL4-VP2 and provide cell-specific binding, and the reporter (GAL4)5-sr39tk (G5-sr39tk), which contains a reporter with five GAL4 repeats to generate a GAL4-responsive sr39tk (13). The two parts are linked in a head-to-head orientation and are inserted into the E1 region of Ad5 to produce Ad5-(PSE-BC)-(GAL4-(VP16)2)-(GAL4)5-sr39tk (AdTSTA-sr39tk), a reporter gene for PET imaging of AR-responsive PSA in vivo. Such a construct can provide ~12-fold amplification (12) via two steps: the PSA regulates the expression of the potent transcription activator GAL4-VP2, which in turn activates the GAL4-responsive reporter gene sr39tk. AdTSTA-sr39tk can function effectively as a gene delivery vehicle and as a lymphotropic agent in prostate cancer and metastasis in vivo (13, 14).
[PubMed]
The preparation of AdTSTA-sr39tk was conducted in several steps (12, 13, 15). The commercial plasmid pBS II SK+ was used as the starting baseline construct pPSE (12). A NotI-flanked PSA enhancer/promoter expression cassette derived from plasmid PSAR2.4k-PCPSA-P-Lux was inserted into the pPSE to produce PSE. The synthetic ARE4 element that contained four key ARE elements was used to generate a duplicated enhancer core (15). The insertion of this duplicated core (C)in the PSE replaced the sequence between -3935 and -2855 in the PSE to produce a plasmid vector PBC. Then a GAL-VP2 fragment was inserted into PBC followed by the creation of a unique NotI site. A NotI fragment, which contained both the PBC promoter and GAL-VP2 gene, was excised from the obtained plasmid PBC-VP2 and cloned into the NotI site of G5-sr39tk to generate the vector PBC-VP2G5-sr39tk (11, 13). A SalI-Notl fragment containing the PBC-VP2G5-sr39tk fragment was derived from the resulting PBC-VP2G5-sr39tk and excised by NotI and partial SalI digestion (13). The produced expression cassette PBC-VP2G5-sr39tk was inserted into the SalI-NotI site of the commercial vector pShuttle, which was then incorporated into the commercial Ad vector AdEasy through homologous recombination to produce AdTSTA-sr39tk. The virus was propagated in 293 cells, purified on a CsCl gradient, and titered with plaque assays on 293 monolayers.
[PubMed]
Burton et al. examined the movement of AdTSTA-sr39tk to lymph nodes in vivo (14). SCID/Beige (natural killer–deficient) mice were implanted with LAPC-9-VEGF-C tumor cells on the right shoulder, and the tumors were allowed to grow for 30 d. After resection of the tumor, the mice were injected with 1 × 108 pfu AdTSTA-sr39tk in 30 μl phosphate-buffered saline in their forepaws followed by an intravenous injection of ~250 μCi [18F]FHBG. Micro-PET images were collected 1 h later for 20 min followed by imaging with micro-CT for 7 min. A positive [18F]FHBG PET signal was observed in the ipsilateral but not in contralateral axillary lymph nodes. Tumor cell-localized expression of sr39tk was found in the respective lymph nodes by immunohistological staining. Burton et al. then incorporated AdTSTA-sr39tk in the lymphoscintigraphy to assess lymph node metastases (14). Mice (n = 6) were implanted with Renilla luciferase (RL)-labeled tumors (LAPC-9-C-GFP-RL; ~1.0 cm in diameter) on their right upper back, and they also received a tumor-directed injection of 5 × 108 pfu AdTSTA-sr39tk. One hour after intravenous injection of ~250 μCi [18F]FHBG, PET/CT images were collected. Robust [18F]FHBG PET signals were detected in the tumor and the draining axillary lymph nodes. Immediately after the PET/CT imaging, ipsilateral and contralateral lymph nodes were removed for quantification of their radioactivity and RL bioluminescence. The radioactivity in the ipsilateral lymph nodes was found to be twice as high as the radioactivity in the contralateral lymph nodes. Also, the measurement of RL bioluminescence in the isolated lymph nodes confirmed the presence of disseminated tumor cells. These results exhibited an effective movement of AdTSTA-sr39tk to the sentinel lymph node and allowed for direct detection of nodal metastasis with PET.
[PubMed]
No publication is currently available.
CA 82214, CA 09056, CA 86306, CA 92131, CA101904, CA 92865, GM 08652
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