C0 2 environment until plaques appeared (approximately 1 to 2 weeks). Plaques were then 
picked and intracellular virus was released by 3 successive cycles of freezing and thawing. After 
clarification by centrifugation, the lysate was used to reinfect 293 cells for a second round of 
plaque purification. Subsequently, amplification to high titer virus was carried out by 
successively infecting plates of 293 cells, CsCl purification and concentration of virus and 
reinfection as described below (Production of High Titer Adenovirus). 
2.3.4 Construction of Infectious AvlLacZ4 Virus: AvlLacZ4 was constructed in a fashion 
similar to AvlCF2 and has an identical general genomic organization. First a nuclear targeted 
lac Z gene was constructed using standard and PCR based cloning methods. The recombinant 
gene consists of the first 4 codons from the SV40 large T antigen followed by codons 127 to 147 
encoding the SV40 large T antigen nuclear targeting signal (Genbank accession # V01380) and 
then codons 6 - 1021 of the E. coli LacZ gene (bases 1302 to 4358), Genbank accession 
#J01636). This gene fragment was then inserted into the EcoRV site of pAvS6 (Figure 11). 
The orientation of the gene coding strand with respect to the plasmid pAvS6 was similar to that 
of pAvS6-CFTR. Final assembly of the infectious, AvlLacZ4 recombinant adenovirus was 
identical to that of AvlCF2 (not shown). 
2.3.5 Construction of Infectious AvlNulll Virus: AvlNulll has a genomic organization 
similar to that of AvlCFl, but does not contain an exogenous cDNA in the expression cassette. 
The details of its construction have been previously described (Crystal et.al., RAC Proposal, 
1992). 
2.4. Production of the Recombinant Adenovirus AvlCF2: The strategy for production of 
the recombinant adenoviral vector AvlCF2 (Figure 12) consists of initially generating a Master 
Cell Bank (MCB) of 293 cells (a human kidney epithelial cell line containing the left 1 1 % of the 
Ad5 genome). Cells from the MCB were then be used to generate a stock of high titer AvlCF2 
viral stock referred to as the Master Viral Bank (MVB). Both the MCB and MVB will be 
characterized with regard to performance in production of adenovirus vector and absence of 
pathogenic contaminants and certified for use in production of biologic material for clinical use 
under FDA approved procedures for Good Laboratory Practice (GLP). 
Aliquots of the MVB will be used to infect aliquots of the MCB as described below to 
generate "Batches" of crude viral lysate (CVL). Batches CVL will be characterized, purified 
and stored as lots of purified infectious virus at a concentration of approximately 10 11 to 10 12 
pfu/ml in a sterile vehicle consisting of 10 mM Tris-HCl, pH 7.4, 1 mM MgCl 2 , 10% (v/v) 
glycerol at -70°C. 
All procedures involved in the production, characterization, packaging, labeling and storage 
of AvlCF2 will be performed in compliance with FDA/GLP regulations. Importantly, quality 
assessment of the products will be performed at every stage of the production process as outlined 
below. 
2.4.1 Production of High Titer Adenovirus: Batches of AvlCF2 virus will be produced by 
infecting 30 plates of 293 cells (15 cm diameter, approximately 70 - 80 % confluent) from the 
MCB with AvlCF2 from the MVB at an multiplicity of infection (MOI) of 10 pfu/cell in 
IMEM, 2% fetal calf serum, 1% glutamine for 90 min with rocking. Medium will then be 
supplemented to 10% fetal calf serum and cells will be cultured for 36 - 40 hr. Infected cells 
will be collected by low speed centrifugation and intracellular virus will be released by 3 cycles 
of freezing and thawing to generate a batch of CVL containing AvlCF2. 
Recombinant DNA Research, Volume 17 
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