Developing recombinant adeno-associated virus producer cell lines through systems synthetic biology

Abstract

Recombinant adeno-associated virus (rAAV) is one of the most widely used vehicles for gene therapy due to its effectiveness in in vivo gene delivery, long-term persistency, and safety. In general, a very large dose of rAAV is required in clinical applications, hence manufacturing is anticipated to be carried out in large scale to meet the demand. Current rAAV production relies on multiple plasmid transfections or helper virus infections, which pose challenges in large-scale manufacturing. Additionally, rAAV produced in current manufacturing processes often has large fractions of empty viral particles without the payload. Innovations in viral vector manufacturing are called for to increase vector titers and enhance vector quality.Our aim is to bring into being a cell line, which has all the essential genetic elements for the synthesis of rAAV vectors, that can be induced to generate the recombinant virus. Since the production does not require any plasmid transfection or virus infection, the process is simple and scalable. Additionally, through tuning of the induction conditions, we can control the expression dynamics of different viral components and modulate the full-to-empty particle ratio of the vector to enhance the product quality. We have taken a synthetic biology approach to establish rAAV producing cell lines by replacing the native transcriptional regulation of viral genes with inducible promoters, organizing the essential viral genes into genetic modules, and integrating those modules into the HEK293 cell genome. The genome module contained a rAAV genome encoding GFP; the replication module encodes the Rep68 protein and helper proteins of adenovirus; and the packaging module provided capsid proteins and the Rep52 protein. After transfection, cells with these three modules integrated into the host genome were screened for rAAV productivity. To facilitate screening, we also developed an assay cell line, which harbors rep and helper genes but has no viral genome nor cap gene, for titration of infectious rAAV. We successfully isolated producers that can generate infectious rAAV upon induction. By optimizing the inducer ratio and induction timing, better titers with high full particle content were achieved. Systems biology approaches, such as transcriptomics and targeted proteomics, were adopted to quantify both cellular and viral transcripts and proteins. Time-course profiles of viral transcripts and proteins revealed different expression dynamics of each cell line and their possible influence on rAAV titers and quality. Inhibitory host responses attenuating rAAV production were also unveiled through multi-omics analysis. These understandings led to further modification of vector design and cultural conditions to enhance productivity. We redesigned a new inducible genome module, which effectively repressed wasteful GFP expression. Later, we introduced more copies of the cap gene into the cell genome. As a result of these strategic modifications, we successfully developed high rAAV producers capable of producing more than 105 vector genome per cell with a percentage of full particles exceeding 40%. Further adaptation of these producers into suspension culture demonstrated their scalability. Taken together, utilizing a systems design and synthetic biology approach, a scalable and controllable rAAV production system was demonstrated. This work exemplifies the crucial role of synthetic biology to engineer next-generation gene therapy manufacturing platforms.