Valérie Jérôme

Gene therapy: designer promoters for tumour targeting

One of the biggest challenges facing cancer therapy is to generate tumour-specific treatment strategies. Gene therapy hopes to achieve this by directing the activity of therapeutic genes specifically to the sites of disease. Of paramount importance for the success of this approach is the availability of tumour-specific delivery systems: both the transductional targeting of the vector vehicle and the restriction of transgene expression to the tumour are promising strategies towards this goal. This review will focus on the recent achievements in the field of transcriptional targeting and the different strategies to improve or design promoters with the desired specificities.

A strategy for enhancing the transcriptional activity of weak cell type-specific promoters

Cell type- and tissue-specific promoters play an important role in the development of site-selective vectors for gene therapy. A large number of highly specific promoters has been described, but their applicability is often hampered by their inefficient transcriptional activity. In this study, we describe a new strategy for enhancing the activity of weak promoters without loss of specificity. The basic principle of this strategy is to establish a positive feedback loop which is initiated by transcription from a cell type-specific promoter. This was achieved by using a cell type-specific pro- moter to drive the simultaneous expression of the desired effector/reporter gene product and a strong artificial transcriptional activator which stimulates transcription through appropriate binding sites in the promoter. Using a VP16-LexA chimeric transcription factor, we show that this approach leads to a 14- to >100-fold enhancement of both the endothelial cell-specific von Willebrand factor promoter and the gastrointestinal-specific sucrase-isomaltase promoter while maintaining approximately 30- to >100-fold cell type specificity.

Cytotoxic T lymphocytes responding to low dose TRP2 antigen are induced against B16 melanoma by liposome-encapsulated TRP2 peptide and CpG DNA adjuvant.

The induction of a potent and specific T cell response is a major challenge in the development of efficacious cancer vaccine strategies. We applied a novel liposomal formulation (AVE3) for efficient delivery of antigenic peptides into APCs of the skin. These liposomes resulted in a long-lasting deposition of encapsulated compounds at the injection site and the draining lymph nodes. Using a peptide from the melanocyte differentiation antigen tyrosinase-related protein (TRP2) 2 we could show that vaccination with liposome-encapsulated peptide in combination with oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) as adjuvant leads to the induction of tumor cell-specific cytotoxic T cells. The most potent immune response was observed when both, TRP2 peptide and CpG ODNs, were encapsulated into AVE3. Importantly, in contrast to vaccination with free TRP2 liposomal TRP2 peptide generated T cells which respond to 1000-fold lower antigen concentration. Using the poorly immunogenic B16 melanoma model we could demonstrate that vaccination with liposomal TRP2 peptide plus CpG ODNs but not vaccination with free peptide or adjuvant alone resulted in tumor protection in subcutaneous and metastatic tumor models. In summary, vaccination with liposome-encapsulated peptide antigen and CpG ODN allows for the in vivo loading and activation of DC, thereby generating reactive CTL populations even against poorly immunogenic self-peptide presenting tumors resulting in a potent anti-tumor immune response.

A dual specificity promoter system combining cell cycle-regulated and tissue-specific transcriptional control

The expression of both proliferation-associated and cell type-specific genes is a hallmark of both cancer cells and tumor endothelial cells. The possibility to combine both features in a single transcriptional control unit would greatly increase the selectivity of vectors used for cancer gene therapy. Previous studies by our laboratory have shown that the transcription of several cell cycle genes is regulated by a novel cell cycle-regulated repressor, termed CDF-1. This repressor functions by blocking in resting cells the transcriptional activation by specific factors binding to the upstream activating sequence (UAS), most notably the CCAAT-box binding factor NF-Y/CBF. Based on this work we have developed a dual specificity promoter system that combines cell type specificity with cell cycle regulation. A chimeric transcription factor (Gal4/NF-Y) consisting of the transactivation domain of NF-Y and the DNA-binding domain of Gal4 is expressed from a tissue-specific promoter. Gal4/NF-Y can bind to a second promoter consisting of a minimal cyclin A promoter with multiple Gal4 binding sites replacing the normal UAS. This leads to the tissue-specific expression of Gal4/NF-Y whose stimulatory activity on the promoter is restrained in resting cells by the recruitment of the CDF-1 repressor to the promoter. The functionality of this system is demonstrated for the specific transcriptional targeting of proliferating melanoma cells, where cell cycle regulation was >20-fold and cell type specificity was >50-fold.

A novel, transformation-relevant activation domain in Fos proteins.

We have previously demonstrated that transformation by Fos is critically dependent on an intact DNA-binding domain (bZip) and a functional N-terminal transactivation motif (N-TM). We now show that a novel motif (C-terminal transactivation motif [C-TM]) near the C terminus also plays an important role in both transformation and the activation of AP1-dependent transcription and that the hydrophobic amino acids in the C-TM are functionally essential. The C-TM is the most crucial element in the C-terminal transactivation domain in Fos, as indicated by its relative strength and context-independent function. The C-TM is clearly different from the previously identified HOB2 domain, located N terminally to the C-TM, and the C-terminally positioned TATA-binding protein-binding domain. We also show that the C-terminal transactivation domain strongly synergizes with the HOB1-like N-TM, even when both domains are present on different proteins within a dimeric complex, and that the C-TM plays a crucial role in this cooperation. These observations can be corroborated in a model in which multiple contacts with the basal machinery are established either to stabilize the transcription complex or to facilitate its sequential assembly.

A synthetic leucine zipper-based dimerization system for combining multiple promoter specificities

One of the biggest challenges facing gene therapy is the development of vectors that direct the activity of therapeutic genes specifically to the sites of disease. To achieve this goal, the restriction of transgene transcription via synthetic promoters that are endowed with multiple specificities represents a particularly promising strategy. Towards this end, we have developed a generally applicable strategy (DCTF system) where a synthetic promoter is driven by an artificial heterodimeric transcription factor whose DNA-binding and transactivating subunits are expressed from two promoters with different selectivity. A crucial determinant of the DCTF system is the heterodimerization interface that should provide for a high affinity interaction without interference by endogenous proteins. Here, we describe such a dimerization system based on engineered Fos and Jun leucine zippers. We show the usefulness of this system for the combination of cell type-specific and cell cycle-regulated transcription and demonstrate its functionality in an in vivo setting.