Development of Novel E1-Complementary Cells for Adenoviral Production Free of Replication-Competent Adenovirus (2006)

Human adenoviruses have been used as highly efficient gene delivery vehicles or oncolytic agents in a variety of clinical and preclinical studies. Recombinant adenoviruses have predominantly been rendered replication defective or conditionally replicating via modifications to the E1 region, such as deletions, mutations, or replacement of the endogenous transcriptional control elements [1Chu R.L. Post D.E. Khuri F.R. Van Meir E.G. Use of replicating oncolytic adenoviruses in combination therapy for cancer.Clin. Cancer Res. 2004; 10: 5299-5312Crossref PubMed Scopus (136) Google Scholar]. However, the E1 gene products are important for directing cellular and viral gene expression to enable a productive virus life cycle. During viral infection, E1A expression pushes the cell cycle into S phase to support viral DNA replication and E1B expression delays cellular apoptosis, thus gaining time for successful viral production [2White E. Regulation of apoptosis by adenovirus E1A and E1B oncogenes.Semin. Virol. 1998; 8: 505-513Crossref Scopus (64) Google Scholar]. Therefore, due to the essential role of E1 genes in adenoviral biology, the helper-independent production of E1-modified adenoviral vectors is assisted by cell lines that produce complementary E1 proteins. Expression of E1A in transfected cells can immortalize rodent and human cells [3Houweling A. van den Elsen P.J. van der Eb A.J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA.Virology. 1980; 105: 537-550Crossref PubMed Scopus (219) Google Scholar, 4Gallimore P.H. Grand R.J. Byrd P.J. Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes.Anticancer Res. 1986; 6: 499-508PubMed Google Scholar]. However, expression of E1A promotes apoptosis, which is inhibited by coexpression of E1B [5Teodoro J.G. Branton P.E. Regulation of apoptosis by viral gene products.J. Virol. 1997; 71: 1739-1746Crossref PubMed Google Scholar, 6White E. Cipriani R. Sabbatini P. Denton A. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.J. Virol. 1991; 65: 2968-2978Crossref PubMed Google Scholar]. Therefore, cotransfection of E1A and E1B is necessary to establish stable cells that express both E1A and E1B in trans. The human embryonic kidney 293 cell line was generated about 30 years ago and since then it has been used as the major host cell for adenoviral production [7Graham F.L. Smiley J. Russell W.C. Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5.J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3512) Google Scholar]. In 293 cells, a continuous adenovirus type 5 (Ad5) DNA fragment containing both E1A and E1B genes (nucleotides 1 to 4344) was integrated into chromosome 19 at 19q13.2 [8Louis N. Evelegh C. Graham F.L. Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line.Virology. 1997; 233: 423-429Crossref PubMed Scopus (158) Google Scholar]. Since many first-generation adenoviral vectors are deleted from nucleotide (nt) 400 to nt 3500, the viral genome may have significant homology to the integrated viral DNA fragment in the host cell DNA. This homology between cellular DNA and viral genome can mediate double crossover recombination [9Murakami P. et al.A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.Hum. Gene Ther. 2002; 13: 909-920Crossref PubMed Scopus (37) Google Scholar]. Therefore, 293-cell-derived viral E1 DNA may rescue the E1 deletion in the viral products and generate replication-competent adenovirus (RCA). This is problematic for large-scale viral production and clinical applications [9Murakami P. et al.A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.Hum. Gene Ther. 2002; 13: 909-920Crossref PubMed Scopus (37) Google Scholar, 10Hehir K.M. et al.Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.J. Virol. 1996; 70: 8459-8467PubMed Google Scholar, 11Smith J.G. Eck S.L. Molecular characterization of an adenoviral vector resulting from both homologous and nonhomologous recombination.Cancer Gene Ther. 1999; 6: 475-481Crossref PubMed Scopus (8) Google Scholar]. The fundamental concern for the presence of RCA in viral products is safety. The FDA has established that the presence of RCA in replication-defective adenoviral vectors must be lower than 1 RCA in 3 × 1010 viral particles (vp) [12Biological Response Modifiers Advisory CommitteeAdenovirus Vector Titer Measurements and RCA Levels.2001Google Scholar]. To reduce the generation of RCA during adenoviral production, other E1-complementary cells that contain a smaller adenoviral E1 DNA fragment to minimize the homology to the viral genome have been developed [13Fallaux F.J. et al.Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors.Hum. Gene Ther. 1996; 7: 215-222Crossref PubMed Scopus (443) Google Scholar, 14Fallaux F.J. et al.New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.Hum. Gene Ther. 1998; 9: 1909-1917Crossref PubMed Scopus (396) Google Scholar, 15Kim J.S. Lee S.H. Cho Y.S. Park K. Kim Y.H. Lee J.H. Development of a packaging cell line for propagation of replication-deficient adenovirus vector.Exp. Mol. Med. 2001; 33: 145-149Crossref PubMed Scopus (13) Google Scholar, 16Gao G.P. Engdahl R.K. Wilson J.M. A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus.Hum. Gene Ther. 2000; 11: 213-219Crossref PubMed Scopus (30) Google Scholar, 17Schiedner G. Hertel S. Kochanek S. Efficient transformation of primary human amniocytes by E1 functions of Ad5: generation of new cell lines for adenoviral vector production.Hum. Gene Ther. 2000; 11: 2105-2116Crossref PubMed Scopus (143) Google Scholar]. The reduction of the sequence common between cell and vector may eliminate homology at least at the 5′ end of the adenoviral vector to reduce significantly recombination mediated by double crossover. For example, the Per.C6 cell line was generated using adenoviral E1 DNA that deleted the 5′ endogenous E1A promoter and DNA 3′ of the E1B 55-kDa coding region [14Fallaux F.J. et al.New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.Hum. Gene Ther. 1998; 9: 1909-1917Crossref PubMed Scopus (396) Google Scholar]. This cell line has limited homology to many E1-deficient adenoviral vectors and therefore significantly reduces RCA during vector production. However, the continuous E1 DNA fragment in the host cell DNA cannot preclude unconventional recombination. Only a short stretch of homology between cellular DNA and the vector genome can mediate recombination and generate recombinants with a rescued E1 region [9Murakami P. et al.A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.Hum. Gene Ther. 2002; 13: 909-920Crossref PubMed Scopus (37) Google Scholar]. Although these recombinants are helper-dependent for a productive viral replication, their presence is still problematic for product qualification and clinical application. The emergence of undesirable recombinants during viral production is also an important consideration for oncolytic viruses. These conditionally replication-competent adenoviruses are developed by viral gene mutation or replacement of transcriptional control elements for essential viral genes. Most of the modifications are in the E1 region and allow the viruses to replicate and kill specifically permissive tumor cells [1Chu R.L. Post D.E. Khuri F.R. Van Meir E.G. Use of replicating oncolytic adenoviruses in combination therapy for cancer.Clin. Cancer Res. 2004; 10: 5299-5312Crossref PubMed Scopus (136) Google Scholar]. Although different permissive tumor cells can be used for production of these replicating viruses, a common production cell line may greatly reduce costs by standardizing process development and qualification. Based on the design of oncolytic adenoviruses such as CG7870, CV890, and CG8840 [18Yu D.C. Chen Y. Seng M. Dilley J. Henderson D.R. The addition of adenovirus type 5 region E3 enables calydon virus 787 to eliminate distant prostate tumor xenografts.Cancer Res. 1999; 59: 4200-4203PubMed Google Scholar, 19Li Y. Yu D.C. Chen Y. Amin P. Zhang H. Nguyen N. Henderson D.R. A hepatocellular carcinoma-specific adenovirus variant, CV890, eliminates distant human liver tumors in combination with doxorubicin.Cancer. Res. 2001; 61: 6428-6436PubMed Google Scholar, 20Zhang J. Ramesh N. Chen Y. Li Y. Dilley J. Working P. Yu D.C. Identification of human uroplakin II promoter and its use in the construction of CG8840, a urothelium-specific adenovirus variant that eliminates established bladder tumors in combination with docetaxel.Cancer Res. 2002; 62: 3743-3750PubMed Google Scholar], E1-complementary producer cells can be used for the production of these viruses despite their specificity for different cancers. However, the use of conventional E1-complementary cells like 293 cells, which have the continuous stretch of wild-type E1A and E1B genes, may generate wild-type RCA. This continuous DNA fragment may also generate other undesirable recombinants that alter the transcriptional control elements for E1A or E1B genes. The presence of these recombinants has a negative impact on viral tropism and potency, as well as product safety, and needs to be eliminated. In this study, we developed novel E1-complementary cells for the production of RCA-free adenoviruses. We used the highly efficient retroviral transduction system rkat43.2 [21Finer M.H. Dull T.J. Qin L. Farson D. Roberts M.R. kat: a high-efficiency retroviral transduction system for primary human T lymphocytes.Blood. 1994; 83: 43-50Crossref PubMed Google Scholar] to generate retroviral vectors for adenoviral E1 expression. First, we constructed plasmids rkat43.2E1A and rkat43.2E1B (Fig. 1A ) by replacing the CD4ζ coding sequences of pRT43.2F3 [21Finer M.H. Dull T.J. Qin L. Farson D. Roberts M.R. kat: a high-efficiency retroviral transduction system for primary human T lymphocytes.Blood. 1994; 83: 43-50Crossref PubMed Google Scholar] with the DNA coding for Ad5 E1A and E1B, respectively. We generated high-titer retroviral supernatants by transient cotransfection of rkat43.2E1A or rkat43.2E1B with MMLV packaging plasmids as previously described [22Roberts M.R. et al.Targeting of human immunodeficiency virus-infected cells by CD8+ T lymphocytes armed with universal T-cell receptors.Blood. 1994; 84: 2878-2889Crossref PubMed Google Scholar]. We then utilized the retroviral supernatants MMLV-E1A and MMLV-E1B to co-infect the human lung carcinoma cell line A549 by spinoculation. We used MMLV-E1A/MMLV-E1B ratios of 1/3, 1/1, and 3/1 to optimize the infection conditions for generating stable cells. Under current study conditions, most of the cells in all three infections died during the first week. We single-cell cloned the remaining cells by limiting dilution and screened them with an E1-deleted, GFP-expressing adenovirus (Ad-GFP), which is capable of replication only in E1-producing cells. We harvested the crude viral lysates and used them to infect HuH7 cells, which we then assayed by FACS for GFP expression (Supplemental Fig. 1). In naïve A549 cells, Ad-GFP did not replicate and the subsequent HuH7 infection revealed only a background level of GFP-positive cells. In contrast, Ad-GFP actively replicated in 293 cells and therefore the subsequent HuH7 infection produced a gene transfer efficiency of 100%. Clones generated from co-infection of E1A/E1B retroviral vectors at a ratio of 1/3 and 1/1 supported Ad-GFP replication, whereas clones transduced with more MMLV-E1A than MMLV-E1B (3/1 ratio) could not. We selected the two clones from the 1/1 population with an efficient level of GFP expression, clones 51 (Ac51) at 91% and 139 (Ac139) at 78%, for further characterization. To ensure that the new clones were free of any replication-competent MMLV sequences, we isolated genomic DNA from naïve A549, Ac51, and Ac139 cells and analyzed it by TaqMan PCR with primers specific for MMLV gag/pol. Both parental A549 cells and adenoviral producer cell clones were negative for MMLV gag/pol sequences (data not shown). To evaluate viral production quantitatively, we tested the new E1-complementary cells, Ac51 and Ac139, and 293 and PerC.6 cells for production of an E1-deficient adenovirus encoding GM-CSF (Ad-GMCSF) (Supplemental Fig. 2A). Compared to the initial infection dose, both Ac51 and Ac139 had over 1000-fold amplification of Ad-GMCSF and produced virus at levels similar to those of 293 and Per.C6 cells (Supplemental Fig. 2B). We maintained the cell lines in continuous culture and reassayed them at weeks 5 and 10 to evaluate the stability of their E1 complementation. Yields of Ad-GMCSF virus were stable for Ac51 and Ac139 cells through 10 weeks of culture and remained comparable to those of 293 and Per.C6 cells (Supplemental Fig. 2B). In contrast to the E1-deficient adenoviruses, the conditionally replicating oncolytic adenoviruses retain some or all of the E1 genes. We evaluated production of two oncolytic adenoviruses, CG8840 and OV945 (Supplemental Fig. 3A), on naïve A549, 293, Ac51, and Ac139 cells and determined virus yield by plaque assay on 293 cells (Supplemental Figs. 3B and 3C). In these studies, the virus production from Ac51 and Ac139 was equivalent to or greater than that from 293 and A549 cells, suggesting that these clones can effectively complement production of E1-modified oncolytic adenoviruses. In further studies, we adapted Ac51 and Ac139 cells to serum-free medium and they supported the production of E1-deficient and oncolytic viruses at levels comparable to those of 293 cells in suspension (Supplemental Figs. 3A and 3B). We analyzed the expression of Ad5 E1 proteins from the new A549-derived E1 cells by Western blot (Fig. 1B). In this study, we visualized E1A, E1B 19-kDa, and E1B 55-kDa proteins with appropriate and them the Ac51, Ac139, 293, and Per.C6 cells. Both Ac51 and Ac139 cells produce of E1A than those of Per.C6 and 293 cells, E1B production in all cell lines is However, the E1B 55-kDa production in both Ac51 and Ac139 cells is significantly lower than that of Per.C6 and 293 cells. Based on the of the retroviral the coding for both E1B and 55-kDa proteins and their is by the retroviral to the for both proteins. from the for the E1B 55-kDa protein may not and thus in 55-kDa expression in the new E1 cells. In 293 or Per.C6 cells, the E1A and E1B genes were transfected as a continuous DNA fragment from the viral E1 gene expression may still the control used in the and E1B 55-kDa protein may be in a more efficient et of human and Ad5 for two tumor at different PubMed Scopus (136) Google Scholar]. both Ac51 and Ac139 have lower production of E1B 55-kDa protein than 293 and Per.C6 cells, is impact on their to parental A549 cells. The E1B 55-kDa products are for these cells to support adenovirus production, suggesting that E1B 55-kDa protein in these cells may have a significant role in viral production. similar E1B to the of E1A was in transfected E1-complementary cells et cell lines from human lung carcinoma A549 cells support the of E1-deleted adenovirus Ther. 1996; Google Scholar]. In addition to the major and other such as the and were by Western the were Fig. The most significant in these new cell lines is the genomic of the E1A and E1B genes by the use of retroviral To the of the E1A and E1B genes in the host cell we analyzed the cellular DNA from Ac51, Ac139, 293, and Per.C6 by PCR and First, we DNA from these cell lines and an A549 control using primers specific for E1A or E1B in Fig. the A549 cells were negative for E1A or E1B sequences, whereas the E1-producing cell lines were for both E1A and E1B genes as However, we selected the primers from both E1A and E1B genes to the continuous E1A to E1B Ac51 and Ac139 have a different from that of 293 and Per.C6 cells. on the of Fig. primers nt and nt a DNA fragment from 293 and Per.C6 cells, not the new E1 cells, that Ac51 and Ac139 cells contain adenoviral E1A and E1B genes. In a blot we DNA from different of Ac51, Ac139, and 293 cells with or it on a and screened it with specific for E1A or E1B genes (Fig. 2B). Ac139 to have two of E1A and two of Ac51 has is a of E1A and two of The also that for both Ac51 and Ac139 at early and that these clones are important in this study was to evaluate the production of RCA in the different cell We used an RCA-free viral of an E1-deficient which the FDA RCA in 3 × 1010 viral in a for the RCA in Ac51, Ac139, 293, and Per.C6 cells. Ad-GMCSF has significant homology to 293 cells, as well as a continuous DNA fragment homologous to Per.C6 cells. Therefore, we that using 293 cells, RCA could be generated at a of 1 RCA in 1 × to 1 × 1010 vector particles J. et al.Characterization of replication-competent adenovirus from large-scale production of a adenoviral Gene Ther. 1999; 10: PubMed Scopus Google Scholar]. In Per.C6 cells, we also unconventional recombinants due to the single short stretch of homology [9Murakami P. et al.A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.Hum. Gene Ther. 2002; 13: 909-920Crossref PubMed Scopus (37) Google Scholar]. In contrast, the genomic of E1A and E1B genes in the cellular DNA of clones Ac51 and Ac139 greatly reduce the generation of RCA or recombinants that may undesirable cytopathic in a To evaluate RCA during viral production, we used the E1-deficient Ad-GMCSF virus to infect the cell lines in a The continuous amplification of first-generation vectors on different cells has previously been used for RCA K.M. et al.Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.J. Virol. 1996; 70: 8459-8467PubMed Google Scholar, 16Gao G.P. Engdahl R.K. Wilson J.M. A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus.Hum. Gene Ther. 2000; 11: 213-219Crossref PubMed Scopus (30) Google Scholar]. In this continuous RCA or other of recombinants that be with Ad-GMCSF to subsequent recombinants with replication efficiency than the parental Ad-GMCSF may be during the and therefore can be with in the We produced Ad-GMCSF for in Ac51, Ac139, 293, and Per.C6 cells. To 1 of the we cell line with × of Ad-GMCSF at an of We crude viral lysates and determined by et for the and of adenoviral Ther. 2000; 7: PubMed Scopus Google Scholar]. We then used these crude lysates for of infections on naïve cells. This amplification was a of The viral production in amplification cycle was × for all cell clones We PCR of the viral crude lysates from 293, Ac51, and Ac139 cells at and to the emergence of RCA during the of the amplification (Fig. 3A The and used in this PCR assay specifically a 293-cell-derived E1 DNA fragment that the E1A promoter and of the E1A The the PCR from the 293 cellular and is in the Ad-GMCSF PCR can be in from 293 cells at and not in Ac51 or Ac139 cells, that RCA has during the viral amplification The of this assay was 1 of wild-type Ad5 DNA in a background of Ad-GMCSF viral and of viral DNA were used in PCR Therefore, we that were at least RCA in 3 × 1010 by in from 293 cells. were in any of Ad-GMCSF viral products from the Ac51 or Ac139 cells. We a PCR assay using a previously specific for the promoter and E1A gene to the viral crude lysates from Per.C6 cells for recombination P. et of replication-deficient E1-positive particles in adenoviral vector Virol. 2004; PubMed Scopus Google Scholar] (Fig. 3B). In this study, viral DNA from and were as described The PCR a DNA was in the Per.C6 cellular DNA and in viral and of the PCR that E1A and the human promoter DNA was in Ad-GMCSF viral the presence of Based on studies, the recombinants most contain E1 sequences from DNA [9Murakami P. et al.A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.Hum. Gene Ther. 2002; 13: 909-920Crossref PubMed Scopus (37) Google Scholar, P. et of replication-deficient E1-positive particles in adenoviral vector Virol. 2004; PubMed Scopus Google Scholar]. Although PCR has that Ac51 and Ac139 not produce RCA during viral production, we a further assay to the RCA of these cells. We further × 1010 virus particles from of the Ad-GMCSF generated on × A549 cells for We crude lysates and assayed them on naïve A549 cells for plaque and To establish the of the we × 1010 Ad-GMCSF particles with wild-type adenovirus and them in in for both 293 and Per.C6 cells, recombinants are in the Ad-GMCSF the presence of RCA or However, in an assay that was to wild-type particles in a background of × 1010 Ad-GMCSF we viral in virus on Ac51 or Ac139 cells, suggesting that both cell lines allow RCA-free adenoviral RCA by of E1-deficient virus on the different cell Ad-GMCSF virus RCA in 3 × 1010 viral was used to infect 293, Ac51, and Ac139 cells in at for viral lysates were harvested and by These viruses were further to naïve cells of the type the conditions for the viral This process was for a of For of × 1010 of viral were on × A549 cells in three 1 to The infection was in with and the were on a for and then with an 30 of medium with for a of were harvested in 10 for viral and crude lysates were in by or wild-type Ad5 into × 1010 of Ad-GMCSF virus and through A549 cells in the the viral plaque of A549 cells were with 1 of viral crude in the infection were and cells were with medium containing for plaque The was and cells were with the were for with an Titer to the viral for A549 cells RCA-free Ad-GMCSF virus RCA in 3 × 1010 viral was used to infect 293, Ac51, and Ac139 cells in at for viral lysates were harvested and by et for the and of adenoviral Ther. 2000; 7: PubMed Scopus Google These viruses were further to naïve cells of the type the conditions for the viral This process was for a of For of × 1010 of viral were on × A549 cells in three 1 to The infection was in with and the were on a for and then with an 30 of medium with for a of were harvested in 10 for viral and crude lysates were in by or wild-type Ad5 into × 1010 of Ad-GMCSF virus and through A549 cells in the For the viral plaque of A549 cells were with 1 of viral crude in the infection were and cells were with medium containing for plaque The was and cells were with the were for with an Titer to the viral for in a new the PCR and assay is that Ac51 and Ac139 cells not produce RCA during the production of E1-deficient adenoviral We also a PCR of the oncolytic viral products produced from Ac51 and Ac139 cells. we used retroviral and primers in a PCR to DNA in OV945 could be suggesting that cellular was in the of OV945 (data not shown). In we have a novel and used retroviral vectors to establish new E1-complementary cells. These cells have adenoviral E1A and E1B genes integrated into genomic are E1 complementary and support the production of and E1-modified adenoviral production. RCA that the new E1 cells not support RCA of these E1-complementary cells to serum-free suspension culture them for large-scale adenoviral with this can be in the at with