Fig. 4.

Targeted gene integration contributes to persistence of gene expression. a A 3′ junction capture PCR of genomic DNA was performed to determine the presence of hAAT-integrated ROSA26 alleles (expected size, 1.7 kb) at 210 days post-injection. Each lane represents amplicons from an individual mouse’s genomic DNA from groups in Fig. 3a. b Serum levels of hAAT at 210 days were compared to initial serum levels at day 7 for individual mice. Each data point represents one mouse. *Significance with pvalue <0.05 (p = 0.0159 and 0.0043, respectively). “N” denotes none. c Liver-extracted DNA was used as a template to quantify hexon to determine the amount of residual viral episomes (p = < 0.0001 and p = 0.0017, respectively). Each dot represents one qPCR replicate with four replicates per mouse. dhAAT copy numbers remaining at 210 dpi were quantified by qPCR (p = 0.1957 and 0.3724, respectively). Each dot represents one qPCR replicate with four replicates per mouse. NS represents non-significance with pvalue >0.05. e The amount of hAAT copies per hexon copy present is shown per group. Each dot represents one mouse, with three mice per group. Error bars are s.d. of the mean. Closed circles represent mice injected with Ad5-EF1α-hAAT (5.0 × 10¹⁰ VP) and Ad5-CMV-EGFP (5.0 × 10¹⁰ VP). Open circles are mice injected with Ad5-EF1α-hAAT (7.5 × 10¹⁰ VP) and Ad5-CMV-EGFP (2.5 × 10¹⁰ VP). Half closed, downwards triangles represent mice injected with Ad5-EF1α-hAAT (2.5 × 10¹⁰ VP) and Ad5-Cas9-gRNA (7.5 × 10¹⁰ VP). Closed triangles are mice injected with Ad5-EF1α-hAAT (5.0 × 10¹⁰ VP) and Ad5-Cas9-gRNA (5.0 × 10¹⁰ VP). Open triangles represent mice injected with Ad5-EF1α-hAAT (7.5 × 10¹⁰ VP) and Ad5-Cas9-gRNA (2.5 × 10¹⁰ VP)