In a recent study published in the journal Nature Biotechnology, researchers describe the pA regulator system based on a ribonucleic acid (RNA)-based switch to regulate mammalian gene expression by modulating a synthetic polyA signal (PAS) cleavage at a transgenic 5′ untranslated region (UTR).
Study: Control mammalian gene expression by modulation of polyA signal cleavage at 5′ UTR. Image Credit: MMD Creative / Shutterstock.com
Current approaches to gene therapy
Gene control in mammalian cells is critical for developing safe and successful gene treatments. Current methods are associated with certain disadvantages, such as adverse immunological responses, limited efficiency, and therapeutic gene overexpression.
Current gene transfer technologies like adeno-associated viruses (AAVs) have difficulty performing conditional and reversible gene control. Toxic ligands, high leakage, and ligand concentrations, as well as small dynamic range are some of the limitations associated with current RNA-based systems.
About the study
Luciferase assays were performed in mammalian cells, followed by RNA extraction from transfected cells for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The researchers imaged stable cell lines by fluorescence microscopy and analyzed them by flow cytometry.
The AAV vector was created, followed by pA regulator-controlled luciferase expression experiments in mice and in vivo bioluminescent imaging. Additionally, pA regulator insertion was achieved by clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) and fluorescence-activated cell sorting (FACS) of the regulator-controlled cluster of differentiation 133 (CD133) single-cell clones.
The researchers discovered ways to incorporate an artificial PAS into structured aptamers, which retained its activity, even when incorporated into structured aptamers. The synthetic PAS was managed by binding to Tc, a United States Food and Drug Administration (FDA)-approved oral medication. A synthetic PAS was added at regions where the cb32 aptamer sequence required slight substitution to form the PAS sequence ‘AAUAAA’ or in the single-stranded region.
Two polyadenylation-enhancing motifs were introduced to the synthetic PAS function. Luciferase activity in HEK-293T cells was measured to determine the cleavage efficiency of the synthesized PAS.
A6 was selected as the foundation for building the biological switch regulated by Tc due to its apparent high cleavage efficiency. The ‘induction in fold’ was calculated based on the proportion of luciferase signals in the presence or absence of Tc to assess Tc-induced expression. Taken together, over 180 constructions were created to systematically assess the impacts of various elements of the Y-shaped structure to improve the configuration of Y16.
A ‘G-quad ‘-mediated and drug-inducible alternative splicing method was developed to manage the synthetic PAS to attain increased sensitivity and dynamic range. Several sequences that added a potential 3′ SS ‘AG’ downstream of the MAZ G-quad were also assessed.
Dose-response curves were generated using Y362 and Y387 as examples to define the complete regulatable range of the pA regulators. The pA regulator was assessed in live mice using an AAV2/9 vector encoding Y387 and the luciferase gene. To this end, mice were injected intraperitoneally with different dosages of Tc to evaluate the in vivo response of the Y387 pA regulator.
The RNA-based switch pA regulator is a unique approach for regulating the expression of mammalian genes by modulating PAS cleavage. A dual mechanism regulates the cleavage triggered by drug binding and comprises aptamer fastening to restrict the cleavage of PAS and drug-elicited alternative splicing to remove the PAS.
Moreover, this technique avoids immunological reactions associated with other systems. As a result, an induction of 900-fold is achieved with a half-maximal effective concentration (EC50) of 0.50 g/ml tetracycline (Tc), which is within the U.S. FDA-authorized dosage range.
The regulator could control luciferase transgenes in mice and CD133 expression by human tissues in a reversible and dose-dependent way, thereby ensuring long-term stability. The pA system enabled the generation of any intact protein as a transgenic product without changing its coding sequence, thus avoiding transgene-specific immune responses that have been observed in other systems. Moreover, the system had a 900-fold regulatable range while reducing baseline leakage expressions to 0.10%.
Tc-binding aptamers may influence gene expression by modulating PAS cleavage. The method involved inserting a synthetic polyA signal into a 5′ UTR, which caused efficient matching messenger RNA (mRNA) molecular cleavage. A small molecule-type ligand blocked cleavage by binding with RNA aptamer sequences, thereby retaining intact mRNA and inducing transgenic expression.
The Y-shaped structure of aptamers could inhibit PAS activity, whereas Tc binding enhances it at the G-quad. This would lead to downstream 3′ SS alternative splicing.
PAS and alternative splicing were strongly linked, and the pA regulator effectively regulated transgenes like the enhanced green fluorescent protein (eGFP). This system was efficient in multiple mammalian cell lines and is compatible with cells and promoters. Flow cytometry showed Tc recognition by the entire cell population, thus making it a ‘portable’ motif.
The study findings highlight the pA regulator system, an RNA-based switch that regulates gene expression by adjusting synthetic polyA signal cleavage in a transgenic 5′ UTR. This technique differs from traditional riboswitch systems, as the PAS is present in the 5′ UTR, combines the effects of numerous aptamers, and uses two processes of Tc binding and alternative splicing. However, the novel system can only use Tc as the inducer ligand, which cannot penetrate all body tissues efficiently.
- Luo, L., Jea, J. D., Wang, Y., et al. (2024). Control mammalian gene expression by modulation of polyA signal cleavage at 5′ UTR. Nature Biotechnology. doi:10.1038/s41587-023-01989-0