A new method has been developed for controlling therapies inside human cells using an RNA-based control switch, which could be used to selectively activate transgenes to trigger the production of therapeutic proteins in specific tissues.
Techniques that have been developed for controlling the translation of endogenous or exogenous RNAs in human cells have limited potential and a more effective method would benefit a variety of biotechnological applications.
While transgenes can be incorporated into human cells using genetic engineering techniques, those genes may need to be activated only in specific cells. If transgenes cannot be selectively activated, there could potentially be dangerous side effects.
Researchers at MIT and Harvard University previously developed a method of controlling genes in specific cells based on RNA sequences, which differ from tissue to tissue. When genes are activated, messenger RNAs (mRNA) carry the instructions for building particular proteins. The researchers developed a technique where an RNA molecule (termed a toehold) was introduced that binds to the ribosome-binding site of mRNA molecules in bacterial cells. When the toehold binds to the mRNA that codes for a specific protein, it is prevented from binding to the ribosome, thus preventing the production of the protein.
The activity of these toeholds can be controlled through a second binding site, which acts as a trigger. A site in the toehold will bind to a different mRNA sequence, and if binding occurs the toehold will release the mRNA that it was blocking which would allow it to be translated into a protein.
The researchers then worked on developing their system to work in non-bacterial (eukaryotic) cells. In eukaryotic cells, gene translation is more complex, so their toehold system could not be easily converted. Instead, the researchers developed a new system based on the mechanisms used by viruses to switch on the production of specific host genes.
The researchers developed eukaryotic toehold switches (eToeholds) that have internal ribosome entry site (IRES) sequences that form inhibitory loops unless a specific trigger RNA (trRNA) is present. When that trRNA is present, the eToeholds anneal to it and that disrupts the inhibitory loops and allows translation. The researchers used naturally occurring IRES from several types of viruses and engineered them to incorporate a sequence that binds to a trRNA.
The researchers optimized the RNA annealing and achieved up to 16-fold induction of transgene expression in mammalian cells, and demonstrated eToeholds can discriminate among viral infection status, presence or absence of gene expression, and cell types based on the presence of exogenous or endogenous RNA transcripts.
This approach has numerous potential applications. The researchers showed their eToeholds could detect mRNA encoding genes from the Zika virus and the SARS-CoV-2 virus, which could potentially be used in engineered T cells that respond to viral mRNA. The researchers also developed eToeholds that can detect mRNA that codes for normal cellular proteins inside mammalian cells, such as heat shock proteins, and could be used to block protein production.
One of the most exciting applications could be in novel cancer therapies. A transgene could be introduced and activated in tumors to produce a toxic protein to kill the tumor cells, but the transgene would remain inactive in healthy cells.
The researchers conducted the study in vitro and are now working on developing a method for introducing the eToeholds in vivo.
You can read more about the research in the paper – RNA-responsive elements for eukaryotic translational control – which was recently published in Nature Biotechnology. DOI: 10.1038/s41587-021-01068-2