AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines

Abstract

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

An In Vitro Study on Prestin Analog Gene in the Bullfrog Hearing Organs

“…We examined the electrophysiological properties from HEK cells transfected with the fPres-EGFP protein fusions by a site-specific gene transfer at the human AAV site 1 (AAVS1) [ 27 – 30 ]. Transgene expression is influenced by the integration site and some random insertions or transient transfections which can interfere with genes or disturb their transcription, while site-specific integration can minimize variations between different cells and constructs [ 31 , 32 ]. We chose the gerbil prestin as a positive control, while cells transfected only with the EGFP-vector were a negative control.…”

Characteristics of BAY 2599023 in the Current Treatment Landscape of Hemophilia A Gene Therapy

“…Mammalian systems include human embryonic kidney (HEK)293 and HeLa cell lines [ 73 , 74 ]. In preclinical studies, adherent HEK293 cells are typically co-transfected with two or three plasmids, encoding the vector genome, rep and cap genes, and adenoviral helper genes.…”

Recent advances in upstream process development for production of recombinant adeno‐associated virus

“…Some common stable cell line strategies are summarized in Table 2. A stable producer cell line can be generated from the major production cell lines such as HEK293 (Abaandou et al, 2021; Selvaraj et al, 2021) and Sf9 (Joshi et al, 2019; Mietzsch et al, 2013), or less frequently reported HeLa (Luo et al, 2017) and A549 cells (Farson et al, 2004). Post GOI and rep/cap element integration, the rAAV production is initiated with the presence of a helper virus.…”