Background

[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 administration (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 imaging, or suicide genes such as herpes simplex virus type 1-thymidine kinase (HSV1-tk) can be used for therapeutic applications (7). As an oxygenase (Mw = 62 kDa), Fluc oxidizes the heterocyclic substrate d-luciferin to oxyluciferin injected into the body and emits light in the wavelength range of 400–620 nm (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) (9). 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 (10, 11). The TSTA system is composed of two parts in general; i.e., an activator BC-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-Fluc (G5-FL), which contains a reporter with five GAL4 repeats to generate a GAL4-responsive Fluc signal (6). 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-Fluc (AdTSTA-FL), an optical agent for imaging AR-responsive PSA. Such a construct can provide ~50-fold amplification (11) via two steps: the PSA regulates the expression of the potent transcription activator GAL4-VP2, which in turn activates a GAL4-responsive reporter Fluc. AdTSTA-FL can function effectively as a gene delivery vehicle and as a lymphotropic agent in prostate cancer and metastasis in vivo (6, 12).

Synthesis

[PubMed]

The preparation of AdTSTA-FL was conducted in several steps (5, 6, 10, 11, 13). The commercial plasmid pBS II SK+ was used as the starting baseline construct pPSE (11). 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 (13). 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 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-FL to generate the vector PBC-VP2G5-FL (10). A SalI-Notl fragment containing the PBC-VP2G5-FL fragment was derived from the resulting PBC-VP2G5-FL and excised by NotI and partial SalI digestion (5). The produced expression cassette PBC-VP2G5-FL 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-FL (6). The virus was propagated in 293 cells, purified on a CsCl gradient, and titered with plaque assays on 293 monolayers.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Zhang et al. examined the activity of AdTSTA-FL in vitro (5). Androgen-dependent prostate cancer cells (LNCaP) were infected with AdTSTA-FL and incubated up to 48 h. The cells were harvested and lysed for assaying activity. GAL4-VP2 and Fluc levels were enhanced significantly in the presence of the synthetic androgen agonist R1881, with a maximum enhancement at 48 h after transcription. Similar studies were conducted in other cells, including MCF7 cells (androgen-expressing breast cancer cells) and PC3 (androgen-negative prostate cancer cells), but only a low basal level of TSTA expression was observed and did not respond to R1881. Thus the AdTSTA-FL responded only to androgen-dependent prostate cancer cells. AdTSTA-FL displayed 10-fold more ligand-induced Fluc activity than the control, AdCMV-FL.

Sato et al. examined the specificity of AdTSTA-FL in vitro on the basis of the measurement of infectivity (6). A variety of cells were used in the study, including LNCaP and LPAC-4 cells, H157 and A549 cells (lung cancer), MCF7 cells, and HepG2 cells (liver cancer). The infectivity in these cells with respect to that in the Hela cells (cervical carcinoma) was evaluated on the basis of their viral DNA uptake, which was 1.7 for LNCaP, 1.6 for H157, 1.5 for MCF7, and 1.1 for LAPC-4 and HepG2. Then the activity of AdTSTA-FL was evaluated in several prostate cancer cell lines, including two androgen-responsive cells (LNCaP and LPAC-4) and two AR-negative cells (DU145 and PC3). With a dose of one plaque-forming unit (pfu, an infectious unit) per cell (1 multiplicity of infection (m.o.i)), the normalized Fluc activity was 4.4-fold lower in LPAC-4 than that in LNCaP, whereas the AR-negative cells exhibited negative results (i.e., ~500-fold lower activity). The luciferase activity in other cancer cells was also measured and normalized to the activity measured for LNCaP, and all had much lower activity: a reduction of 2.9-fold was found in A549, 12-fold in H157, 45-fold in HepG2, 60-fold in Hela, and 200-fold in MCF7.

Burton et al. assessed the accumulation of AdTSTA-FL in lymph nodes ex vivo (12). Mice (n = 3) were injected with 1 × 10⁸ pfu AdTSTA-FL in 20 μl phosphate-buffered saline at their forepaws. Their draining lymph nodes (the axillary and brachial nodes) were dissected 24 h after injection. The nodal capsules were disrupted for extraction of the adenoviral particles. The transduction luciferase activity was found in ipsilateral axillary and brachial lymph nodes but not in the contralateral nodes. The number of viral genomes transported to and retained in the brachial node was ~60,000 copies, which was equivalent to ~0.1% delivery efficiency. Burton et al. also examined the transverse of AdTSTA-FL to regional lymph nodes in the mouse prostate Pten tumor suppressor gene knockout model (Pten^-/-). Ten-week-old Pten^-/- mice with enlarged carcinoma prostate glands were injected with 1 × 10⁸ pfu AdTSTA-FL on their dorsolateral lobe. One hour later, ~1% of injected AdTSTA-FL was found in the periaortic lumbar nodes, with a smaller amount in more distant mesenteric and renal lymph nodes.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

1.

Douglas J.T. Adenoviral vectors for gene therapy. Mol Biotechnol. 2007;36(1):71–80. [PubMed: 17827541]

2.

Majhen D. , Ambriovic-Ristov A. Adenoviral vectors--how to use them in cancer gene therapy? Virus Res. 2006;119(2):121–33. [PubMed: 16533542]

3.

Kuhlmann K.F. , Gouma D.J. , Wesseling J.G. Adenoviral gene therapy for pancreatic cancer: where do we stand? Dig Surg. 2008;25(4):278–92. [PubMed: 18635930]

4.

Nettelbeck D.M. , Jerome V. , Muller R. Gene therapy: designer promoters for tumour targeting. Trends Genet. 2000;16(4):174–81. [PubMed: 10729833]

5.

Zhang L. , Johnson M. , Le K.H. , Sato M. , Ilagan R. , Iyer M. , Gambhir S.S. , Wu L. , Carey M. Interrogating androgen receptor function in recurrent prostate cancer. Cancer Res. 2003;63(15):4552–60. [PubMed: 12907631]

6.

Sato M. , Johnson M. , Zhang L. , Zhang B. , Le K. , Gambhir S.S. , Carey M. , Wu L. Optimization of adenoviral vectors to direct highly amplified prostate-specific expression for imaging and gene therapy. Mol Ther. 2003;8(5):726–37. [PMC free article: PMC2820502] [PubMed: 14599805]

7.

Johnson M. , Sato M. , Burton J. , Gambhir S.S. , Carey M. , Wu L. Micro-PET/CT monitoring of herpes thymidine kinase suicide gene therapy in a prostate cancer xenograft: the advantage of a cell-specific transcriptional targeting approach. Mol Imaging. 2005;4(4):463–72. [PMC free article: PMC2835410] [PubMed: 16285908]

8.

Paulmurugan R. , Gambhir S.S. Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem. 2005;77(5):1295–302. [PMC free article: PMC4154832] [PubMed: 15732910]

9.

Sadowski I. , Ma J. , Triezenberg S. , Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988;335(6190):563–4. [PubMed: 3047590]

10.

Zhang L. , Adams J.Y. , Billick E. , Ilagan R. , Iyer M. , Le K. , Smallwood A. , Gambhir S.S. , Carey M. , Wu L. Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer. Mol Ther. 2002;5(3):223–32. [PubMed: 11863411]

11.

Iyer M. , Wu L. , Carey M. , Wang Y. , Smallwood A. , Gambhir S.S. Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci U S A. 2001;98(25):14595–600. [PMC free article: PMC64727] [PubMed: 11734653]

12.

Burton J.B. , Johnson M. , Sato M. , Koh S.B. , Mulholland D.J. , Stout D. , Chatziioannou A.F. , Phelps M.E. , Wu H. , Wu L. Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer. Nat Med. 2008;14(8):882–8. [PMC free article: PMC2811163] [PubMed: 18622403]

13.

Wu L. , Matherly J. , Smallwood A. , Adams J.Y. , Billick E. , Belldegrun A. , Carey M. Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vectors. Gene Ther. 2001;8(18):1416–26. [PubMed: 11571582]