Efficient Transformation of Primary Human Amniocytes by E1 Functions of Ad5: Generation of New Cell Lines for Adenoviral Vector Production

Abstract

Primary human cells are relatively refractory to transformation by adenoviral E1 functions. For almost two decades, human embryonic kidney (HEK)-derived 293 cells have been the only E1-complementing cell line suitable for production of E1-deleted adenoviral vectors. More recently, new vector production cell lines have been derived from human embryonic retina (HER) cells, a cell type that is difficult to obtain. We were surprised to find that readily available primary human amniocytes are efficiently transformed by adenoviral E1 functions. We selected cell lines that allow high-titer production of recombinant adenoviral vectors. The generation of replication-competent adenovirus (RCA) during production, caused by homologous recombination between vector and cellular DNA, was excluded by designing the transforming plasmid to lack sequence overlap with current adenoviral vectors. In addition, we generated an infectious plasmid that can be used for convenient generation of first-generation adenoviral vectors in Escherichia coli and that matches the E1 complementation in the new production cell lines.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…Contamination with RCA is unacceptable for clinical applications and must be avoided during production steps. In order to minimize the RCA occurrence, E1 complementing cell lines of the next generation, such as PER.C6 human embryonic retinoblasts, 23 N52.E6 human amniocytes 24 or HeLa-derived GH329 cells, 25 were established. These cells contain a minimal E1 gene fragment with no sequence overlap with the rAd genome.…”

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

“…Unfortunately, this cell line is only available through licensing. Yields from the PER.C6 cell line are similar to those achieved in 293 and 911 cells; however, as overlap between the genome and the E1 sequence has been eliminated, this clone, as well as the N52.E6 cell line, does not generate RCAs (Fallaux et al 1998;Schiedner et al 2000). Another cell line is A549, a lung carcinoma cell.…”

“…Unfortunately, homologous recombination between the left terminus of first-generation Ad vector or helper-virus DNA and partially overlapping E1 sequences in the genome of 293 cells may lead to the emergence of replicative competent adenoviruses (RCA), which contain E1 genes (Lochmuller et al 1994). To overcome this problem, alternative host cell lines have been developed by reducing these overlapping sequences, as for 911 cells, or by eliminating any overlap, as for N52.E6 (Schiedner et al 2000) or PER.C6 (Fallaux et al 1998) cells. The 911 cell line exhibits similar frequencies of homologous recombination to 293 cells, although vector yields are three times higher in 911 cells (Fallaux et al 1996).…”

Application of a disease-regulated promoter is a safer mode of local IL-4 gene therapy for arthritis

“…dl70-3 were prepared according to the AdEasy system (Stratagene, La Jolla, CA, USA), with the exception that recombinant viral particles were produced in E1 transformed N52E6 amniocyte cells. 47 Replication-deficient (E1 deleted) Ad5.CMV-mIL-4 was produced by infection of N52E6 cells with Ad5E1mIL-4. Viruses were purified by two consecutive CsCl 2 gradient purifications and stored in small aliquots at À80 1C in buffer containing 25 mM Tris, pH 8.0, 5 mM KCl, 0.2 mM MgCl 2 , 137 mM NaCl, 730 mM Na 2 HPO 4 , 0.1% (w v À1 ) ovalbumin and 10% (v v À1 ) glycerol.…”