Infectious Delivery of 120-Kilobase Genomic DNA by an Epstein–Barr Virus Amplicon Vector
Authors: Robert E. White, Richard Wade-Martins, Michael R. James
Journal: Molecular Therapy (2002)

It has been shown in a wide variety of contexts that persistent gene expression can best be obtained by using the genomic locus of a transgene. However, the size of most genomic loci precludes their use in current viral gene therapy vectors. Large transgene capacity and extrachromosomal persistence make Epstein–Barr virus (EBV) a promising vector to deliver genomic transgenes for gene therapy. We constructed an EBV amplicon vector that contains the EBV lytic origin of replication, the terminal repeats for viral packaging, and the EBV latent origin of replication for episomal persistence. This vector was able to deliver inserts of 60–123 kb to B-cell lines in culture in three steps. First, clonal packaging cells lines were generated that produce infectious amplicons at a titer of ∼3–4×106 transducing units/ml after concentration. Second, we show infectious vector delivery to the Loukes B-cell line and three different EBV-immortalized lymphoblastoid cell lines. This infectious delivery system was 2000 times more efficient than transfection in B cells. Third, clonal cell lines from infection of Loukes contained persistent episomes of recircularized infectious vector. This first demonstration of infectious delivery of 120 kb of genomic DNA shows the potential of this high-capacity vector system. It has been shown in a wide variety of contexts that persistent gene expression can best be obtained by using the genomic locus of a transgene. However, the size of most genomic loci precludes their use in current viral gene therapy vectors. Large transgene capacity and extrachromosomal persistence make Epstein–Barr virus (EBV) a promising vector to deliver genomic transgenes for gene therapy. We constructed an EBV amplicon vector that contains the EBV lytic origin of replication, the terminal repeats for viral packaging, and the EBV latent origin of replication for episomal persistence. This vector was able to deliver inserts of 60–123 kb to B-cell lines in culture in three steps. First, clonal packaging cells lines were generated that produce infectious amplicons at a titer of ∼3–4×106 transducing units/ml after concentration. Second, we show infectious vector delivery to the Loukes B-cell line and three different EBV-immortalized lymphoblastoid cell lines. This infectious delivery system was 2000 times more efficient than transfection in B cells. Third, clonal cell lines from infection of Loukes contained persistent episomes of recircularized infectious vector. This first demonstration of infectious delivery of 120 kb of genomic DNA shows the potential of this high-capacity vector system. IntroductionSeveral recent reports have shown that persistent long-term transgene expression can be achieved in vitro [1Wade-Martins R. White R.E. Kimura H. Cook P. James M.R. Stable correction of a genetic deficiency in human cells by a 115 kb episomal genomic transgene.Nat. Biotechnol. 2000; 18: 1311-1314Crossref PubMed Scopus (69) Google Scholar] and in vivo [2Schiedner G. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity.Nat. Genet. 1998; 18: 180-183Crossref PubMed Scopus (555) Google Scholar, 3Stoll S.M. Epstein–Barr virus/human vector provides high-level long-term expression of α1-antitrypsin in mice.Mol. Ther. 2001; 4: 122-129Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 4Rowntree R.K. An element in intron 1 of the CFTR gene augments intestinal expression in vivo.Hum. Mol. Genet. 2001; 10: 1455-1464Crossref PubMed Scopus (55) Google Scholar] by using the genomic locus of a gene. In contrast to conventional cDNA expression cassettes, such genomic sequences allow expression to be driven from native promoters, including tissue-specific promoters with alternative transcriptional start sites. All potential cis-regulatory elements in upstream, downstream, and intronic DNA are also included within the vector. The presence of the full intron and exon structure further ensures that the full range of the gene's splice variants are appropriately produced. A complete genomic DNA locus, comprising all the regulatory regions necessary for persistent, regulated expression, can span over 100 kb of chromosomal DNA [5Li Q. Harju S. Peterson K.R. Locus control regions—coming of age at a decade plus.Trends Genet. 1999; 15: 403-408Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 6Blackwood E.M. Kadonaga J.T. Going the distance: a current view of enhancer action.Science. 1998; 281: 60-63Crossref PubMed Scopus (608) Google Scholar]. However, the majority of current gene therapy vectors, including retroviruses, lentiviruses, and adeno-associated virus, cannot deliver more than 10 kb of exogenous DNA. The "gutless" adenovirus is larger, with a maximum theoretical capacity of 36 kb, and has been used for the delivery of a 19-kb genomic transgene [2Schiedner G. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity.Nat. Genet. 1998; 18: 180-183Crossref PubMed Scopus (555) Google Scholar]. However, none of the current widely used vector systems can accommodate inserts of > 100 kb, the size needed to deliver many complete genomic loci.Epstein–Barr virus (EBV) is a 172-kb human 7-herpesvirus that persists asymptomatically as an extrachromosomal (episomal) element in the B lymphocytes of > 90% of the human population. Long-term episomal persistence is caused by the EBV nuclear antigen (EBNA-1) binding to the origin of plasmidial replication (oriP) to mediate nuclear retention and cell-cycle-synchronous replication of the EBV episome [7Yates J. Warren N. Reisman D. Sugden B. A cis-acting element from the Epstein–Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells.Proc. Natl. Acad. Sci. USA. 1984; 81: 3806-3810Crossref PubMed Scopus (487) Google Scholar, 8Yates J.L. Warren N. Sugden B. Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells.Nature. 1985; 313: 812-815Crossref PubMed Scopus (979) Google Scholar, 9Rawlins D.R. Milman G. Hayward S.D. Hayward G.S. Sequence-specific DNA binding of the Epstein–Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region.Cell. 1985; 42: 859-868Abstract Full Text PDF PubMed Scopus (341) Google Scholar, 10Adams A. Replication of latent Epstein–Barr virus genomes in Raji cells.J. Virol. 1987; 61: 1743-1746PubMed Google Scholar]. EBNA-1 mediates the initiation of replication within the dyad symmetry (DS) element of oriP [11Gahn T.A. Schildkraut C.L. The Epstein–Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication.Cell. 1989; 58: 527-535Abstract Full Text PDF PubMed Scopus (226) Google Scholar], while nuclear retention of the episome is driven by EBNA-1 bound to the family of repeats (FR) [12Krysan P.J. Haase S.B. Calos M.P. Isolation of human sequences that replicate autonomously in human cells.Mol. Cell. Biol. 1989; 9: 1026-1033Crossref PubMed Scopus (216) Google Scholar, 13Leight E.R. Sugden B. EBNA-1: a protein pivotal to latent infection by Epstein–Barr virus.Rev. Med. Virol. 2000; 10: 83-100Crossref PubMed Scopus (158) Google Scholar]. In the presence of EBNA-1, plasmids containing oriP are stably maintained as extrachromosomal elements in primate and canine cell lines [8Yates J.L. Warren N. Sugden B. Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells.Nature. 1985; 313: 812-815Crossref PubMed Scopus (979) Google Scholar]. EBV episomes carrying up to 660 kb of genomic DNA have also been shown to be stably maintained in both human [14Simpson K. McGuigan A. Huxley C. Stable episomal maintenance of yeast artificial chromosomes in human cells.Mol. Cell. Biol. 1996; 16: 5117-5126Crossref PubMed Scopus (76) Google Scholar, 15Westphal E.M. Sierakowska H. Livanos E. Kole R. Vos J.-M. A system for shuttling 200-kb BAC/PAC clones into human cells: stable extrachromosomal persistence and long-term ecotopic gene expression.Hum. Gene Ther. 1998; 9: 1863-1873Crossref PubMed Scopus (41) Google Scholar, 16Wade-Martins R. Frampton J. James M.R. Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells.Nucleic Acids Res. 1999; 27: 1674-1682Crossref PubMed Scopus (65) Google Scholar] and murine [17Krysan P.J. Calso M.P. Epstein–Barr virus-based vectors that replicate in rodent cells.Gene. 1993; 136: 137-143Crossref PubMed Scopus (56) Google Scholar] cell lines, and most recently in vivo in mice [3Stoll S.M. Epstein–Barr virus/human vector provides high-level long-term expression of α1-antitrypsin in mice.Mol. Ther. 2001; 4: 122-129Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. Recent work in our laboratory has shown that genomic loci are functionally expressed from EBV episomes over prolonged periods of cell culture [1Wade-Martins R. White R.E. Kimura H. Cook P. James M.R. Stable correction of a genetic deficiency in human cells by a 115 kb episomal genomic transgene.Nat. Biotechnol. 2000; 18: 1311-1314Crossref PubMed Scopus (69) Google Scholar]. This collective body of work demonstrates that the EBV episome is an excellent vehicle for mediating long-term persistent expression of a genomic DNA transgene.At 172 kb in size, EBV has sufficient transgene capacity to accommodate genomic loci for infectious delivery. Amplicon plasmids carrying the EBV lytic origin of replication (oriLyt), the terminal repeats (TR), and oriP can be packaged into infectious virions in the presence of a helper virus [18Hammerschmidt W. Sugden B. Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein–Barr virus.Cell. 1988; 55: 427-433Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 19Hammerschmidt W. Sugden B. Genetic analysis of immortalizing functions of Epstein–Barr virus in human B lymphocytes.Nature. 1989; 340: 393-397Crossref PubMed Scopus (428) Google Scholar, 20Zimmerman J. Hammerschmidt W. Structure and role of the terminal repeats of Epstein–Barr virus in processing and packaging of virion DNA.J. Virol. 1995; 69: 3147-3155PubMed Google Scholar]. EBV amplicons containing these elements have been used to deliver reporter genes and cDNA constructs to B-cell lines [21Banerjee S. Livanos E. Vos J.-M.H. Therapeutic gene delivery in human Blymphoblastoid cells by engineered non-transforming infectious Epstein–Barr virus.Nat. Med. 1995; 1: 1303-1308Crossref PubMed Scopus (71) Google Scholar, 22Sun T.Q. Vos J.-M. Engineering 100–300-kb DNA as persisting extrachromosomal elements in human cells using human artificial episomal chromosomes system.Methods Mol. Genet. 1996; 8: 167-188Crossref Scopus (4) Google Scholar]. However, such systems have so far failed to take advantage of the full potential transgene capacity of EBV amplicon vectors.In this study, we describe the construction of a bacterial artificial chromosome (BAC)-based EBV amplicon vector. We used this vector to deliver genomic DNA inserts of 60 kb and 123 kb to a range of B-cell lines in vitro. We transfected each construct into the Burkitt's lymphoma (BL) cell line, P3HR1-HH514-C16 (hereafter referred to as HH514), which carries a nontransforming strain of EBV, to produce packaging cell lines. We used these lines to generate amplicon stocks of titers up to ~ 3–4 × 106 transducing units (t.u.)/ml. We were able to deliver the amplicons by infection to both EBV immortalized lymphocyte cell lines (LCLs) and the EBV-negative BL cell line Loukes. Finally, after antibiotic selection of infected Loukes cells, we were able to show episomal persistence of recircularized infectious vector in clonal cell lines. This is the first demonstration of the infectious delivery and subsequent stable propagation of such a large piece of human genomic DNA by an EBV amplicon system.ResultsDesign and Construction of VectorThe BAC-based EBV amplicon vector (pEBV-BAC) used in this study for the delivery of large genomic inserts is shown in Fig. 1A. The vector with the genomic DNA insert was assembled in two stages. First, the genomic DNA insert was cloned into the NotI sites of a precursor vector, which lacks the oriLyt sequences. In the second stage an I-PpoI fragment containing oriLyt and the kanamycin resistance gene was inserted and cloned by double antibiotic selection to create the complete amplicon vector. The original identification of oriLyt described both a minimal lytic origin and a larger region that exhibits wild-type replication activity [18Hammerschmidt W. Sugden B. Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein–Barr virus.Cell. 1988; 55: 427-433Abstract Full Text PDF PubMed Scopus (280) Google Scholar]. We used the larger region in an attempt to maximize the efficacy of the packaging system. The internal repeat region (IR2) of oriLyt contains multiple NotI sites, necessitating this two-stage cloning strategy. Because I-PpoI has a 15-bp recognition site and cuts extremely rarely in the human genome [23Lowery R. Hung L. Knoche K. Bandziulis R. Properties of I-Ppo I: a rare cutting intron-encoded endonuclease.Promega Notes. 1992; 38: 8-12Google Scholar], this strategy is suitable for the routine cloning of oriLyt into the vector carrying any genomic insert. This strategy inserts oriLyt into the vector in either orientation. All experiments were performed with clones in which the relative orientation of oriP and oriLyt is the same as in the wild-type virus.Two genomic DNA inserts were placed into the pEBV-BAC amplicon vector for infectious transfer. The first insert was determined to be 59,888 bp (referred to as the 60-kb insert) by end-sequencing and comparison to the human genomic sequence. The second insert of 123,463 bp (referred to as the 123-kb insert) was cloned from the fully sequenced BAC 255A7 (GenBank acc. no. AF069291) and contains the complete (> 50 kb) genomic locus of the Nijmegen breakage syndrome (NBS1) gene and > 30 kb of sequence at both the 3' and 5' ends of the gene. The amplicons carrying the 60-kb and 123-kb inserts were designated pEBV-BAC-60 and pEBV-BAC-123, respectively.Figure 1B shows the general strategy, comprising three stages, for working with our EBV amplicon system. First, clonal packaging cell lines are created that carry the vector maintained as an EBV-based episome. Second, these packaging lines are induced to activate the EBV lytic cycle and the amplicon vectors are packaged as infectious EBV virions. Third, the virions are concentrated, titered, and delivered by infection to the target cell.Generation of Packaging Cell LinesThe first stage of the process is to generate a packaging cell line for each amplicon construct. We transfected 1 μg of pEBV-BAC-60 DNA into 1 × 106 HH514 cells, a BL cell line carrying a nontransforming strain of EBV [24Heston L. Rabson M. Brown N. Miller G. New Epstein–Barr virus variants from cellular subclones of P3J-HR-1 Burkitt lymphoma.Nature. 1982; 295: 160-163Crossref PubMed Scopus (84) Google Scholar, 25Rabson M. Heston L. Miller G. Identification of a rare Epstein–Barr virus variant that enhances early antigen expression in Raji cells.Proc. Natl. Acad. Sci. USA. 1983; 80: 2762-2766Crossref PubMed Scopus (64) Google Scholar]. At 48 hours after transfection only 20–40 cells (0.004%) expressed green fluorescent protein (GFP) as seen by fluorescence microscopy, indicating a very low transfection efficiency, characteristic of most B-cell lines. The cells were diluted into four 96-well plates and selected with hygromycin B. Clonal cell lines were expanded and a plasmid rescue assay was used to isolate episome from low-molecular-weight DNA preparations. We identified three clonal lines carrying pEBV-BAC-60, which gave in excess of 1000 bacterial colonies on agar plates containing chloramphenicol and kanamycin after episome rescue. Rescued episome was analyzed by NotI digestion of DNA from the bacterial colonies. All of the colonies recovered from all three cell lines contained intact pBAC-EBV-60 (Fig. 2A). Quantitative Southern blot analysis of the three packaging lines showed the pEBV-BAC-60 episome to be maintained at a copy number of ~ 35–40 copies/cell (data not shown).FIG. 2Analysis of clonal cell lines created to package pEBV-BAC-60. (A) Episome rescued from HH514 cell lines carrying pEBV-BAC-60. Low-molecular-weight DNA from clonal cell lines was electroporated into bacteria. DNA prepared from bacterial colonies carrying rescued episome was digested with Noti and analyzed on a pulsed-field gel. Eight rescued plasmids were analyzed from each HH514 clone. All rescued plasmids were the same as the parental plasmid (lane M). S, DNA size markers with size shown to left. The bands corresponding to the genomic insert, vector, and parts of oriLyt are indicated, and are of the expected sizes. (B) GFP expression in pEBV-BAC packaging cell clone 1 cells 48 hours post-induction analyzed by fluorescence microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To package the EBV amplicon into infectious virions, it is necessary to induce the lytic cycle of EBV. We have found that HH514 cells are most efficiently induced by treatment with both o-13-tetraphorbol acetate (TPA) and n-butyrate (data not shown). Chemical induction of the three pEBV-BAC-60 packaging cell lines resulted in a dramatic increase in GFP expression within 48 hours, as seen by fluorescence microscopy (Fig. 2B); this is a good indirect indication that the EBV lytic cycle of replication is efficiently amplifying the amplicon.Preparation and Analysis of pEBV-BAC-60 Virion DNAFive days after induction of the lytic cycle, the virus was harvested from the culture supernatant. Analysis of cells and virus by Gardella gel electrophoresis [26Gardella T. Medveczky P. Sairenji T. Mulder C. Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis.J. Virol. 1984; 50: 248-254Crossref PubMed Google Scholar] showed a reduction in circular episomal DNA in the cells, and a distinct band representing linear virion DNA in the viral supernatant (data not shown). Infectious virions were concentrated from the cellular supernatant 100-fold by ultracentrifugation. To analyze the virus in more detail, DNA was extracted in agarose plugs from 50 μl of concentrated virion stock. The DNA was resolved by pulsed-field gel electrophoresis (PFGE) with and without I-PpoI digestion and transferred to nylon membrane by Southern blot. Two hybridization probes were used to determine the structure of the packaged virions. First, a pBAC108L probe showed that the undigested linear pEBV-BAC-60 virion DNA is ~ 170 kb in size, consistent with the predicted 172 kb (Fig. 3A). Digestion with I-PpoI yields bands consistent with the predicted amplicon dimer (Fig. 3B). Second, an EBNA-1 probe, which also detects the HH514 helper virus, revealed bands consistent with the structure shown in Fig. 3B. Following hybridization with the EBNA-1 probe, the blot was analyzed on a Phosphorimager to compare yields of the helper and recombinant viruses. Quantitative comparison of signal intensities revealed that packaged pEBV-BAC-60 virion made up 3–6% of total signal. Because there are two copies of EBNA-1 in the amplicon, which is packaged as a dimer, this represents 1.5–3% of total viral yield from the three clonal lines. This level is consistent with data reported from other EBV amplicon systems [27Wang J. Vos J.-M.H. Development of a competitive PCR method for physical titration of recombinant EBV vector in a helper-dependent packaging system.Mol. Ther. 2001; 3: 976-983Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar].FIG. 3Analysis of virion DNA. (A) DNA extracted from viral particles in agarose was run on a pulsed-field gel, either undigested (lanes U) or after digestion (lanes D) with I-PpoI. The gel was blotted and hybridized with probes for pBAC (left) and EBNA-1 (right). The upper bands after EBNA-1 hybridization to digested DNA show the presence of the helper virus, which is not cut by I-PpoI. Lower bands are those that carry the vector region of the amplicon. The relative intensities of the 150- to 170-kb bands (helper) and the 73/81-kb bands (amplicon), as measured by Phosphorimager analysis, are a measure of relative yield of helper virus and EBV-BAC-60 amplicon in the virion preparation. (B) The size of the bands of the predicted structure of recombinant dimer virus. After digestion with I-PpoI, the two bands derived from the recombinant virus are apparent using each probe: the central 81-kb band that would be produced by digestion of the intact vector, and the terminal band of either 8 or 73 kb generated by linearization at the terminal repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Infection of B CellsWe used 50 μl of concentrated virion to infect ~ 5 × 105 cells from the EBV-negative BL cell line, Loukes. The cells were assayed 48 hours postinfection for transduction by the virion GFP marker gene by microscopy and flow cytometry (Fig. 4A). Defining an infected cell to be one with a fluorescence level > 99.5% of uninfected cells (region M2 in Fig. 4), we observed that 30–40% of the 5 × 105 cells were infected by 50 μl of concentrated virus. This equates to an pEBV-BAC-60 amplicon titer of ~ 3–4 × 106 t.u./ml in concentrated virion stocks. This is likely to be an underestimate of the infection because, from the profile of green fluorescence, it appears that the entire cell population is greener than the uninfected cells, a shift that does not occur after infection with HH514 virus alone. A subset of the cells (~ 10%, as described by region M4) clearly express a considerably higher level of GFP than the majority. These are the cells that are visibly infected under fluorescent microscopy. It is also apparent that amplicon preparations that gave stronger signals by pulsed-field analysis (Fig. 3) infected a higher proportion of cells.FIG. 4Amplicon infection of B cells. (A) Loukes cells were infected with pEBV-BAC-60 virions, from HH514 clones 1, 2, and 3. Cells were analyzed by fluorescence microscopy and by flow cytometry. Region M2 encompasses the cells that are greener than 99.5% of uninfected cells, which defines a cell as "infected." Region M4 encompasses those cells that are considerably greener than the majority of the cell population and are the cells that are green under fluorescence microscopy. The cytometry data were used to calculate the pEBV-BAC-60 titer expressed as transducing units/ml. (B) LCLs infected with virus purified from pEBV-BAC-60-HH514 clone 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The pEBV-BAC-60 amplicon was then tested for its ability to infect three lymphocyte cell lines (LCLs) immortalized by the B95-8 strain of EBV. LCLs made from patients are a common resource in the study of genetic disorders, and the ability of the pEBV-BAC amplicon system to infect LCLs would give the vector system broad applicability to gene expression studies using complete genomic DNA loci. Three LCLs derived from patients with the recessive disorder ataxia telangiectasia (A-T) were infected. Fluorescence microscopy and flow cytometry showed that the cell lines exhibited different levels of infection (Fig. 4B). Judging infection by the M2 criterion, IAT-9.3, IAT-16.3, and IAT-23.6 were infected with 46%, 36%, and 55% efficiency, respectively. However, a much smaller proportion of cells fell in the M4 region for the LCLs than for the Loukes cells, and so fluorescence microscopy considerably underestimates the efficiency of infection of LCLs (Fig. 4B).The Episomal Genomic Transgene Is Stably Carried in Cell Lines Derived by InfectionClonal cell lines were derived from the pEBV-BAC-60-infected Loukes cells by limiting-dilution cloning under hygromycin B selection. Plasmid rescue assays were used to analyze the episomes present in selected lines. NotI digestion and PFGE analysis of rescued episome from three lines showed that they carried episomes that gave the same pattern of NotI digestion as the parental plasmid, pEBV-BAC-60 (Fig. 5A). A 21-kb NotI fragment representing the vector was present in all but one of the rescued plasmids.FIG. 5Analysis of plasmids rescued from three Loukes clones expanded following pEBV-BAC-60 infection. Cell lines are named LI-C3 (for Loukes infected with clone 3) followed by a cell line-specific coordinate. DNA prepared from four bacterial colonies following plasmid rescue from each cell line are run either undigested (lanes U) or following digestion with Noti (lanes D) on a pulsed-field gel. The pattern of bands is compared with the original plasmid pEBV-BAC-60 digested with NotI, and with undigested p172 (172 kb) and p88 (88 kb). (A) The samples were resolved by PFGE; the gel was then stained with ethidium bromide and photographed. (B) The gel was transferred to nylon membrane by Southern blotting and hybridized to the PCMV-CFP-PRSV-Hygr cassette. Undigested DNA runs on the gel in a supercoiled form (migrating more slowly) and as linear DNA, as a result of damage sustained during DNA preparation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that the rescued plasmid is a dimer of pBAC-EBV-60, as would be expected if recircularization had occurred at the EBV TRs postinfection, we analyzed undigested rescued amplicon. Undigested plasmid was not visible by ethidium bromide staining, so the gel was blotted and hybridized to a vector-specific probe (Fig. 5B). The undigested plasmid DNA formed two bands, representing supercoiled (upper band) and linear (lower band) DNA. When the pattern of bands from undigested rescued plasmids was compared to those of a 172-kb plasmid (p172), it was seen that the majority of rescued plasmids were pEBV-BAC-60 dimers, of a total size of ~ 178 kb (Fig. 5B).This is consistent with the previous data (Fig. 3) showing packaging of pEBV-BAC-60 as a dimer in EBV virions.Packaging and Delivery of a 123-kb Genomic DNA InsertThe previous data show that the general strategy outlined in Fig. 1B can be applied to use the pEBV-BAC system to deliver a 60-kb insert as a dimer. Notably, the process of delivery of pEBV-BAC-60 by infection to B cells was ~ 2000 times more efficient than by transfection, which delivered DNA to < 0.005% of cells, comparing both by fluorescence microscopy. We were next interested in delivering a 123-kb genomic DNA insert containing the complete NBS1 locus. HH514 cells were transfected with the vector pEBV-BAC-123 (total size 152 kb; Fig. 1A), and a number of clonal packaging lines were isolated. Figure 6A shows plasmid rescue analysis from one of the lines. Quantitative Southern blotting of the pEBV-BAC-123 packaging line showed the episome to be present at ~ 17 copies/cell (data not shown). Chemical stimulation of three pEBV-BAC-123 packaging lines using TPA and n-butyrate induced the EBV lytic cycle and packaging of amplicon into virions. DNA was isolated from virions and resolved by PFGE, both undigested and digested with NotI, Southern blotted, and hybridized using GFP as a probe (Fig. 6B). The pattern of bands observed with both probes is identical for each clone and consistent with the expected bands from the 152-kb amplicon packaged and linearized at the TRs (Fig. 6C). Some virions have also been packaged at 175 kb, which may be a product of recombination during packaging. This is implied by the presence of a 19-kb NotI fragment that is not expected in a monomer virus. Finally, the virion stock was used to infect Loukes cells (Fig. 6D). Analysis of the infection by cytometry showed pEBV-BAC-123 amplicon titer to be 1–2 × 105 t.u./ml in concentrated virion stock.FIG. 6Packaging and infectious delivery of the 123-kb NBS1 insert. (A) Plasmid rescue from one of the clonal HH514 packaging cell lines created for pEBV-BAC-123 shows the episome to be present and unrearranged. (B) Southern blot analysis of pEBV-BAC-123 virion DNA. DNA was extracted from virions prepared from three pEBV-BAC-123 packaging lines, resolved by PFGE either undigested (lanes U) or following digestion with NotI (lanes D), transferred to nylon membrane, and hybridized with the vector-specific GFP DNA probe. Hybridization of a BAC probe to the digested DNA highlighted an ~8-kb band as well as the 19-kb band shown above (not shown). (C) The size of the bands of the predicted structure of recombinant virus. After digestion with NotI, fragments of 8 kb and 11 kb are predicted to be released from the termini of the virus. Failure to linearize at the TR will produce a 19-kb band (the sum of the terminal bands) after NotI digestion. The genomic organization of the 123 kb is shown, approximately to scale. The NBS1 gene is adjacent to the ht41 gene as shown. While the entire open reading frame of ht41 is contained within the genomic insert, its first exon and its promoter are missing. (D) pEBV-BAC-123 virions prepared from all three packaging lines were used to infect Loukes cells, and vector delivery was inferred from GFP expression in target cells.Vie