Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors

Abstract

Robust and accurate quantification of recombinant adeno-associated virus (rAAV) vectors' infectivity is essential for pre-clinical and clinical development of AAV gene therapy programs. The industry standard method for rAAV titration is the 50% tissue culture infectious dose (TCID50) assay using HeLa-based cell lines that stably encode the rep and cap genes from AAV serotype 2. Co-infection with wild-type (WT) adenoviruses provides the helper functions for expression of these genes, and the use of quantitative PCR (qPCR)/droplet digital PCR (ddPCR) serves as the endpoint method for the detection of infectious events. However, TCID50 assays using these HeLa-based rep cap trans-complementing cell lines have traditionally been regarded as challenging due to high variability, stability of the integrated genes, and safety concerns associated with the use of WT helper viruses. Here we developed a novel method for infectious titration of rAAV using our vector "tetracycline-enabled self-silencing adenovirus" (TESSA); we engineered it to deliver and express the AAV2 rep genes and adenoviral helper functions for rAAV genome replication, independent of the cell type. This approach allows the infectious titration of rAAV serotypes in cell lines permissive to adenovirus but without the production of adenoviral particles for improved safety, therefore benefiting GMP analytical requirements for rAAV gene therapies.

Keywords: TCID50; TESSA; TREAT assay; infectious titer; rAAV.

Graphical abstract

Figure 1

TESSA-E1-Rep enables rAAV replication (A) Adenovirus MLP is modified to include a repressor-binding site (tetracycline operator, TetO), and the repressor (TetR) is encoded under MLP transcription. This creates a doxycycline-controllable negative feedback loop regulating the expression of the adenovirus structural genes. (B) rAAV genome replication in A549 cells. Cells were infected with rAAV2-EGFP (50 GC/cell) only or co-infected with the indicated viruses (10 TCID50/cell). rAAV genome replication determined by EGFP-qPCR.

Figure 2

Overview and qualification of the TREAT assay for infection titration of rAAV via the TCID50 method (A) Layout of the TREAT assay for rAAV titration with internal assay controls. (B) Protocol timeline from cell seeding to harvest and endpoint detection via endpoint qPCR.

Figure 3

Linearity of the TREAT assay The log of detected DNA copy number at each dilution was plotted against the concentration of the rAAV for linear fitting, with an R² value of 0.9997.