Volume 9, Issue 6, Pages 804-817 (June 2004) A Conditionally Replicating Adenovirus for Nasopharyngeal Carcinoma Gene Therapy  Marie C Chia, Wei Shi, Jian-Hua Li, Otto Sanchez, Craig A Strathdee, Dolly Huang, Pierre Busson, Henry J Klamut, Fei-Fei Liu  Molecular Therapy  Volume 9, Issue 6, Pages 804-817 (June 2004) DOI: 10.1016/j.ymthe.2004.03.016 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Schematic of the construction of adv.oriP.E1A. The E1A transcriptional unit was cloned downstream of the oriP-basal CMV promoter into the pΔE1SP1A shuttle vector. A novel recombinant adenovirus was generated in 293 cells after homologous recombination of the pΔE1SP1A vector with pJM17. This CRA hence does not contain E1B the sequence. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Selective expression of E1A in EBV-positive cells. EBV-positive C666-1 and EBV-negative CNE-1 and CNE-2Z NPC cell lines were mock infected (lanes 1) or infected with either adv.oriP.Luc (lanes 2) or adv.oriP.E1A (lanes 3–7) with cell lysates analyzed for expression of the 41-kDa E1A protein product. CNE-1, CNE-2Z, and control C666-1 lysates (lanes 1 and 2) were obtained 48 h postinfection. C666-1 cell lysates were prepared at 8, 24, 30, 48, and 144 h (lanes 3–7, respectively) postinfection with adv.oriP.E1A. Cell lysates from 293 cells, which constitutively express E1A, provided the positive control. All three NPC cell lines are easily transfectable by adv (>90% transfection efficiency with 2 pfu/cell). Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Effects of adv.oriP.E1A on human cell lines. (A) Lack of cytopathic effects in EBV-negative cell lines. A549 (lung carcinoma), MDA-MB-231 (breast adenocarcinoma), SaOS-2 (osteosarcoma), CNE-2Z (nasopharyngeal carcinoma), and KS-1 (nasopharyngeal fibroblast) cells were infected with 1, 10, or 25 pfu/cell of adv.oriP.E1A, fixed, and stained 4 days postinfection. There is no evidence of a cytopathic effect in these cells. All cells are efficiently transduced by the adenovirus as demonstrated by β-galactosidase expression after 10 pfu/cell of adv.CMV.B-gal. (B) Top: Dose-dependent decrease in cell viability as determined by MTT assay in C666-1 cells. EBV-positive C666-1 cells were infected with either adv.oriP.E1A or adv.oriP (25 pfu/cell) and assayed for cell viability 7 days postinfection. Radiation (6 Gy) was administered 24 h postinfection. Experiments were conducted three independent times and are reported as means ± SEM. Bottom: Cells were infected with 10 pfu/cell of adv.oriP.E1A, fixed, and stained at 3, or 5 days postinfection. Mock-infected cells grew to confluency, in contrast to adv.oriP.E1A-infected cells, which died. (C) Wild-type adv.5 elicits cytotoxicity earlier than adv.oriP.E1A. C666-1 cells were infected with either adv.oriP.E1A or wild-type adv.5 with 2.5, 5, 10, 25, or 50 pfu/cell. Cell viability was assessed 1, 4, or 7 days postinfection. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Effects of adv.oriP.E1A on human cell lines. (A) Lack of cytopathic effects in EBV-negative cell lines. A549 (lung carcinoma), MDA-MB-231 (breast adenocarcinoma), SaOS-2 (osteosarcoma), CNE-2Z (nasopharyngeal carcinoma), and KS-1 (nasopharyngeal fibroblast) cells were infected with 1, 10, or 25 pfu/cell of adv.oriP.E1A, fixed, and stained 4 days postinfection. There is no evidence of a cytopathic effect in these cells. All cells are efficiently transduced by the adenovirus as demonstrated by β-galactosidase expression after 10 pfu/cell of adv.CMV.B-gal. (B) Top: Dose-dependent decrease in cell viability as determined by MTT assay in C666-1 cells. EBV-positive C666-1 cells were infected with either adv.oriP.E1A or adv.oriP (25 pfu/cell) and assayed for cell viability 7 days postinfection. Radiation (6 Gy) was administered 24 h postinfection. Experiments were conducted three independent times and are reported as means ± SEM. Bottom: Cells were infected with 10 pfu/cell of adv.oriP.E1A, fixed, and stained at 3, or 5 days postinfection. Mock-infected cells grew to confluency, in contrast to adv.oriP.E1A-infected cells, which died. (C) Wild-type adv.5 elicits cytotoxicity earlier than adv.oriP.E1A. C666-1 cells were infected with either adv.oriP.E1A or wild-type adv.5 with 2.5, 5, 10, 25, or 50 pfu/cell. Cell viability was assessed 1, 4, or 7 days postinfection. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Replication of adv.oriP.E1A in C666-1 cells. (A) Southern blot analysis of E1A shows time-dependent increase in adenoviral DNA. EBV-positive C666-1 cells were mock infected (lane 1) or infected with adv.oriP (lane 2) or 1 pfu/cell adv.oriP.E1A (lanes 3–6) or wild-type adv.5 (lanes 7–9). DNA was isolated at 24, 48, or 72 h postinfection, digested with EcoRI, and probed for E1A. (B) Increase in E1A expression in C666-1 cells over time. Quantitative real-time PCR was conducted to assess E1A gene expression over time. EBV-positive C666-1 cells were infected with 1 pfu/cell of either adv.oriP.E1A or the control virus adv.oriP for 1 h at 37°C. Cellular and viral DNA was extracted using the QiaAMP DNA extraction assay. Ct values were normalized to β-actin expression and compared to values obtained at time 0. PCR was performed in triplicate. (C) Western blot analysis of increasing fiber knob protein expression over time. C666-1 cells were mock infected (lane 1) or infected with either adv.oriP.E1A (lanes 2–7, top) or adv.oriP (lanes 2–7, bottom) and cell lysates were analyzed for expression of the 62-kDa fiber knob protein product. C666-1 cell lysates were prepared at 4, 8, 12, 24, 48, or 72 h (lanes 2–7, respectively) postinfection with adv.oriP.E1A or adv.oriP. β-Actin was used as loading control (41 kDa). Increase in fiber knob protein expression is indicative of adenoviral replication. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Lack of tumor formation after ex vivo treatment with adv.oriP.E1A. EBV-positive C666-1 cells were infected in vitro with 15 pfu/cell of either adv.oriP.E1A (50 or 100%) or adv.oriP for 1 h. After overnight incubation at 37°C, cells were injected intramuscularly into SCID mice. Tumor plus leg diameter was then followed three times weekly until 15 mm or for humane endpoints. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Significant delay of tumor growth in vivo. EBV-positive (A) C666-1 or (B) C15 xenograft tumors were established in SCID mice, and once they reached 0.3–0.4 g, animals were randomized into one of four groups: (1) control, no treatment; (2) RT alone, 2 × 4 Gy (C666-1), 2 × 2 Gy (C15); (3) i.t. adv.oriP.E1A; (4) i.t. adv.oriP.E1A + RT. Treatment schedule was as indicated, with each injection comprising 1 × 109 pfu in 100 μl volume. Statistical significance was determined using the one-way ANOVA. (A) Control vs adv.oriP.E1A + RT, P = 0.002. (B) Control vs adv.oriP.E1A + RT, P = 0.02. At least two independent experiments were conducted for each xenograft model. (C) Change in E1A expression in C666-1 tumors over time. Quantitative real-time PCR was used to assess the increase in E1A gene expression over time. Tumor-bearing SCID mice were injected i.t. with 1.4 × 107 pfu of adv.oriP.E1A or PBS. Tumors were removed 4, 8, or 24 h postinjection and assessed for E1A gene copy number. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Minimal systemic toxicity of adv.oriP.E1A in vivo. (A) Hematoxylin and eosin-stained sections of liver, heart, spleen, and kidney from mice treated with systemic administration of PBS control, adv.oriP, or adv.oriP.E1A. (B) Lung inflammation and occlusion of the alveolar space was observed after high doses (109 pfu) of systemic administration of adv.oriP.E1A. Molecular Therapy 2004 9, 804-817DOI: (10.1016/j.ymthe.2004.03.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions