Entwicklung rekombinanter Clostridium sporogenes-Sporen als neuer Therapieansatz für die Behandlung nekrotisierender Tumore

Genetically modified anaerobic clostridia open a new possibility of treatment of solid tumours with promising potential. The immunologically well tolerated spores of these organisms only germinate in hypoxic regions of massive tumours. Proteolytic clostridia in general are able to colonize tumours efficiently. A destructive effect of colonization can be highly increased by the recombinant production of reactive agents directly in the tumour, minimizing possible adverse side effects. CPE is a toxin of Clostridium perfringens and it was shown to target claudin receptors, which are massively overexpressed in pancreatic carcinoma cell lines. CPE thus binds specifically to cancer cells, forms pores, and ultimately causes cell death. A Clostridium acetobutylicum strain was transformed with a shuttle vector carrying the gene for CPE, fused with the amyP signal peptide sequence, and controlled by a constitutive clostridial promoter. This strain is able to produce and secrete up to 500 ng/ml of CPE into the surrounding medium but is not able to colonize in vivo solid tumours efficiently. Therefore, it was the aim of this work to adapt the constitutive expression system to Clostridium sporogenes. Unfortunately, the unwanted expression of this fusion protein in E. coli caused by the used constitutive promoter leads to the death of the host. Thus, attempts were made to use different inducible promoter systems and to silence the expression in E. coli using antisense RNA against the mRNA of the signal peptide. Additionally, new clostridial signal peptides sequences were fused to cpe gene. These constructs were characterized and tested for the further use in C. sporogenes