Steroid mediated gene delivery: focus on androgens
The use of gene therapy to treat genetic disease states has increasingly become the focus of research. The ideal gene delivery system should have at least the following characteristics: the ability to target cells specifically, the capacity to transduce a large number of cells regardless of their mitotic status, and the potential to be synthetic and of low toxicity.
Recently we have developed a new procedure to transfer genes. We proposed to target nuclear steroid receptors to achieve a receptor-dependent enhanced expression of transgene DNA. This strategy, termed steroid-mediated gene delivery (SMGD), facilitates the nuclear uptake of transfected DNA with the help of glucocorticoid receptors (GR). Nuclear receptors such as GR are natural cytoplasm-nucleus shuttles that have been proposed to actively trafficking via interacting with microtubuli. To this aim we synthesized a construct consisting of the synthetic steroid dexamethasone, a chemical spacer and a DNA-binding moiety, which was finally coupled to a reporter gene. The first DNA-binder was psoralene (Rebuffat et al, Nat Biotechnol. 2001 Dec; 19(12): 1155- 1161) and the optimized version contained peptide nucleic acid (PNA) (Rebuffat, FASEB J. 2002 Sep;16 (11): 1426-1428). Using this ligand-DNA-construct we showed that cells translocated and expressed transgene DNA. The transport and expression was specific for cells with, but not cells without GR, an effect even more pronounced in growth arrested than in proliferating cells.
These results were the first proof of priniple, that a chemically modified steroid ligand can be used for cell specific DNA delivery in vitro. A patent covering this strategy was accepted in Australia, New Zealand, USA and is in the nationalization phase in Japan, Canada and Europe.
In the present application we propose to target androgen receptors (AR) by SMGD. For that purpose we propose to synthesize a bifunctional steroid derivative consisting of an androgen moiety linked to a PNA clamp through a chemical spacer. The PNA will allow to link androgens to a defined position in a plasmid without interfering with the expression of the genes in the plasmid. The androgens first to be considered for the construct will be dihydrotestosterone, a steroid with a high affinity for both AR and sex hormone binding globulin (SHBG) and 7alpha-methyl-19-nortestosterone (7alpha-MENT) with a high affinity for AR but not for SHBG.
In order to ascertain that the constructs maintain their affinity and specificity for the AR, receptor binding studies, translocation and transactivation assays the cells with different expression levels of AR or AR-green fluorescent protein (AR-GFP) will be performed. The derivatives will be linked to a plasmid containing the gene of Herpes simplex virus type I thymidine kinase (HSV-TK) and/or firefly luciferase (LUC). The predicted enhanced HSV-TK activity in AR expressing cells will make these cells sensitive to gangciclovir (GCV). This effect will be investigated in cells with and without AR in vitro and in vivo in mice with AR-expressing prostate cancer or AR expressing orthotopic transitional cell carcinoma in the bladder. The expression of LUC will allow sequential monitoring of bioluminescence in vivo.
If successful, the present investigation will enhance the trafficking of DNA from the cytoplasm to the nucleus in AR-expressing cells, an effect with potential utility for clinical applications aiming at the selective macromolecular treatment of target tissues that express AR such as prostate cancer.
Swiss National Science Foundation, Project no. 107870