678. Increasing AAV Vector Yield By Riboswitch-Mediated Attenuation of Toxic Transgene Effects in HEK-293 Producer Cells

Abstract

Adeno-associated virus (AAV) vectors are among the most promising viral vector systems for human gene therapy and have also been used extensively to investigate gene function in preclinical research. Achievement of high-titer vector yields regardless of the packaged transgene hence is a key goal of process optimization. However, transgenes expressed by constitutive, ubiquitously active promoters (such as the CMV promoter) often impair producer cell performance due to cytotoxic, anti-proliferative or other unknown effects, resulting in low vector yield.In our study, we explored artificial aptazyme riboswitches as novel RNA-intrinsic tools to regulate vector transgene activity during AAV production in HEK-293 cells. To this end, we integrated the guanine-responsive GuaM8HDV aptazyme in the UTR of a standard AAV transgene cassette and explored switching behavior under AAV production conditions.Our results demonstrate that transgene expression can be efficiently attenuated by a single addition of guanine in a routine medium exchange step during AAV production, thereby decreasing producer cell performance-impairing transgene effects such as cytotoxicity. In a proof-of-concept study using transgenes with both known (e.g. pro-apoptotic) as well as unknown modes of producer cell impairment, AAV vector yields could be boosted up to 23-fold with the riboswitch approach as compared to conventional vectors. As expected, this effect worked independently of the AAV serotype/capsid variant used. Importantly, GuaM8HDV-harboring AAV vectors preserved functionality (i.e. the ability to express the transgene) in vivo, as demonstrated in a mouse model of AAV-TGFb1-induced pulmonary fibrosis. Contrary to other inducible systems for gene expression control, the riboswitch system presented here does not require expression of additional transcription factors, but works in a small-molecule regulatable, RNA-intrinsic manner. Moreover, the riboswitch sequence only occupies ≈100 base pairs of plasmid space – a particular advantage for AAV vectors. Finally, this approach should in principle be expandable to other viral vector systems produced in mammalian cell culture such as Adenoviral vectors.Thus, we propose riboswitches as novel tools to foster transgeneindependent, high-titer production of viral vectors in mammalian cell culture systems. Moreover, our study further supports exploration of this technology for other purposes such as studying transgene dynamics and controlling transgene expression as a safety module in next-generation gene therapy vectors.