There is an urgent need for improved therapies for inoperable metastatic colon cancer. Gene-directed enzyme prodrug therapy (GDEPT) using adenovirus vectors works well in preclinical models of this disease, but successful clinical application is hampered by an inability to construct vectors that express at high levels in infected tumor cells but not in infected normal cells. Constitutive activation of beta-catenin-dependent gene expression is almost certainly a key causative event in the genesis of colon and some other cancers. Here we have exploited this oncogenic defect to design a synthetic promoter, CTP1, that, in contrast to currently available tumor-selective promoters, is both highly active in cancer cells and highly cancer-cell-specific. CTP1 directs high-level beta-galactosidase expression in freshly isolated biopsies of secondary colon cancer, but is not detectably active in associated normal liver tissue. We also demonstrate that CTP1 can direct high-level, tumor-specific therapeutic gene expression in vivo. Intratumoral injection of an adenovirus vector encoding Escherichia coli nitroreductase driven by CTP1 efficiently sensitized SW480 xenografts to the prodrug CB1954, whereas systemic vector and prodrug administration produced no apparent signs of toxicity. CTP1 may form the basis for effective, targeted gene therapy of metastatic colon cancer and other tumors with deregulated beta-catenin/T cell factor.

ABSTRACT

Expression of the transcription unit early region 2 (E2) is of crucial importance for adenoviruses because this region encodes proteins essential for viral replication. Here, we demonstrate that the E1A 12S protein of the oncogenic adenovirus serotype 12 activates the E2 promoter in dependence of the N terminus and the conserved region 1. Activation is mediated through a cAMPresponse element that is bound by CREB-1 and ATF-1. Moreover, the Ad12 E2 promoter is inducible by protein kinase A and repressed by either a dominant-negative cAMP-response element-binding protein (CREB) mutant or the highly specific protein kinase A inhibitor protein underscoring the participation of CREB-1/ATF-1 in promoter activation. E1A 12S binds to CREB-1 and ATF-1 in dependence of the N terminus and CR1 and is recruited to the E2 cAMP-response element through both cellular transcription factors. Most interestingly, point mutations revealed that E1A 12S domains essential for binding to CREB-1/ATF-1 and for activation of the Ad12 E2 promoter are also essential for binding to the CREB-binding protein. Due to these data and results obtained in DNA-dependent protein-protein interaction assays, we propose a model in which the cAMP-independent activation of the Ad12 E2 promoter is mediated through a ternary complex consisting of CREB-1/ATF-1, E1A 12S , and CREB-binding protein, which assembles on the E2 cAMP-response element.

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

Association with the cellular coactivators p300 and CBP is required for the growth-regulatory function of adenoviral (Ad) early region 1A (E1A) proteins. E1A regions necessary for these interactions overlap with domains involved in the induction of tumours in immunocompetent rodents through highly oncogenic Ad12. Differences in the association of cellular factors with the respective E1A domains of Ad12 and nononcogenic Ad2 might therefore be involved in serotype-specific oncogenicity. We analyzed the interaction of the Ad12 E1A 235R protein with p300 and CBP. Here we demonstrate that in the case of Ad12, but not Ad2/5, amino acids (aa) 1-29 of E1A proteins are sufficient to bind the p300-C/H3 domain in vivo and wild-type p300 in vitro. The conserved arginine-2, which is essential for the interaction between Ad2 E1A and p300, was dispensable for the Ad12 E1A 235R-p300 interaction in vitro. In addition to the p300-C/H3 region, we identified a second domain within p300 (aa 1999-2200) binding to the 235R protein. Contrary to p300, the amino-terminus and CR1 are necessary to associate with CBP. The aa 1-29 of the 235R protein but not CR1 are essential for the repression of colTRE-driven gene expression. This repression function is strictly dependent on p300 but not on CBP.