NOVEL pA REGULATOR SYSTEM THAT TURNS GENE OFF/ON IN CUE: A BREAKTHROUGH IN PRECISION CONTROL OF SAFE GENE THERAPY

 

Gene therapy is a technique that alters the genes of a person to cure or treat disease. They work on several mechanisms like replacing a disease-causing gene, inactivation of a disease-causing gene, and introducing a modified or new gene into the body. 

Gene therapy has held promise across the world for many years. But why can't we see that many gene therapies coming out and being used in treating patients? The answer lies in the principle of the therapeutic window. The therapeutic window defines the dosage between the minimum effective therapeutic concentration and the minimum toxic concentration. So staying on the therapeutic window is crucial, as overexpression and underexpression can lead to toxicity and no therapeutic effects. However, there is no proper knowledge of making gene therapy safe. Most of the gene therapy is not approved by the FDA as those systems use regulatory proteins that act as foreign substances in the human body triggering an immune response. 

Recently a paper “Control of mammalian gene expression by modulation of polyA signal cleavage at 5′ UTR” published in Nature Biotechnology has reported an effectively regulating gene expression that holds promising results that can fill the bridge between clinical trials and real-time application of gene therapies. 

The paper describes the pA regulator, an RNA-based switch that controls mammalian gene expression by synthetic polyA signal (PAS) cleavage introduced in the 5′ UTR of RNA. 

The RNA is engineered to have a Synthetic polyA signal (PAS) at the beginning of the RNA than at the end. The mRNA can form an aptamer structure that regulates the on and off of the gene. The on and off of the gene is due to the binding of the small molecules. When the small molecules bind to the aptamer, it prevents the PAS from cleaving from the sequence which is then translated into the desired protein. In the absence of the small molecule, the PAS is recognised in translation and the mRNA gets cleaved leaving the coding genes which halts the production of protein.

To enhance polyadenylation, a GU-rich region and a G-rich motif MAZ are introduced. These engineered RNA motifs operate within the same RNA molecule and are described as cis-acting. This means that their regulatory effects are confined to the specific RNA molecule in which they are located, contributing to the overall control of gene expression through the modulation of cleavage and polyadenylation processes.

Synthetic polyA signal (PAS) - red, GU-rich region - green, G-rich motif MAZ - blue

To test it, the small molecule was considered as tetracycline. When tetracycline binds to the aptamer region, it masks the PAS by which translation proceeds. In the absence of tetracycline, the PAS is recognized and cleaved at the start of mRNA by which the coding genes are cleaved. 

Unlike the tet-on system that requires one promoter to generate the regulatory transactivator protein and a second promoter to express the transgene, the pA regulator is a compact single promoter system that can be implemented in a single plasmid or viral vector

To add on, the pA regulator has a compact single promotor system that can be implemented in a single plasmid or vector when compared to a tet-on system (Tetracycline-controlled transcriptional regulation system) that requires one promoter to generate the regulatory transactivator protein and a second promoter to express the gene. 

Another advantage of the pA system is that it doesn't generate any additional amino acid sequence to the transgene coding sequence which could promote transgene-specific immune responses. The pA regulator controls the expression of genes in a dose-dependent and reversible manner, suitable for effective gene therapy than CRISPR/ cas9 DNA editing which is irreversible. 

To test the pA regulator approach in contexts to control expression of transgenes in human cells and live mice, and also to control endogenous gene expression in the human genome. They have effectively controlled the luciferase transgene in live mice and the endogenous CD133 gene in human cells, in a dose-dependent and reversible manner with long-term stability.

The authors have outlined the integration of this technology into clinical gene therapy, where a gene is administered to compensate for a patient's disorder caused by a malfunctioning gene. In this scenario, the patient's received gene incorporates the described switch, enabling physicians to regulate the production of the therapeutic protein. If the patient requires a minimal amount of the protein, they would only need a small dose of tetracycline, activating the therapeutic gene at a proportional level. Conversely, if a higher protein dosage is necessary, an increased tetracycline dose would enhance protein production. To halt the therapeutic protein's production, the patient can simply cease taking tetracycline, returning the switch to its default off position in the absence of the drug.

So the authors state that this could be an opportunity for biological studies as well as clinical applications such as ex vivo and in vivo gene and cell therapy.

REFERENCE

Luo L, Jea JD-Y, Wang Y, Chao P-W, Yen L. Control of mammalian gene expression by modulation of polya signal cleavage at 5′ UTR. Nature Biotechnology. 2024; doi:10.1038/s41587-023-01989-0 

IMAGE CREDIT

Nature biotechnology. 2024, doi:10.1038/s41587-023-01989-0

European Pharmaceutical Review, 2023, https://images.app.goo.gl/ReKWUBsNuh46SjpW7