Directed Evolution of Riboswitches in Mammalian Cells for External Control of Therapeutic Gene Expression

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Therapeutic gene transfer shows high promise for the treatment of cancer. Replication-defective and more recently replication-competent/oncolytic viruses are used as gene transfer vectors. The latter combine tumor cell lysis with amplification of the transferred therapeutic gene. External control of therapeutic gene expression is desirable or even required to enable timing and dosing in patients, and as a safety measure, especially for replicating vectors. We propose artificial aptazymes, ligand-dependent self-cleaving ribozymes, as innovative tool for regulation of therapeutic gene expression. Aptazymes act RNA intrinsically, independent of regulatory protein-nucleic acid interactions, are non-immunogenic and of small size. These are key advantages compared to the widely used inducible promoters, which were also reported to lose regulation at high copy numbers, e.g. after virus genome amplification. Using a model aptazyme with previously unreached but still suboptimal induction rates in mammalian cells, we have recently optimized aptazyme positioning in mammalian transcription units. Importantly, we have shown that down-regulation of gene expression is fully functional in both replication-deficient and oncolytic adenovirus vectors, independent of virus replication and spread. The aims of the proposed project are (i) to identify by directed evolution ON- and OFF-switch aptazymes with high induction rates in mammalian cells and (ii) to exploit these switches for regulating the expression of tumor-targeted cytotoxic ligands by oncolytic adenoviruses (OAds). For directed evolution, we will generate high diversity aptazyme libraries by randomizing the linker sequence between ribozyme and ligand-binding RNA domain. Aptazymes with high induction rates will then be isolated by sequential positive and negative selection of aptazyme-controlled GFP and suicide gene expression, respectively, using stably transduced cells. Both ON- and OFF-switch aptazymes responsive to different ligands will be isolated by selection in presence or absence of the ligand. The resulting aptazymes will be sequenced and characterized in transfection studies. Next, we will exploit the identified aptazymes for regulating the expression of the natural death ligand TRAIL and ImmunoRNases by OAds. ImmunoRNAses show parakrine toxicity targeted by an antibody domain to desirable tumor surface molecules. We have recently demonstrated expression of functional immunoRNase by OAds, but expression control is required to reduce interference with virus replication. Here we will investigate in cell cultures and animal tumor models both aptazyme control and activity of TRAIL and EGFR-specific ImmunoRNase encoded by OAds in optimized transgene cassettes. Finally, we will analyze combined oncolysis and death ligand therapy. In conclusion, we will identify riboswitches with high potential for gene regulation in laboratory research and biomedicine and establish a novel regimen for cancer treatment.