Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals

Abstract

Synthetic riboswitches that can regulate gene expression by a small molecule recognized by an RNA aptamer in mammalian cells have various potential applications in biotechnology and medicine. However, the variety of small molecules and their cognate aptamers that have been demonstrated to function in mammalian cells is limited. The currently available aptamer-ligand pairs also require high small molecule concentrations to enable gene regulation, making them less desirable for industrial and biomedical applications. We conducted in vitro selection of RNA aptamers against a small molecule ASP7967 whose structure is closely related to ASP2905, a known inhibitor of potassium voltagegated channel sub-family H member 3 (KCNH3). One of the aptamers selected (AC17−4) was found to be functional in HEK293 cells, and it was used to design aptazyme-based riboswitches that can activate gene expression (>10-fold) in the presence of ASP2905 or ASP7967 at as low as 5 μM in the culture medium. An aptazyme-based riboswitch was successfully used to regulate human erythropoietin expression in mice injected with an adeno-associated virus (AAV8) vector using orally administered ASP7967. Furthermore, by combining aptazyme-based and exon-skipping riboswitch mechanisms, an ON/OFF ratio approaching 300 was achieved with a low basal expression level in cultured cells.

Synthetic Gene Circuits for Regulation of Next‐Generation Cell‐Based Therapeutics

“…Numerous proof‐of‐concept mouse studies have demonstrated the functionality of aptazyme‐mediated regulation of AAV‐based transgene expression in vivo. [ 11,118,121,122 ]…”

A comparative survey of the influence of small self-cleaving ribozymes on gene expression in human cell culture

“…Until now, aptazymes for mammalian cells are mostly based on the HHR and are predominately derived from HHR sequences from S. mansoni [ 20 , 42–45 ] or the satellite tobacco ringspot virus (sTRSV) [ 46 , 47 ]. Lately, aptazymes based on the HDV [ 43 , 48 ], pistol [ 49 , 50 ] and twister [ 43 , 51 , 52 ] ribozyme were developed. Artificial ribozyme-based riboswitches do not rely on the expression of specific protein factors, do not interfere with cellular mechanisms, and are easy to implement by inserting them into untranslated regions (UTRs) of a gene of interest.…”

“…Artificial ribozyme-based riboswitches do not rely on the expression of specific protein factors, do not interfere with cellular mechanisms, and are easy to implement by inserting them into untranslated regions (UTRs) of a gene of interest. Multiple proof-of-principle studies have demonstrated that ribozymes and aptazymes can be used to regulate therapeutically relevant biological outcomes such as T-cell proliferation [ 47 ], adeno- and AAV-vectored transgene expression in cell culture, as well as mice [ 43 , 44 , 50 , 53–56 ] and direct regulation of viral gene expression and replication [ 57 ]. In these studies, the transferability of aptazymes to different genetic contexts is substantiated.…”

Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches

“…Recently, we reported an RNA aptamer AC17-4 that binds to ASP2905 and its derivative ASP7967. 16 Originally ASP2905 was identified as a small molecule inhibitor of potassium channel Kv12.2 in mammalian cells, 17 and we repurposed it as a membrane-permeable ligand for mammalian riboswitches. 16 We expected ASP2905 to exhibit permeability across the liposome lipid bilayer and decided to utilize the AC17-4 RNA aptamer to develop new riboswitches.…”