It must be stressed that among the innovative aspects of the proposed research is not only the production of an antitumor ribonuclease, but the production of a human antitumor ribonuclease agent. This will be obtained by conferring the therapeutic potential of well known non-human antitumor ribonucleases, such as the bovine and the amphibian enzymes mentioned above, to human ribonucleases devoid of antitumor action. A non-cytotoxic human ribonuclease is already available in one of the proposers' laboratory, and its cDNA has been cloned and expressed. Through mutagenesis and engineering of the DNA coding for this inactive human ribonuclease, recombinant mutant proteins will be obtained with the structural features essential for exerting an antitumor action. The production of the engineered proteins will be followed by the determination of their biological properties, and the mutants with the highest antitumor action will be analysed by NMR for the determination of their structures. This will in turn direct the programming of second generation mutants.
DNA and protein engineering will be carried out by the proponent from Italy; the antitumor and biological assays will be performed by the proponent from the Czech Republic; the determinations of RNase mutant structures by NMR will be carried out by the Spanish proponent, while the Hungarian proponent will promote innovative NMR methodologies for the analysis of the dynamics of RNases.
The success of the experimental approach we propose is very probable, as in the laboratory of one of the proponents has been already demonstrated that a biologically inactive ribonuclease can be transformed by directed mutagenesis in an antitumor ribonuclease. Furthermore, the work will be judiciously distributed among the different groups, each already well established in its respective scientific field.
The aim of the research as proposed is to produce a ribonuclease(s) which can be effectively and safely employed in tumor therapy. This will be carried out by applying basic as well as advanced biotechnology methodologies and innovative therapeutic ideas.
The first original observation on the antitumor action of a ribonuclease has been obtained in the laboratory of one of the proponents, working with bovine seminal ribonuclease. The laboratories of two of the proponents have been active for years in the chemical and biological characterization of this enzyme, a very effective antitumor and antimetastatic agent, for which some of the structural determinants essential for the antitumor action have been already defined. However, a biotech firm in the U.S.A. has obtained in the last few years a headstart in the general field of antitumoral RNases with a ribonuclease from an amphibian source, which has been recently admitted to Phase III clinical trials. This has been mainly due to the attention of the American general public and of the economic market to innovative research in medicine. Thus the exploitation of the expertise and of the research data already available to the proponents from EU and CCE countries undoubtedly merits the attention of the EC as a funding institution.
Funding SchemeCSC - Cost-sharing contracts
277 21 Libechov