Adenovirus-mediated tissue-targeted expression of a Caspase-9-based artificial death switch for the treatment of prostate cancer

Clinical experience with suicide gene therapy for prostate cancer using fir st-generation approaches has provided a basis for developing improved strat egies. Given the low proliferation rate exhibited by prostate cancer, one i mprovement would be to develop suicide genes that effectively kill both div iding and nondividing cells. A second improvement would be to restrict cyto toxicity to prostate cancer cells, limiting injury of nondiseased tissue. H ere we describe a novel approach to achieving both goals based on: (a) the use of a small, but potent, prostate-specific composite promoter, ARR(2)PB, based on the rat probasin gene; and (b) the use of a powerful artificial d eath switch, called inducible caspase-9 (iCaspase-9). ARR(2)PB includes two copies of the androgen response region (ARR), each containing two androgen receptor (AR)-binding sites, placed upstream of the probasin promoter elem ents necessary for basal transcription. Because iCaspase-9 contains two bin ding sites for the dimeric ligand, AP20187, administration of chemical indu cers of dimerization leads to aggregation and caspase activation, followed by rapid apoptosis in both dividing and nondividing cells. Using both reage nts, we constructed two novel adenoviruses (ADVs), ADV.ARR(2)PB-iCasp9 expr essing iCaspase-9 and control ADV.ARR(2)PB-EGFP expressing enhanced green f luorescent protein (EGFP). We demonstrate that tissue specificity is not sa crificed in an ADV backbone because the marker protein, EGFP, is expressed in R1881-stimulated ADV.ARR(2)PB-EGFP-transduced LNCaP cells but not in AR( -) PC-3, 293, HuH-7, U-87, and MCF-7 cells. Similarly, Pro-iCaspase-9 is ex pressed in ADV.ARR(2)PB-iCasp9-infected LNCaP cells after R1881 administrat ion and is activated after AP20187 administration. In vitro experiments rev ealed rapid and efficient iCaspase-9-induced apoptosis of LNCaP cells in bo th an R1881- and AP20187-dependent manner. Only 28, 8, and 0.5% survival of LNCaP cells was seen at multiplicities of infection of 2, 10, and 25, resp ectively. Furthermore, at a multiplicity of infection of 10, extraordinary sensitivity to AP20187 was seen (IC50, similar to3 pm). In vivo experiments showed that ADV.ARR(2)PB-iCasp9 induced apoptosis in LNCaP but not in HuH- 7 xenograft tumors in an AP20187-dependent manner. Furthermore, a simple i. p. injection of AP20187 dramatically suppressed LNCaP tumor growth in nude mice and led to a significantly increased host survival. This study demonst rates the feasibility of using tissue-specific expression of cell cycle-ind ependent iCaspases as a nonmutagenic alternative modality for prostate canc er suicide gene therapy.