Volume 10, Issue 1, Pages 150-161 (July 2004) Optimization of a synthetic β-catenin-dependent promoter for tumor-specific cancer gene therapy  Kai S. Lipinski, Hakim A. Djeha, Jonathan Gawn, Suzanne Cliffe, Norman J. Maitland, Daniel H. Palmer, Andrew Mountain, Alistair S. Irvine, Christopher J. Wrighton  Molecular Therapy  Volume 10, Issue 1, Pages 150-161 (July 2004) DOI: 10.1016/j.ymthe.2004.03.021 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Substitution of the basal SV40 promoter from CTP1 with a TATA box increases the β-catenin dependence. (A) Composition of the CTP2 promoter. From CTP1 the basal SV40 promoter was exchanged with the Ad5 E1B TATA box. The spacing between the five Tcf sites is indicated and is the same as for CTP1. d = 46 indicates the distance from the most proximal Tcf site to the start of the TATA box in base pairs. (B) Permissive SW480 (left) and nonpermissive HeLa (right) cells were transfected with the indicated luciferase reporter constructs. Luciferase activity was measured from cell lysates after 48 h. Fold activation is relative to the promoterless plasmid pGL3basic. A representative experiment of three experiments is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Variation of the distance between Tcf-binding sites and the distance of the sites to the TATA box significantly alters promoter activity. (A, top) Arrangement of Tcf sites along the DNA helix axis for CTP2, TcfA, TcfB, and TcfC with the assumption that one round (360°) of the DNA helix corresponds to about 10.4 bp. Number 1 is the most proximal and 5 is the most distal Tcf site. (Bottom) SW480 cells were transfected with the indicated luciferase reporter constructs. Luciferase activity was measured from cell lysates after 48 h. A representative experiment is shown. (B, top) The distance between the most proximal of the TcfC Tcf sites and the E1B TATA box was varied (d = 25/46/140/499 bp, respectively) and its effect on promoter activity determined. Note that the construct with d = 46 is identical to TcfC-E1BTATA in A. (Bottom) SW480 cells were transfected with the indicated luciferase reporter constructs. Luciferase activity was measured from cell lysates after 48 h. The activity of TcfC-(d = 46)-E1BTATA-Luc was set as 100%. The promoter from the most active construct (d = 25 bp) was named CTP3. A representative experiment of three experiments is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 The optimized promoter CTP4 gives the best activity/specificity profile. (A) Composition of the CTP4 promoter. Numbers indicate the base pairs between the Tcf sites and the distance from the end of the most proximal site to the start of the TATA box. (B) Permissive SW480 and nonpermissive HeLa cells were transiently transfected using the indicated luciferase reporter constructs. Luciferase activity was monitored after 24 h from whole-cell lysates. Experiments were done in triplicate, and the means and SD are shown. A representative experiment of two experiments is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 CTP4 shows improved activity in permissive SW480 and HepG2 cells. Several independent virus preparations of Ad.CMV-LacZ, Ad.CTP1-LacZ, Ad.CTP3-LacZ, and Ad.CTP4-LacZ were compared to analyze preparation dependence. (A) SW480 and (B) HepG2 cell were infected at an m.o.i. of 200 and cell lysates were prepared after 48 h and analyzed for β-gal expression. A representative experiment of three is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Promoter breakthrough from CTP4 is reduced to less than mock levels in nonpermissive cells. Activity of CMV, CTP1, CTP3, and CTP4 in nonpermissive (A) HeLa cells, (B) dendritic cells, and (C) adenoviral vector producer cell lines PER.C6, 293, and 911, testing promoter activity if adenoviral replication occurs, is shown. Cells were infected with the indicated viral constructs at an m.o.i. of (A) 200, (C) 100, or (B) as indicated. Cell lysates were prepared 48 h later and analyzed for β-gal expression. A representative experiment of three is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Breakthrough activity from CTP1 promoter but not CTP4 results in inhibition of luciferase protein translation/expression. A CMV-Luc reporter plasmid (1.0 μg) was cotransfected with pGL3basic, CTP1-DTA, or CTP4-DTA plasmid (each 1.0 μg) into nonpermissive HeLa cells. Luciferase activity was measured after 48 h from whole-cell lysates. Assays were done in triplicate showing the mean ± SD. A representative experiment of two experiments is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 An adenoviral vector, Ad.CTP4-DTA, expressing DTA from the CTP4 promoter, specifically inhibits protein production. SW480 and HeLa cells were first infected with Ad.CMV-Luc (m.o.i. = 50) and after 24 h cells were infected with either Ad.CTP4-DTA or Ad.CTP4-LacZ (m.o.i. = 50). Luciferase activity was analyzed from whole-cell extracts after 48 h. Data were done in triplicate showing the means ± SD. A representative experiment of two experiments is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 8 Ad.CTP4-DTA kills specifically permissive, β-catenin-deregulated tumor cell lines. SW480, HepG2, HeLa, HNX14C, and HUVEC were infected with Ad.CTP4-DTA (hatched) or Ad.CTP4-LacZ control virus (black) at the indicated m.o.i. After 4 days cell viability was analyzed using the MTS substrate assay (Promega) and reading the OD450 inversely correlating to cell death. Within one experiment infections were done in triplicate of which the mean OD450 values are presented. One representative experiment of at least two is shown. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 9 CTP4 activity in human primary prostate and ovarian tumor cultures. (A–C) Primary prostate and (D and E) ovarian tumor cultures were infected with 500 pfu per cell of either Ad.CMV-LacZ (A, D) or Ad.CTP4-LacZ (B, E). 24 h later cultures were analyzed for LacZ expression by X-gal staining. (C) The prostate culture was also stained for β-catenin after viral infection. One representative tumor of two positive independent prostate samples is shown. The shown prostate tumor was classified as prostate carcinoma, grade gleason 7 (4 = 3) with positive bone scan. The clinical grading of the ovarian tumor sample is not known. Molecular Therapy 2004 10, 150-161DOI: (10.1016/j.ymthe.2004.03.021) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions