Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in HEK-293 packaging cells

Abstract

Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs)

“…293pTP cells were derived from HEK-293 cells and overexpression of human Ad5 pTP gene as previously characterized [50]. The cell lines are maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA), containing 100 U/ml penicillin and 100 mg/ml streptomycin, at 37°C in 5% CO 2 as described [51-53].…”

Functional Characteristics of Reversibly Immortalized Hepatic Progenitor Cells Derived from Mouse Embryonic Liver

“…The coding regions of Cre recombinase and firefly luciferase (FLuc) were PCR amplified and subcloned into an adenoviral shuttle vector and subsequently used to generate recombinant adenoviruses in our recently engineered packaging line 293pTP [57], resulting in adenoviruses Ad-Cre and Ad-FLuc, which also express GFP as a marker for monitoring infection efficiency. Analogous adenovirus only expressing GFP (Ad-GFP) was used as a control [44,58,59,60,61,62,63,64,65,66].…”

Engineering the Rapid Adenovirus Production and Amplification (RAPA) Cell Line to Expedite the Generation of Recombinant Adenoviruses

“…We previously showed that overexpression of human Ad5 pTP in HEK-293 cells can enhance the amplification and packaging of adenovirus [16]. However, the possible contributions of adenoviral genes, which are known to participate in viral gene expression and DNA replication, have not been comprehensively evaluated.…”

“…Adenoviral lysate samples were prepared in five-fold serial dilution and used to infect subconfluent HEK-293 cells seeded in 6-well cell culture plates as described [11, 16]. The numbers of GFP or RFP-positive cells were counted at 36-48h post infection.…”

“…Several host factors such as OCT1 and NF1 also play important roles in adenoviral DNA replication [15]. We have recently demonstrated that overexpression of human Ad5 pTP in HEK-293 cells (i.e., 293pTP) can enhance adenovirus amplification and production [16]. …”