SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

To overcome the low efficiency of gene therapy, we combined a conditionally replicating adenovirus (CRAd) and an adenoviral vector with a therapeutic gene. CRAd has an oncolytic activity in cancer cells with abnormal Rb activity and helps the replication of therapeutic genes incorporated in the E1-deleted adenovirus. We investigated the anticancer effect of a combination of CRAd and adenovirus carrying tumor necrosis factor-related apoptosis inducing ligand (ad-TRAIL). We expected to see increased gene expression in cancer cells as well as an antitumor effect. With the combined application of CRAd and ad-luciferase in head and neck cancer cell lines, we observed considerably increased luciferase activity that was 10- to 50-fold greater than with ad-luciferase alone. The combination of CRAd and ad-TRAIL showed significant suppression of growth in cell lines and increased the sub-G1 portion of cells 30-fold compared to any single treatment. The expression of TRAIL was highly amplified by the combined treatment and was accompanied by expression of molecules related to apoptosis. In a xenograft animal model, mice treated with CRAd and ad-TRAIL showed complete regression of established tumors, whereas mice treated with CRAd or ad-TRAIL alone did not. In conclusion, this combined strategy using CRAd and adenovirus carrying a therapeutic gene increased the gene transfer rate and enhanced antitumor effects. We expect that this combination strategy could be extended to a multitarget cancer gene therapy by combining multiple adenoviruses and CRAd. (Cancer Sci 2009)

The low efficiency of gene transfer is one of the most difficult problems in the development of the practical application of gene therapy against cancer. To overcome this problem, we have used a strategy combining a conditionally replicating adenovirus (CRAd), designated Δ24RGD, producing a mutant E1 and an adenovirus expressing a therapeutic gene.(1,2) Theoretically, E1 protein of CRAd can permit viral replication in cancer cells with a damaged pRb/p16 pathway. Thus, CRAd can selectively replicate in tumor cells and induce oncolysis.(3–5) The Δ24RGD contains an Arg-Gly-Asp (RGD) motif that is known to interact with the αv integrin, allowing the adenoviral fiber to enter to cells without the coxsackie adenoviral receptor.(6)

Comparative studies of CRAd and replication-defective viruses have shown greater expression of integrated transgenes for CRAd. However, clinical studies with CRAd failed to show significant results due to the limited gene payload capacity of the vector. Based on this limitation, we combined an E1-deleted replication-defective adenovirus containing a therapeutic gene with CRAd. We have previously shown that a replication-defective adenovirus with an E1 deletion could replicate when combined with CRAd, which supplied E1 in trans.(1,2)

Tumor necrosis factor-related apoptosis inducing ligand is a cytokine of the tumor necrosis factor family and induces apoptosis in transformed cell lines but not in normal cells.(7,8) Most preclinical trials of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) have used soluble TRAIL (sTRAIL). However, some cancer cells have shown resistance to sTRAIL.(9) Additionally, sTRAIL has shown difficulty in inducing apoptosis in certain head and neck squamous cell carcinoma (HNSCC) cell lines.(10) This difficulty has been attributed to the expression of a decoy receptor,(11) loss of caspase,(12) and production of inhibitors.(13–15) Many studies have attempted to increase TRAIL sensitivity by adding an anticancer agent or transferring the p53 gene.(16) In a previous report, we substituted sTRAIL for adnovirus carrying TRAIL (ad-TRAIL) to solve the problem of resistance. Ad-TRAIL induced apoptotic cell death in several cancer cell lines resistant to sTRAIL such as A549, LNCaP, and SKOV3.(17) However, in the SNU-1041 HNSCC cell line, ad-TRAIL was ineffective in inducing cell death.

In this study, we combined CRAd and replication-defective ad-TRAIL to overcome TRAIL resistance and enhance the efficiency of cancer gene therapy in HNSCC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cell lines.  Human HNSCC cell lines SNU-1041, SNU-1066, and SNU-1076 were purchased from the Korean Cell Line Bank (Seoul, Korea). Cell lines were cultured in RPMI-1640 medium with 10% FBS and gentamycin (Gibco BRL, Grand Island, NY, USA).

Recombinant adenovirus.  The Ad-Easy system was kindly provided by T-C He (Howard Hughes Medical Institute, Baltimore, MD, USA) and Δ24RGD CRAd was generously gifted by David Curiel, (University of Alabama, Tuscaloosa, AL, USA). Δ24RGD contains a 24-bp deletion in the CR2 region of E1A and an RGD-4C modification of the fiber gene of E3. The CR2 domain is responsible for binding pRb to allow adenovirus-infected cells to enter the S phase of the cell cycle. Therefore, an adenovirus with a deletion in this region can only replicate in cells that are defective in the pRb/p16 pathway; pRb binding is not necessary. Moreover, the RGD-4C motif in the E3 region enables the virus to infect cells without binding to the coxsackie adenovirus receptor.

We previously described the construction of an adenovirus expressing TRAIL.(17) Adenoviruses were amplified in HEK293 cells and prepared by a standard CsCl method. We used the CMV promoter for ad-TRAIL, ad-luciferase, and ad-lacZ. The adenovirus titers were measured by the TCID50 method and the acquired value was recalculated as pfu/mL.

Gene expression measurement by Western blot and FACS analysis.  Cultured cells were rinsed with PBS, suspended in lysis buffer (0.5% NP40, 50 mm Tris-Cl, 150 mm NaCl, 1 mm DTT, 1% sodium deoxycholate, 0.1% SDS, 1 mm EDTA, 1 mm PMSF, 0.1 M aprotinin, and 1 M pepstatin A) and incubated at 4°C for 30 min. The cell lysates were then centrifuged at 16 300 g for 20 min at 4°C. An appropriate amount of each supernatant (determined by protein assay) was mixed with 4× sample loading buffer and denatured for 10 min at 70°C. The denatured protein samples were fractionated on 4–12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA, USA), transferred onto nitrocellulose membranes (Schleicher & Schuell, Dachen, Germany) and incubated with Tris-buffered saline containing 0.1% Tween-20, and 5% non-fat dry milk. The membranes were then incubated with TRAIL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bcl-2, caspase 8, caspase 9, poly(ADP-ribose) polymerase (PARP), and caspase 3 (Cell Signaling Technology, Danvers, MA, USA) antibody. We identified TRAIL expression with phycoerythrin-conjugated anti-TRAIL (BD Pharmingen, San Jose, CA, USA) and by FACSCalibur (BD Biosciences, San Jose, CA, USA). Experiments were repeated at least three times.

Analysis of apoptosis by sub-G1 fraction determination.  To measure the degree of apoptosis, the number of cells in sub-G1 was determined. Cells were plated in a 6-well plate (5 × 105 cells/well), incubated for 24 h, then various amounts of ad-TRAIL and Δ24RGD were transduced for 48 h separately and combined. The cells were fixed in 70% ice-cold ethanol and suspended with propidium iodide (20 μg/mL; Sigma, Seoul, Korea) and ribonuclease A (5 μg/mL; Sigma-Aldrich). Flow cytometry was carried out on a FACSCalibur device (BD Biosciences) and data were analyzed using CellQuest Pro software (BD Biosciences). Experiments were repeated at least three times.

Luciferase assay.  Cells were seeded in a 24-well plate (3 × 105 cells/well). After 24 h incubation, adenoviruses were transduced separately and combined. At 48 h, luciferase activity was measured using Tropix lysis buffer and a 1420 luciferase counter (Perkin Elmer, Waltham, MA, USA) according to the manufacturer’s instructions. Experiments were repeated at least three times.

LacZ staining.  Cells were transduced with ad-LacZ alone or ad-LacZ and CRAd together. After 48 h of transduction, the efficiencies of LacZ gene transfer were measured by X-Gal staining. Experiments were repeated at least three times.

In vitro analysis of cytotoxic effects.  The in vitro cytotoxic effects of the adenovirus were analyzed by determining the viability of the cells using crystal violet staining (Sigma-Aldrich). Cells were plated in a 6-well plate (5 × 105 cells/well), and infected 24 h later. After 72 h, cells were stained with 1% crystal violet in 70% ethanol for 30 min followed by washing twice and air-drying. Experiments were repeated at least three times.

Combined treatment with CRAd and ad-TRAIL in an animal model.  Male BALB/c nude mice were purchased from Japan SLC (Hamamatsu, Japan). All experiments were approved by Institutional Animal Care and Use Committee (IACUC) of the Clinical Research Institute, Seoul National University Hospital (Seoul, Korea) (accredited by the Association for Assessment and Accreditation of Laboratory Animal Care). We established an HNSCC xenograft model by s.c. injection of SNU-1041 cells (2 × 106 cells/mouse). Seven days later, when the tumor diameter reached approximately 5 mm, ad-luciferase (2 × 108 pfu/site), CRAd+ad-luciferase (1 × 108 + 1 × 108 pfu/site), ad-TRAIL + CRAd (1 × 108 + 1 × 108 pfu/site) and ad-TRAIL + CRAd (1 × 108 + 1 × 108 pfu/site) were injected intratumorally on days 0, 2, 4, and 7. Tumor volume was measured using the formula: 0.5 × length × width2. The ANOVA test was used for statistical analysis.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Expression of reporter genes increased by combination of CRAd.  To identify increased gene expression in the E1-deleted replication-incompetent adenovirus, we tested the combinatorial application of CRAd with ad-luciferase, ad-LacZ, and ad-GFP (Fig. 1). We observed a significant increase in luciferase activity of 10- to 50-fold over transduction with ad-luciferase alone. Similar results were observed for ad-lacZ and ad-GFP. These findings suggest that CRAd could facilitate replication of an inserted gene in a replication-deficient adenovirus, as previously reported.(2)

image

Figure 1.  Enhanced expression of luciferase, LacZ, and GFP by combination of conditionally replicating adenovirus (CRAd). (A) Increased luciferase expression from adenovirus carrying luciferase (ad-luci; 5 moi) transduced cells with co-transduction of CRAd (1 moi). Luciferase activity was measured 48 h after virus infection. RLU, relative light units. (B) Increased LacZ expression in ad-LacZ (5 moi) transduced cells with co-transduction of CRAd (1 moi). Cells were stained 48 h after infection. (C) Increased GFP expression in ad-GFP (5 moi) transduced cells with co-transduction of CRAd (1 moi). We counted the number of cells in the FL1 fraction. The numbers in each graph represent the mean values. MOCK, no infection.

Download figure to PowerPoint

Combination with ad-TRAIL and CRAd.  The anticancer effect of ad-TRAIL was weak in HNSCC cells, as shown in Figure 2(A). When cancer cells were treated with ad-TRAIL in the range of 10–30 moi, no cytotoxic effect was observed, similar to treatment with ad-luciferase. However, we found significant enhancement of ad-TRAIL cytotoxicity when combined with CRAd. Figure 2(B) shows the growth suppression effects of the combination of ad-TRAIL and CRAd in SNU-1041, SNU-1066, and SNU-1076 cell lines. Treatment with the combination of ad-TRAIL and CRAd resulted in a profound antiproliferative effect in all three HNSCC cell lines, as shown by crystal violet staining. As indicated by propidium iodide staining, the combination of CRAd and ad-TRAIL also resulted in a significant 30-fold increase in the number of cells in the sub-G1 phase when compared to single transduction of ad-TRAIL (Fig. 2C). These results suggest that a synergistic anticancer effect could be obtained by combining the replication-defective adenovirus with even a small amount of CRAd.

image

Figure 2. Induction of apoptosis by combination of adenovirus carrying tumor necrosis factor-related apoptosis inducing ligand (ad-TRAIL) and conditionally replicating adenovirus (CRAd) in head and neck cancer cell lines. (A) Weak cytotoxicity of ad-TRAIL in SNU-1041 cells. Ad-luciferase (ad-luci) and ad-TRAIL were used in the range of 10–30 moi. Crystal violet staining was carried out after 72 h of transduction. (B) The combination of ad-TRAIL (5 moi) and CRAd (2 moi) in all three cell lines: SNU-1041 (upper left corner); SNU-1066 (upper right corner); and SNU-1076 (bottom left corner). Ad-luci was used as a control at the same number of viral pfu for the single treatment. (C) Increased sub-G1 portion with combined treatment. The numbers in each graph indicate the percentage of cells in the sub-G1 phase. The data are representative of three independent experiments. MOCK, no infection.

Download figure to PowerPoint

CRAd increased expression of TRAIL in a replication defective adenovirus.  To investigate whether the anticancer effects were caused by increased expression of TRAIL, we evaluated TRAIL gene expression by Western blot and FACS analysis. As shown in Figure 3(A), the combination of ad-TRAIL and CRAd produced a much higher level of TRAIL expression than single transduction. Addition of CRAd resulted in a significantly increased (3- to 5-fold) number of cells expressing TRAIL when compared to treatment with ad-TRAIL alone (Fig. 3B). We also monitored the apoptosis pathway by checking caspase 3, caspase 8, caspase 9, PARP, and Bcl-2 expression by Western blotting. Significant upregulation of cleaved caspase 3, caspase 8, and PARP was observed (Fig. 3A). Caspase 9 was observed in CRAd conditions only. Additionally, expression of the anti-apoptotic molecule Bcl-2 decreased when ad-TRAIL and CRAd were combined. These findings clearly indicate that the combination of ad-TRAIL and CRAd resulted in increased TRAIL expression, which subsequently led to activation of the apoptotic pathway.

image

Figure 3.  Increased expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and detection of molecules related to apoptosis. (A) Western blot analysis showed expression levels of adenovirus carrying TRAIL (ad-TRAIL; 2.5 moi) combined with conditionally replicating adenovirus CRAd (2 moi). The expression of TRAIL, caspase 8, caspase 9, cleaved caspase 3, poly(ADP-ribose) polymerase (PARP), and Bcl-2 was detected. MOCK, no treatment. (B) Analysis of TRAIL gene expression by FACS. The numbers in each graph represent the percentage of cells showing increased TRAIL expression.

Download figure to PowerPoint

Anticancer effect on HNSCC xenografts in vivo.  In the xenograft model, tumors grew continuously in all groups except for the ad-TRAIL + CRAd group, in which the tumor volume gradually decreased (Fig. 4). On day 11, most mice treated with ad-TRAIL + CRAd showed a significant reduction in tumor volume. Furthermore, 75% of the combination group showed complete regression of the tumor at the endpoint of the experiment.

image

Figure 4. Combined treatment with adenovirus carrying tumor necrosis factor-related apoptosis inducing ligand (ad-TRAIL) and conditionally replicating adenovirus (CRAd) strongly suppressed tumor growth in vivo. Tumors were established by injecting SNU-1041 head and neck carcinoma cells (2 × 106cells/mouse) into nude mice (eight mice/group). Adenovirus carrying luciferase (ad-luci) was used as a control at the same number of viral pfu for the single treatment. Intratumoral injections of: ad-luci (2 × 108 pfu); ad-TRAIL (1 × 108 pfu) + ad-luci (1 ×108 pfu); CRAd (1 × 108 pfu) + ad-luci (1 × 108 pfu); and ad-TRAIL (1 × 108 pfu) + CRAd (1 × 108 pfu) were given on days 0, 2, 4, and 7. Data are represented as mean ± SD (P = 0.01).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Of the many obstacles that hinder gene therapy for cancer, low gene transfer efficiency is considered one of the most difficult. Gene transfer efficiency has not been satisfactory at the cellular or tumor levels. By combining a replication-defective TRAIL-expressing adenovirus with CRAd, we have established an effective technique for increasing the expression of therapeutic genes as well as increasing the oncolytic efficiency. After viruses infect cancer cells, the cells undergo lysis caused by the oncolytic action of CRAd. The viruses, and hence the killing effect, then leave the dead cancer cells and spread to adjacent tumor cells. At the same time, the mutant E1 produced by CRAd assists the adenovirus containing the therapeutic gene to produce more protein through enhanced replication.(2) Another potential benefit of this combination is the possible reduction of the viral dose, as shown in this experiment. One of the main causes of gene therapy side-effects is the high dose of virus required. The other benefit of this study is transferring virus directly to HNSCC cells. Most viruses cannot pass through the liver when injected systemically. However, we were able to infect the target tissue directly by intratumoral infection.

To strengthen the antitumor effect of CRAd, several modifications have been attempted. CRAd carrying therapeutic genes such as TRAIL,(18) herpes simplex thymidine kinase,(19,20) and p53(21) in its E1 or E3 region have shown enhanced antitumor effects.(22–25) CRAd that can replicate in cells expressing a specific protein have also been introduced. For example, CRAd containing a telomerase-derived promoter selectively replicated and destroyed telomerase-positive cancer cells(26,27) and CRAd containing a COX-2 promoter had a promising result in an in vivo tumor model.(28,29)

Our strategy is different from those using therapeutically armed adenoviruses because we used another replication-defective adenovirus to carry a therapeutic gene instead of inserting the therapeutic gene into CRAd. This study is advantageous because it enables replication-defective adenoviruses carrying a therapeutic gene to replicate within tumor cells by using the mutant E1 from the co-transduced CRAd. That is, CRAd produces mutant E1 in cancer cells allowing the replication of an E1-deleted adenovirus if both viruses were transduced in the same cancer cell. This strategy made it easier to break resistance against TRAIL. Many studies have been reported about TRAIL resistance in cancer cells. Both SNU-1041 and FaDu(10) cancer cells showed TRAIL resistance. Our strategy has the potential to overcome this resistance phenomenon through the addition of CRAd. In Figure 2(A), ad-TRAIL did not have a significant anticancer effect on HNSCC cells. However, the addition of a small amount of CRAd broke TRAIL resistance and led to the killing of tumor cells. Additionally, we tried multitargeting combination gene therapy, such as ad-TRAIL + ad-p63 + CRAd or ad-TRAIL + ad-TK + CRAd, in our unpublished data. In this trial, the triple combination with a small viral dose (ad-TRAIL + ad-p63 + CRAd or ad-TRAIL + ad-TK + CRAd) had an anticancer effect that was superior to single-gene treatments. Such combinations can block more than one signaling pathway and reduce the chance of developing a substitute survival mechanism. If one signaling pathway is blocked, cancer cells can use other pathways for survival.

In the xenograft model of the present study, before day 7 the CRAd only group and the ad-TRAIL + CRAd group showed reduced tumor volumes. However, after stopping virus injection on day 7, the tumor volume increased in the CRAd only group. In contrast, the ad-TRAIL + CRAd group showed continuous reduction of tumor volume. The combination group (ad-TRAIL + CRAd) kept longer and more consistent tumor regression than any single treatment group because it had the ability of oncolysis by CRAd plus prolonged TRAIL gene expression, whereas the CRAd-only group was only able to kill tumor cells by oncolysis. Our study indicates that this combination strategy is effective and could be applied to the clinic. The authors believe that the strategy of combining CRAd with other adenoviruses carrying certain therapeutic genes represents an important future direction for gene therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Supported by Korea Science and Engineering Foundation grant number 2005-01159 (to M-W Sung) and 2008-00728(to C-T Lee).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • 1
    Lee CT, Park KH, Yanagisawa K et al. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus. Cancer Res 2004; 64: 66605.
  • 2
    Lee CT, Lee YJ, Kwon SY et al. In vivo imaging of adenovirus transduction and enhanced therapeutic efficacy of combination therapy with conditionally replicating adenovirus and adenovirus-p27. Cancer Res 2006; 66: 3727.
  • 3
    Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med 1997; 3: 63945.
  • 4
    Heise C, Hermiston T, Johnson L et al. An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat Med 2000; 6: 11349.
  • 5
    Kirn D. Replication-selective oncolytic adenoviruses: virotherapy aimed at genetic targets in cancer. Oncogene 2000; 19: 66609.
  • 6
    Suzuki K, Fueyo J, Krasnykh V, Reynolds PN, Curiel DT, Alemany R. A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency. Clin Cancer Res 2001; 7: 1206.
  • 7
    Wiley SR, Schooley K, Smolak PJ et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995; 3: 67382.
  • 8
    Griffith TS, Lynch DH. TRAIL: a molecule with multiple receptors and control mechanisms. Curr Opin Immunol 1998; 10: 55963.
  • 9
    Zhang L, Fang B. Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther 2005; 12: 22837.
  • 10
    Ozören N, Fisher MJ, Kim K et al. Homozygous deletion of the death receptor DR4 gene in a nasopharyngeal cancer cell line is associated with TRAIL resistance. Int J Oncol 2000; 16: 91725.
  • 11
    Lee SH, Shin MS, Kim HS et al. Alterations of the DR5/TRAIL receptor 2 gene in non-small cell lung cancers. Cancer Res 1999; 59: 56836.
  • 12
    Eggert A, Grotzer MA, Zuzak TJ, Wiewrodt BR, Ikegaki N, Brodeur GM. Resistance to TRAIL-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression. Med Pediatr Oncol 2000; 35: 6037.
  • 13
    Griffith TS, Chin WA, Jackson GC, Lynch DH, Kubin MZ. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells. J Immunol 1998; 161: 283340.
  • 14
    Kim K, Fisher MJ, Xu SQ, El-Deiry WS. Molecular determinants of response to TRAIL in killing of normal and cancer cells. Clin Cancer Res 2000; 6: 33546.
  • 15
    Leverkus M, Neumann M, Mengling T et al. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res 2000; 60: 5539.
  • 16
    Jang SH, Seol JY, Kim CH et al. Additive effect of TRAIL and p53 gene transfer on apoptosis of human lung cancer cell lines. Int J Mol Med 2004; 13: 1816.
  • 17
    Seol JY, Park KH, Hwang CI et al. Adenovirus-TRAIL can overcome TRAIL resistance and induce a bystander effect. Cancer Gene Ther 2003; 10: 5408.
  • 18
    Ren XW, Liang M, Meng X et al. A tumor-specific conditionally replicative adenovirus vector expressing TRAIL for gene therapy of hepatocellular carcinoma. Cancer Gene Ther 2006; 13: 15968.
  • 19
    Wildner O, Blaese RM, Morris JC. Therapy of colon cancer with oncolytic adenovirus is enhanced by the addition of herpes simplex virus-thymidine kinase. Cancer Res 1999; 59: 4103.
  • 20
    Nanda D, Vogels R, Havenga M, Avezaat CJ, Bout A, Smitt PS. Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus-tymidine kinase. Cancer Res 2001; 61: 874350.
  • 21
    Geoerger B, Vassal G, Opolon P et al. Oncolytic activity of p53-expressing conditionally replicative adenovirus AdΔ24-p53 against human malignant glioma. Cancer Res 2004; 64: 57539.
  • 22
    Hawkins LK, Johnson L, Bauzon M et al. Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region. Gene Ther 2001; 8: 112331.
  • 23
    Hawkins LK, Hermiston TW. Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region. Gene Ther 2001; 8: 113241.
  • 24
    Hawkins LK, Hermiston T. Gene delivery from the E3 region of replicating human adenovirus: evaluation of the E3B region. Gene Ther 2001; 8: 11428.
  • 25
    Hermiston TW, Kuhn I. Armed therapeutic viruses: strategies and challenges to arming oncolytic viruses with therapeutic genes. Cancer Gene Ther 2002; 9: 102235.
  • 26
    Huang TG, Savontaus MJ, Shinozaki K, Sauter BV, Woo SL. Telomerase-dependent oncolytic adenovirus for cancer treatment. Gene Ther 2003; 10: 12417.
  • 27
    Huang Q, Zhang X, Wang H et al. A novel conditionally replicative adenovirus vector targeting telomerase-positive tumor cells. Clin Cancer Res 2004; 10: 143945.
  • 28
    Davydova J, Le LP, Gavrikova T, Wang M, Krasnykh V, Yamamoto M. Infectivity-enhanced cyclooxygenase-2-based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment. Cancer Res 2004; 64: 431927.
  • 29
    Kanerva A, Bauerschmitz GJ, Yamamoto M et al. A cyclooxygenase-2 promoter-based conditionally replicating adenovirus with enhanced infectivity for treatment of ovarian adenocarcinoma. Gene Ther 2004; 11: 5529.