Evaluation of life cycle defective adenovirus mutants for production of adeno‐associated virus vectors

Abstract

Background Adeno‐associated virus‐based vectors are efficient and safe drug candidates for different in vivo gene therapy applications. With increasing numbers of clinical studies based on AAV2 vectors that include not only rare, but also common diseases as a therapeutic target, there is an increased demand for the development of improved production technologies. Methods In the present study, we compared two life cycle defective adenovirus mutants as helper viruses for AAV2 vector production. They had deletions either in the gene coding for the preterminal protein (pTP) that is expressed early in the viral life cycle and is essential for genome replication or in the gene coding for the 100K protein, a protein with many functions, one of which is involved in virus assembly. AAV2 vector production efficiencies were evaluated by analyzing genome‐containing particles using a real‐time polymerase chain reaction and functional units were investigated by transduction assays. Results Somewhat contrary to our expectations, the ∆100K mutant virus showed only a moderate efficiency as a helper virus for AAV2 vector production, whereas the replication‐deficient ∆pTP mutant supported AAV2 production almost as efficiently as adenovirus wild‐type. We also showed that a temperature shift to 32°C together with extended incubation times improved AAV2 vector productivity. Conclusions The present study indicates the advantages of using a ∆pTP mutant adenovirus rather than adenovirus wild‐type as a helper virus for AAV2 production and also indicates that temperature shifts to lower temperatures may improve AAV2 vector production rates.

AAV production in stable packaging cells requires expression of adenovirus 22/33K protein to allow episomal amplification of integrated rep/cap genes

“…As discussed previously, Krüger-Haag et al showed that an adenovirus helper deleted of the 100K gene was unable to efficiently promote rep/cap amplification from an AAV packaging cell line 20 . Interestingly, the same authors showed that an adenovirus deleted of the pre-terminal protein (pTP) was fully capable of inducing rep/cap amplification and rAAV production.…”

“…As Kruger-Haag et al showed that while the 100K-deleted adenovirus was unable to support rAAV production, an observation consistent with TESSA-E1 during MLP repression, the pTP-deleted adenovirus successfully promoted rep/cap DNA amplification despite this pTP-mutant being unable to undergo DNA replication with concomitant poor MLP activation and only limited 22/33K 20 . This apparently contradictory finding led us to hypothesize that, perhaps, sufficient 22/33K proteins are expressed from its internal L4P but can be titrated away from the CARE-amplification process by large amounts of replicating adenovirus genomes within the cell.…”

“…Recently, it was shown that an adenovirus deleted of 810 bp within the L4 100K gene failed to promote amplification of the stably integrated rep/cap genes, and high titre rAAV production from an A549-based stable AAV packaging cell line 20 . As the 100K protein is expressed from the MLP, we assessed whether it comprises the missing ‘late’ adenovirus protein from TESSA-E1, during MLP repression, that is required to facilitate rep/cap DNA amplification 21 .…”

“…Interestingly, the same authors showed that an adenovirus deleted of the pre-terminal protein (pTP) was fully capable of inducing rep/ cap amplification and rAAV production. Adenovirus deleted for the pTP gene is incapable of DNA replication and shows undetectable levels of late mRNAs 20,22 . Specifically, the pTP is essential for initiation of adenovirus www.nature.com/scientificreports/ genome replication, a prerequisite for activation of the MLP and expression of late virus genes, including the 100K protein.…”

SOD2 overexpression in bone marrow‑derived mesenchymal stem cells ameliorates hepatic ischemia/reperfusion injury

“…Empty adenovirus vectors (Ad.null) and vectors encoding SOD2 (Ad.SOD2) were purchased from Shanghai Liangtai Biotechnology Company, and transductions were performed as previously described ( 21 ). To yield the adenovirus, the linearized construct DNA was transfected into 293 cells (American Type Culture Collection) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.…”