Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Abstract

Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

“…Using the Lipofectamine-3000 kit (Thermo Fisher Scientific), cells were transfected with 35 ng of plasmid DNA and subsequently supplied with stimulation media containing increasing amounts of tetracycline. In case of Gua-responsive constructs, cells were calcium-phosphate transfected by mixing 70 ng DNA with 10 µL 300 mmol/l CaCl 2 and 10 µL 2× HBS buffer (50 mmol/l HEPES, 280 mmol/l NaCl, 1.5 mmol/l Na 2 HPO 4 ) and adding 20 µL of this mix per well of a 96-well plate 3 . The media was changed to Gua-containing media approximately 4 h after addition of the transfection mix.…”

“…Self-cleavage within the 3′-UTR can be exploited for the post-transcriptional control of gene expression by decreasing mRNA stability via conditional poly(A) tail cleavage. Utilization of riboswitches to control gene expression has been demonstrated in cells [2][3][4] , C. elegans 5 and in a viral vectormediated fashion in mice 6,7 . In addition to ribozyme-based switches, further synthetic mechanisms for gene expression control have been described in recent years including splicing control 8 and microRNA switches 9 .…”

Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

“…In contrast to the translational block imposed by the TRiP system described here, others have attempted to suppress vector transgene expression by utilizing transcriptional control293637 or through use of a ligand-induced riboswitch38. Employment of transcriptional repressor systems may be limited in the magnitude of repression due to upstream read-through from the 5′LTR during generation of full length packageable RNA (for example, retroviral vectors), or by ITR-driven genome replication (for example, AAV/Adeno vectors).…”

Ligand‐dependent ribozymes

“…Utilizing the HDV ribozyme as expression platform the group of Yokobayashi generated a NOR logic gate in mammalian cells by inserting theophylline‐ and guanine‐dependent HDV aptazymes into the 3′‐UTR of the reporter gene . The guanine switch was subsequently applied for optimizing transgene expression in an AAV construct . An alternative strategy to control gene expression in mammalian cells consists in coupling a ligand‐dependent HHR to a pri‐miRNA sequence .…”