plaques was plaque-purified a second time and re-scored by restriction digest and southern analysis. 
One of the positive plaques from the second purification was amplified, purified by cesium chloride 
gradient ultracentrifugation, desalted, and stored in the -80 °C freezer in 10 % glycerol in PBS. Viral 
stocks were then tested for the ability to confer ganciclovir sensitivity to rat glioblastoma cell lines. 
IV. C Sequence Analysis of Recombinant Virus, H5.010RSVT/C 
H5.010RSVTK r viral DNA was isolated from the purified viral preparation obtained after two rounds of 
plague purification. Selected areas of the viral genome one being sequenced by an approved FDA facility 
in compliance with GLP (Good Laboratory Procedures). 
Two areas of the genome are being subjected to complete sequence analysis including 1) the 5' end of the 
genome spanning the 5' ITR, the entire minigene cassette including RSV promoter, the TK cDNA, and 2) 
the region surrounding the E3 deletion. Regions to be sequenced will be subcloned as overlapping 
restriction fragments into pBluescript II (Stratagene) and pGem5Zf (Promega). Nested deletion clones 
will be generated in both directions for each of the subclones using a modified exolll SI nuclease 
procedure. These deletion clones will be size selected to provide complete coverage of each strand and 
sequenced using the dideoxynucleotide termination procedure. Internal sequencing primers will be 
synthesized and used to close gaps between contigs and to fill in any single-stranded regions. 
IV. D Strategy for Characterization of Clinical Grade Adenovirus - Quality Assurs 
and Control 
A four-stage test program has been designed to assess the master cell bank, the seed lot, the product 
intermediate (cell lysate) and the purified product (virus). The 293 cells used to produce the 
adenovirus will be characterized prior to infection for possible microbial, adventitious viral and select 
specific human viral contaminants. Testing of the adenovirus preparation used to infect the cells will 
include assays for microbial contaminants and adventitious virus. After expansion of the infected cells, 
the cell lysate will be evaluated for microbial contaminants. Product testing of the purified product for 
endotoxin, microbial contaminants, extraneous toxins and infectious adenovirus completes the test 
battery. Each step will be carefully tested for the presence of wild type adenovirus. The majority of 
tests will be performed by Microbiological Associates Inc. (MAI), an independent laboratory that has 
provided contract research and safety assessment for the pharmaceutical industry. 
Similar to the generation of recombinant retroviral gene therapy reagents, adenoviral reagents have 
been constructed to provide "cassette" oriented approaches to their production. A recombinant 
adenoviral vector was constructed from a modified adenovirus type 5 in which the minigene of interest 
is inserted in place of El a and Elb. This vector is cotransfected along with the large fragment of the 
enzyme-restricted Ad5 DNA into 293 cells. 293 cells are a human kidney cell line that contains a 
functional El a gene and provides a trans-acting El a protein in order to allow for homologous infectious 
virions and formation of plaques. A Master Cell Bank (MCB) of 293 cells have been previously 
established and evaluated for performance, in terms of production of recombinant adenoviruses and for 
the absence of other pathogenic contaminants. Individual plaques are isolated and functional DNA would 
be identified by restriction cleavage and Southern blot analysis. The MCB is then infected with the 
crude viral lysates and plaque-purified a second time in order to generate a seed lot using 293 cells. 
The purified seed lot lysate is subjected to safety testing. The MCB is then plated again and infected with 
the certified viral seed lot. Lysates are harvested from the infected cells and virus is purified from the 
lysate and cryopreserved. Individual production lots are extensively evaluated. The purified 
adenoviral DNA is then subjected to sequencing. 
Additional details on the adenovirus production process and quality contol measures is provided in 
Appendix VI. G, page 35. 
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