Introduction of DNA encoding the A fragment of diphtheria toxin (DT-A) into mammalian cells is lethal if the DNA is expressed. Expression can be controlled by cis -acting transcriptional regulatory elements so as to kill specific target cells in culture, and in transgenic mice. Our eventual goal is to develop this concept for cancer therapy by usurping the gene expression program of particular cancer cells to direct their self- destruction by DT-A expression. Critical to this approach will be the development of efficient delivery vehicles for toxin gene constructs. As with currently conceived gene replacement therapy, recombinant viruses appear to offer the most promising vehicles. However, transient expression from a non-integrating virus may be more appropriate for toxin gene therapy than the use of a viral vector with an efficient integration mechanisms, such as a retrovirus. Therefore, this proposal deals with the development of gene delivery vehicles based on the autonomous parvoviruses (APVs), which do not integrate their DNA efficiently into that of the host cell. These viruses have a 5 kb single strand DNA genome, most of which can be replaced (using plasmid clones) by foreign DNA while retaining competence for replication and packaging in virions (for which only the short terminal palindromes are essential).
Our specific aims, are first, to develop a system for generating helper-free recombinant APVs capable of transducing and expressing a reporter gene. This will employ a Lu lll recombinant genome and an H1-derived helper construct whose expression in cells will be inducible by a """"""""genetic switch"""""""" mechanism. Inducibility is necessary because continuous expression of APV proteins appears toxic. Both constructs will initially be inserted in an autonomously replicating (EBO) plasmid. Alternatively, one or other construct will be chromosomally integrated if this proves necessary of efficient inducible production of recombinant virus.
The second aim i s to impose tissue-specific expression on the transduced reporter with appropriate enhancer/promoter sequences in the virus. Elastase and alpha-1 protease inhibitor gene regulatory sequences will thus be used as models for targeting (to cultured exocrine pancreatic cells and hepatoma cells, respectively). These experiments will provide the basis for subsequent insertion of a DT-A coding sequence in the recombinant viruses with purpose of efficiently killing specific target cells by DT-A expression. An efficient system of generating helper-free, recombinant parvoviruses will not only advance the development of toxin gene therapy for cancer but will also expand the range of viral vehicles available for other gene transfer applications.
