Fig. 4: Propagation of rAAV using TESSA-RepCap in HEK293 cells.

a Representative fluorescent images of HEK293 cells infected with (i) rAAV2-EGFP (50 GC/cell, control) or (ii) rAAV2-EGFP with TESSA-RepCap2 (MOI of 25). Imaged at 48 hpi. Scale bar, 2000 μm. Data representative of at least three independent experiments. b Production of rAAV2-EGFP using TESSA in 1 L stir-tank bioreactor culture of suspension HEK293 cells. Cells were co-infected with the TESSA2.0 vectors (TESSA-AAV and TESSA-RepCap2, each at an MOI of 25) or TESSA-RepCap2 (MOI of 25) co-infected with rAAV2-EGFP (HF-derived), at 50 GC/cell. DNAse-resistant genomes were quantified by EGFP-specific qPCR at 96 hpi and presented as GC per mL of cell culture. Data are N = 2 (mean ± SD) technical measurements. c Transduction potency of rAAV2 (produced using the TESSA2.0 vectors, each at an MOI of 25, or co-infection of rAAV2-EGFP at 50 GC/cell with TESSA-RepCap2) as determined in HEK293 cells using a TCID50 assay. GC/TCID50 ratio shown on the right axis was determined by comparing against the EGFP-specific qPCR titre of DNAse-resistant rAAV particles. Data are N = 3 (mean ± SEM) biological replicates (ns, analysed by one-way ANOVA with Tukey’s multiple comparisons). d Assessment of adenovirus contamination in crude rAAV2 preparations derived from TESSA2.0, or via passage of rAAV2 using TESSA-RepCap2, using a TCID50 assay. Data are N = 3 (mean ± SEM) biological replicates. Analysed by one-way ANOVA with Tukey’s multiple comparisons. e Western blot of AAV2 capsid proteins from particles produced in HEK293 cells using TESSA2.0 (± doxycycline), or via co-infection of rAAV2 particles (produced from TESSA2.0) at 50 GC/cell with TESSA-RepCap2 (MOI of 25). AAV2 reference standard material (RSM, ATCC VR-1616) is also shown. Data representative of at least three independent experiments. f Assessment of DNA contaminants in rAAV2 stocks produced via TESSA2.0, HF, or co-infection of TESSA-RepCap2 (MOI of 25) with escalating rAAV2 vector doses (produced using TESSA2.0). Impurities were determined in DNAse-resistant rAAV2 by qPCR against the adenovirus packaging signal Ψ (TESSA2.0-derived rAAV) or AmpR (HF-derived rAAV). Data are the percentage of adenovirus or AmpR DNA compared to EGFP. Titre of rAAV2 is shown on the right axis. Data presented are N = 3 (mean ± SD) biological replicates. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons. ****p ≤ 0.0001. Production yield of (g) rAAV5 (h) rAAV6 (i) rAAV8 (j) rAAV9 encoding EGFP from HEK293 cells co-infected with rAAV2 vectors (50 GC/cell) and TESSA-RepCap5 (100 GC/cell), TESSA-RepCap6 (100 GC/cell), TESSA-RepCap8 (100 GC/cell), or TESSA-RepCap9 (75 GC/cell), respectively. DNAse-resistant particles quantified by EGFP-specific qPCR at 96 hpi and compared to HF approach. Data presented are N = 3 (mean ± SEM) biological replicates. Statistical significance was calculated using an unpaired t-test (two-tailed). **p ≤ 0.01, ***p = 0.0007.