Construction of an rAAV Producer Cell Line through Synthetic Biology

Abstract

Recombinant adeno-associated viruses (rAAV) are important gene delivery vehicles for gene therapy applications. Their production relies on plasmid transfection or virus infection of producer cells, which pose a challenge in process scale-up. Here, we describe a template for a transfection-free, helper virus-free rAAV producer cell line using a synthetic biology approach. Three modules were integrated into HEK293 cells including an rAAV genome and multiple inducible promoters controlling the expression of AAV Rep, Cap, and helper coding sequences. The synthetic cell line generated infectious rAAV vectors upon induction. Independent control over replication and packaging activities allowed for manipulation of the fraction of capsid particles containing viral genomes, affirming the feasibility of tuning gene expression profiles in a synthetic cell line for enhancing the quality of the viral vector produced. The synthetic biology approach for rAAV production presented in this study can be exploited for scalable biomanufacturing.

Keywords: HEK293; adeno-associated virus; biomanufacturing; synthetic biology.

Figure 1

(A) Gene modules and schematic diagram of rAAV2 producer cell line construction. GSTA denotes the GeneSwitch transactivator gene; rtTA3 denotes the reverse Tet transactivator gene; CymR denotes the Cym Repressor; DD denotes the destabilization domain. (B) GFP fluorescence images of RM4 assay cells transduced with rAAV2-GFP produced by producer cell line PC-2. (C) Relative transcript levels of total cap and spliced VP2/3 referencing to GAPDH in traditional triple transfection of HEK293 cells and induced PC-2 cells. Data represent mean and standard deviation of triplicate qPCR wells. (D) Transducing titers of rAAV obtained through PC-2 cells transiently transfected with viral genes. (E) New packaging modules: control (PM-C), with consensus splice donor (PM-D), with intron-less AAV2 cap CDS called VP123 (PM-E), with VP123 and T2A-linked CymR (PM-F). (F) Transducing titers of rAAV obtained by PC-2 cells transiently transfected with new packaging modules. For D and F, the relative titer was in reference to the titer of induction only, and data represent mean and standard deviation of cell counts from multiple images. **: p < 0.01 as determined by a two-tailed, two-sample t-test.

Figure 2

(A) Schematic diagram of capsid-enhanced rAAV producer cell line construction. (B) Transducing titers of rAAV obtained through the induction of stable cell pools. Data represent mean and standard deviation of cell counts from multiple images. (C) GFP fluorescence images of RM4 assay cells transduced with rAAV2-GFP produced by Pf3 or Pf6 clones. (D) Intracellular copy numbers of viral genes from each module, and total intracellular virus genomes before induction (−) and 96 h after induction of PM and RM (+). (E) Relative transcript levels of viral genes in Pf3 and Pf6 cells before induction (−) and at the end of production (+). (F) Virus titers obtained through the induction of Pf3 and Pf6 cells: physical titers measured by qPCR, and transducing titers measured by flow cytometry using RM4 assay cells. (G) Pf3 cell line stability. Physical titer and specific physical titer measured for Pf3 cells induced at 3 and 33 doublings in culture. For D, E and physical titer data, data represent mean and standard deviation of triplicate qPCR wells. **: p < 0.01 as determined by a two-tailed, two-sample t-test. ns: p > 0.05 as determined by a two-tailed, two-sample t-test.

Figure 3

(A) Schematic diagram of a synthetic rAAV2 packaging cell line construction. (B) Relative transcript levels of viral genes in RP6 and RP7 packaging cells before induction (−) and at 96 h after induction (+). (C) rAAV titers obtained through the transfection and induction of RP6 and RP7 cells: physical titers measured by qPCR and transducing titers measured by flow cytometry using RM4 assay cells.

Figure 4

(A) Transcripts per million of viral genes and GAPDH expressed in Pf3 cells at 96 h postinduction measured by RNA sequencing. (B) Absolute quantification of viral proteins expressed in Pf3 cells at 96 h postinduction measured by parallel reaction monitoring (PRM). (C) Effect of varying dox–cumate ratios on particle titer and full capsid content. Presented numbers for dox and cumate represent the actual concentration of the inducers used, in μg/mL, and the mifepristone concentration was kept at 2.5 nM. Data represent mean and standard deviation of three independent replicates. **: p < 0.01 as determined by a two-tailed, two-sample t-test.

Figure 5

(A) Specific physical titers of rAAV produced from traditional triple transfection, transposon-module triple transfection, and Pf3 production methods. (B) Relative transcript levels of viral genes at the end of production of the three production methods. Data represents mean and standard deviation of triplicate qPCR wells. **: p < 0.01 as determined by a two-tailed, two-sample t-test. ns: p > 0.05 as determined by a two-tailed, two-sample t-test.