Infectious Delivery of 120-Kilobase Genomic DNA by an Epstein–Barr Virus Amplicon Vector  Robert E. White, Richard Wade-Martins, Michael R. James  Molecular Therapy  Volume 5, Issue 4, Pages 427-435 (April 2002) DOI: 10.1006/mthe.2002.0557 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 General strategy for the delivery of genomic DNA by an EBV amplicon vector. (A) The EBV amplicon plasmid used in this study contains CFP, hygromycin phosphotransferase (Hygr), EBNA-1, and the EBV elements oriP, the terminal repeats packaging signals (TR), and oriLyt with adjacent internal repeat region IR2. The constructs were built by inserting the genomic DNA inserts at the NotI sites into a precursor vector, followed by the addition of the oriLytI-PpoI fragment. (B) An outline of the strategy used to create packaging clonal cell lines and for production of infectious EBV amplicons. Molecular Therapy 2002 5, 427-435DOI: (10.1006/mthe.2002.0557) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Analysis 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. Molecular Therapy 2002 5, 427-435DOI: (10.1006/mthe.2002.0557) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Analysis 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. Molecular Therapy 2002 5, 427-435DOI: (10.1006/mthe.2002.0557) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Amplicon 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. Molecular Therapy 2002 5, 427-435DOI: (10.1006/mthe.2002.0557) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Analysis 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. Molecular Therapy 2002 5, 427-435DOI: (10.1006/mthe.2002.0557) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Packaging 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. Molecular Therapy 2002 5, 427-435DOI: (10.1006/mthe.2002.0557) Copyright © 2002 American Society for Gene Therapy Terms and Conditions