Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors

The calponin (basic or hi) gene, normally expressed in maturated smooth mus cle cells, is aberrantly expressed in a variety of human soft tissue and bo ne tumors. In this study, rye show that expression of the calponin gene in human soft tissue and bone tumor cells is regulated at the transcriptional level by the sequence between positions -260 and -219 upstream of the trans lation initiation site. A novel conditionally replicating herpes simplex vi rus-1 vector (d12.CALP) in which the calponin promoter drives expression of ICP4, a major trnas-activating factor for viral genes was constructed and tested as an experimental treatment for malignant human soft tissue and bon e tumors. In cell culture, d12.CALP at low multiplicity of infection (0.001 plaque-forming unit/cell) selectively killed calponin-positive human synov ial sarcoma, leiomyosarcoma, and osteosarcoma cells. For ill vivo studies, 10 animals harboring SK-LMS-I human leiomyosarcoma cells were randomly divi ded and treated twice on days 0 and 9 intraneoplastically with either 1 x 1 0(7) plaque-forming units of d12.CALP/100 mm(3) of tumor volume or with med ium alone. The viral treatment group showed stable and significant inhibiti on of tumorigenicity with apparent cure in four of five mice by day 35. Rep lication of viral DNA demonstrated hy PCR amplification and expression of t he inserted LacZ gene visualized by 5-bromo-4-chloro-3-indolyl-beta -D-gala ctopyranoside side histochemistry was associated with oncolysis of d12.CALP -treated tumors, while sparing normal vascular smooth muscle cells. In mice harboring two SK-LMS-1 tumors, replication of d12.CALP was detected in a n ontreated tumor distant from the site of virus inoculation. These results i ndicate that replication-competent virus vectors controlled by the calponin transcriptional regulatory sequence may be a new therapeutic strategy for treatment of malignant human soft tissue and bone tumors.