Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

The cloning and reconstitution of a bovine adenovirus type 2 E3 deletion mutant and the sequencing and analysis of the early 4 region

Recombinant Adenoviruses (Ads) have been shown to have potential applications in three areas: gene therapy, high level protein expression and recombinant vaccines.' At least three different locations within the Ad genome can be deleted and subsequently used for the insertion of foreign sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4) regions. Viral vectors of this type have been well studied in Human Ads 2 and 5, however one has not yet been constructed for Bovine Adenovirus Type 2 (BAV2). The E3 region is located between 76.6 and 86 m.u. on the r-strand and is transcribed in a rightward direction. The gene products of the Early 3 region (E3) have been shown to be non-essential for viral replication, in vitro, but are required for host immunosurveillance. This study represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A deletion of 1800bp was made within the E3 region of BAV2 and the thymidine kinase gene was subsequently inserted in the deleted area . . The plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate homologous recombination with the wild type BAV2 to produce a mutant. Southern Blotting and Hybridization results suggest the presence of a BAV2 E3 deletion mutant with thymidine kinase sequences present. The E4 region of Human Adenovirus types 2 and 5 is located at the extreme right end of the genome (91.3 map units - 99.1 map units) and is transcribed in a leftward direction giving rise to a complicated set of differentially spliced mRNAs. Essentially there are 7 open reading frames (ORFs) encoding for at least 7 polypeptides. The gene products encoded by the E4 region have been shown to be essential for the expression of late viral genes, host cell shutoff and normal viral growth. We have cloned and sequenced the right end segment between 90.5 map units and 100 map units of the BAV2 genome. The results show several open reading frames which encode polypeptides exhibiting homology to three polypeptides encoded by the E4 region of human adenovirus type 2. These include the 14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction enzyme map, and ORF map of the E4 region could be very useful in future molecular manipulation of this region and could possibly explain the slow growth rate of BAV2 in MDBK cells.

Isolation and characterization of genomic DNA sequences that enhance the stability of plasmid DNA in mammalian cells /

The ease of production and manipulation has made plasmid DNA a prime target for its use in gene transfer technologies such as gene therapy and DNA vaccines. The major drawback of plasmid however is its stability within mammalian cells. Plasmid DNA is usually lost by cellular mechanisms or as a result of mitosis by simple dilution. This study set out to search for mammalian genomic DNA sequences that would enhance the stability of plasmid DNA in mammalian cells.Creating a plasmid based genomic DNA library, we were able to screen the human genome by transfecting the library into Human Embryonic Kidney (HEK 293) Cells. Cells that contained plasmid DNA were selected, using G418 for 14 days. The resulting population was then screened for the presence of biologically active plasmid DNA using the process of transformation as a detector.A commercially available plasmid DNA isolation kit was modified to extract plasmid DNA from mammalian cells. The standardized protocol had a detection limit of -0.6 plasmids per cell in one million cells. This allowed for the detection of 45 plasmids that were maintained for 32 days in the HEK 293 cells. Sequencing of selected inserts revealed a significantly higher thymine content in comparison to the human genome. Sequences with high A/T content have been associated with Scaffold/Matrix Attachment Region (S/MAR) sequences in mammalian cells. Therefore, association with the nuclear matrix might be required for the stability of plasmids in mammalian cells.