Volume 11, Issue 4, Pages 563-577 (April 2005) Viral and nonviral factors causing nonspecific replication of tumor- and tissue-specific promoter-dependent oncolytic adenoviruses  Almudena Hurtado Picó, Xiaomin Wang, Isaac Sipo, Ulrike Siemetzki, Jürgen Eberle, Wolfgang Poller, Henry Fechner  Molecular Therapy  Volume 11, Issue 4, Pages 563-577 (April 2005) DOI: 10.1016/j.ymthe.2004.10.021 Copyright © 2004 Terms and Conditions

Fig. 1 Schematic illustration of adenovector constructs. Abbreviations: 5′ITR, left inverted terminal repeat of adenovirus 5 plus adenoviral left side sequences; TetO7, seven direct repeats of a 42-bp sequence containing the tet operator, which is the natural binding site of TetR; HRE, human vascular endothelial growth factor 5′ flanking sequence containing a hypoxia-responsive element; AFP, α-fetoprotein promoter; SPB, pulmonary surfactant protein B promoter; CEA, carcinoembryonic antigen promoter; 2hE-Tyr, human tandem tyrosinase enhancer; Tyr, tyrosinase promoter; CMVmin, minimal immediate CMV promoter, lacking the CMV enhancer (encompasses the sequence between −53 and +75 of the human CMV IE1 gene promoter region); luc, luciferase cDNA; E1A, adenoviral E1A cDNA; E1AΔpRB, adenoviral E1A cDNA with deletion of amino acids 122–129; polyA, bovine growth hormone poly(A) signal; a-polyA, artificial poly(A) signal; 3′ITR, right inverted terminal repeat of adenovirus 5. Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 2 Specificity and strength of tissue- and tumor-specific promoters. Cells were transduced with 2.5 × 103 p/c of the respective ttsP-rdAdV and the control AdV Ad5TRE-luc. For the Tet-On system, cells were cotransduced with 2.5 × 103 p/c each of Ad5TRE-luc and the transactivator vector Ad5CMVrtTA-M2, and luciferase expression was induced by adding 1 μg/ml doxycycline. Luciferase reporter gene activity was determined 24 h later. All ttsP's with the exception of CEA showed strictly target-cell-specific activity. With the exception of hTyr2E/P the activity of the ttsP's did not even reach the level of the CMVmin promoter in Ad5TRE-luc. Mock, nontransduced cells. Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 3 Differential Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB replication in nontarget HeLa cells. (A) Determination of adenoviral DNA replication. Cells were infected with 2 × 103 and 4 × 103 p/c Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB, respectively, and adenoviral DNA replication was determined by competitive PCR after 24 h, as described under Material and Methods. aU, arbitrary units. (B) Determination of cytotoxicity. HeLa cells were infected with various titers of Ad5TetO7CEA-E1AΔpRB, Ad5TetO7SPB-E1AΔpRB, or the control rdAdV Ad5TRE-iGFP. Cells were stained with crystal violet 3 days after infection. mock, nontransduced cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 4 Influence of 5′-terminal adenoviral E1A enhancers, transcriptional regulator elements, and E1A-13S on the activity of ttsP's. Four different tyrosinase-, three AFP-, and two CEA- and SPB-promoter luciferase reporter gene constructs were transfected into HeLa cells (A) without or (B) with cotransfected pTRE-13S, which drives E1A-13S from the CMVmin promoter. Luciferase activities were determined 48 h after transfection. (A) Activity of the ttsP's. For comparison, the activities of constructs without regulator elements and without 5′-terminal adenoviral E1A enhancers were set to 1. (B) E1A-13S-mediated transactivation of ttsP's. Increase in luciferase activity is shown as the ratio of ttsP-driven luciferase activity in the presence of E1A-13S to ttsP-driven luciferase expression in the absence of E1A-13S. The complete experiment was performed three times, of which each time showed triple values. Plasmid transfection efficiency was controlled by competitive PCR as described under Material and Methods. Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 5 Mechanisms involved in inhibition of ttsP activity by tTS. (A) Dox-dependent suppression of ttsP activity. Cells were cotransduced with Ad5CMVtTS in combination with Ad5TetO7CEA-luc, Ad5TetO7SPB-luc, Ad5TetO7hTyr2E/P-luc, or Ad5TetO7[HRE]AFP-luc (each 2.5 × 103 p/c). Decrease in luciferase activity was determined after 24 h and is given by the ratio of activity in the presence vs in the absence of Dox (1 μg/ml). Silencing of ttsP activity could not be determined when luciferase activity in the presence of Dox was already comparably high compared to that found in nontransduced cells (*). (B) tTS inhibits E1A-mediated transactivation of ttsP's. HeLa cells were transfected with 1.3 μg/well pAd5TetO7[HRE]AFP-luc and infected after 24 h with 2.5 × 103 p/c of Ad5CMVtTS and 5 × 102 p/c of a RCA (E1A protein is driven by the native E1A promoter) or with 5 × 102 p/c of the control Ad5TRE-iGFP. Dox was added (1 μg/ml) and luciferase activities were determined 24 h later. Luciferase activities were reduced more strongly by tTS-mediated Dox-dependent silencing in the presence of E1A (3.4-fold) than in its absence (1.63-fold), indicating that tTS not only silences the [HRE]AFP promoter but also inhibits its E1A-mediated transactivation. Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 6 Doxycycline-dependent inhibition of ttsP-RRCA replication. (A and B) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB cytotoxicity. HeLa cells were infected with 2.5 × 103 p/c Ad5CMVtTS and (A) with 2–8 × 103 p/c of Ad5TetO7CEA-E1AΔpRB or (B) with Ad5TetO7SPB-E1AΔpRB. tTS binding to the TetO7 was inhibited by daily application of 1.5 μg/ml Dox. Top: Cells were stained with crystal violet 3 days (Ad5TetO7CEA-E1AΔpRB) or 4 days (Ad5TetO7SPB-E1AΔpRB) after infection. Bottom: Cells were trypsinized and stained with trypan blue, and living cells were counted. (C) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB DNA replication. HeLa cells were infected with 2 × 103 p/c Ad5CMVtTS and 4 × 103 p/c Ad5TetO7CEA-E1AΔpRB or Ad5TetO7SPB-E1AΔpRB and incubated for 24 h in the presence or absence of Dox (1 μg/ml). Thereafter, cells were harvested and adenoviral DNA replication was determined by competitive PCR as described under Material and Methods. Adenoviral DNA load is given in arbitrary units (aU). (D) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB replication. HeLa cells were infected with 2.5 × 103 p/c Ad5CMVtTS and with 2 × 103 p/c Ad5TetO7CEA-E1AΔpRB or with Ad5TetO7SPB-E1AΔpRB for 2 h. After a wash fresh medium was added and cells were incubated in the presence or absence of Dox (100 ng/ml). Dox was added daily directly into the medium. Ad5TetO7CEA-E1AΔpRB-infected cells were harvested 2 days after and Ad5TetO7SPB-E1AΔpRB-infected cells 3 days after infection, when CPE became visible in cells incubated in the presence of Dox. Virus titers were determined by plaque assay in HEK 293 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 6 Doxycycline-dependent inhibition of ttsP-RRCA replication. (A and B) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB cytotoxicity. HeLa cells were infected with 2.5 × 103 p/c Ad5CMVtTS and (A) with 2–8 × 103 p/c of Ad5TetO7CEA-E1AΔpRB or (B) with Ad5TetO7SPB-E1AΔpRB. tTS binding to the TetO7 was inhibited by daily application of 1.5 μg/ml Dox. Top: Cells were stained with crystal violet 3 days (Ad5TetO7CEA-E1AΔpRB) or 4 days (Ad5TetO7SPB-E1AΔpRB) after infection. Bottom: Cells were trypsinized and stained with trypan blue, and living cells were counted. (C) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB DNA replication. HeLa cells were infected with 2 × 103 p/c Ad5CMVtTS and 4 × 103 p/c Ad5TetO7CEA-E1AΔpRB or Ad5TetO7SPB-E1AΔpRB and incubated for 24 h in the presence or absence of Dox (1 μg/ml). Thereafter, cells were harvested and adenoviral DNA replication was determined by competitive PCR as described under Material and Methods. Adenoviral DNA load is given in arbitrary units (aU). (D) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB replication. HeLa cells were infected with 2.5 × 103 p/c Ad5CMVtTS and with 2 × 103 p/c Ad5TetO7CEA-E1AΔpRB or with Ad5TetO7SPB-E1AΔpRB for 2 h. After a wash fresh medium was added and cells were incubated in the presence or absence of Dox (100 ng/ml). Dox was added daily directly into the medium. Ad5TetO7CEA-E1AΔpRB-infected cells were harvested 2 days after and Ad5TetO7SPB-E1AΔpRB-infected cells 3 days after infection, when CPE became visible in cells incubated in the presence of Dox. Virus titers were determined by plaque assay in HEK 293 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 6 Doxycycline-dependent inhibition of ttsP-RRCA replication. (A and B) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB cytotoxicity. HeLa cells were infected with 2.5 × 103 p/c Ad5CMVtTS and (A) with 2–8 × 103 p/c of Ad5TetO7CEA-E1AΔpRB or (B) with Ad5TetO7SPB-E1AΔpRB. tTS binding to the TetO7 was inhibited by daily application of 1.5 μg/ml Dox. Top: Cells were stained with crystal violet 3 days (Ad5TetO7CEA-E1AΔpRB) or 4 days (Ad5TetO7SPB-E1AΔpRB) after infection. Bottom: Cells were trypsinized and stained with trypan blue, and living cells were counted. (C) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB DNA replication. HeLa cells were infected with 2 × 103 p/c Ad5CMVtTS and 4 × 103 p/c Ad5TetO7CEA-E1AΔpRB or Ad5TetO7SPB-E1AΔpRB and incubated for 24 h in the presence or absence of Dox (1 μg/ml). Thereafter, cells were harvested and adenoviral DNA replication was determined by competitive PCR as described under Material and Methods. Adenoviral DNA load is given in arbitrary units (aU). (D) tTS-mediated inhibition of Ad5TetO7CEA-E1AΔpRB and Ad5TetO7SPB-E1AΔpRB replication. HeLa cells were infected with 2.5 × 103 p/c Ad5CMVtTS and with 2 × 103 p/c Ad5TetO7CEA-E1AΔpRB or with Ad5TetO7SPB-E1AΔpRB for 2 h. After a wash fresh medium was added and cells were incubated in the presence or absence of Dox (100 ng/ml). Dox was added daily directly into the medium. Ad5TetO7CEA-E1AΔpRB-infected cells were harvested 2 days after and Ad5TetO7SPB-E1AΔpRB-infected cells 3 days after infection, when CPE became visible in cells incubated in the presence of Dox. Virus titers were determined by plaque assay in HEK 293 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions

Fig. 7 Mechanisms of nonspecific ttsP-RRCA replication in nontarget cells—approaches for overcoming this problem. (A) Enhancement of ttsP activity by E1A enhancers. (B) Influence on ttsP by transcriptional regulator sequences (TR). (C) Feedback transactivation of ttsP by E1A-13S. (D) Transactivation of adenoviral promoters by E1A-13S. (E) Amplification of ttsP's by de novo synthesis of adenoviral genomes. (F) Enhancement of feedback loop. (G) ttsP-RRCA replication in nontarget cells. Molecular Therapy 2005 11, 563-577DOI: (10.1016/j.ymthe.2004.10.021) Copyright © 2004 Terms and Conditions