Scientists have developed an oligonucleotide gene switch that can regulate the expression of therapeutic or cell-killing genes inserted into viruses used for gene therapy or oncolytic virotherapy. Developed by researchers at the German Cancer Research Center (DKFZ) and the University of Konstanz, the aptazyme switch, which is only about 100 base pairs long, is inserted into the genetic code of the engineered virus adjacent to the transgene gene, such that both switch and target gene are transcribed together into a single mRNA. Adding an endogenous substance that binds directly to the mRNA signals the switch to trigger the mRNA to chop itself up, preventing the protein from being produced.
The DKFZ’s Dirk Nettelbeck, Ph.D., and colleagues pinched their idea from nature, which has already evolved riboswitches that in bacteria, for example, regulate gene expression in response to the binding of metabolites, second messengers, or toxic agents. Their artificial construct comprised a synthetic ligand-dependent self-cleaving ribozyme, inserted into the 5’- and/or 3’-UTR of the transcription unit. Its design effectively kept expression of the transgene off in the presence of the exogenous trigger, in this case theophylline, but allows its expression in the absence of theophylline.
The team successfully tested their aptazyme switches in adenovirus vectors, adeno-associated viruses, and oncolytic adenoviruses, and demonstrated that the switch allowed up to a 10-fold increase in transgene expression.
They claim the achievement provides a level of regulatory control over the use of engineered viruses that hasn’t to date been possible. Although inducible promoters are widely used to control gene expression in other cell types, both in a research and potentially therapeutic setting, the constructs are generally just too big to be used in viruses.
“This was the first proof that RNA switches work in viruses at all,” Dr. Nettelbeck explains, who admits further improvements in the basic technology are bound to follow. “The construction of RNA switches is extremely variable. Once the technology is fully developed, we will be able to better equip and regulate viruses for many therapeutic applications.”
The investigators describe their work in Nucleic Acids Research, in a paper titled "Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses."