The final stored preparation to be used in the clinical protocol consists 
of AdCFTR at a titer of 10 u to 10 12 pfu/ml in 10 mM Tris-Cl, pH 7.4, 1 mM 
MgCl z , and 10X glycerol. The preparation is stored at -70* until use. Prior 
to in vivo administration, the preparation is thawed and diluted to the 
required concentration in a final buffer of 10 mM Tris-Cl, pH 7.4, 1 mM 
MgCl 2 , and 3.3% glycerol. 
Quality control measures to be used are designed to assay the 293 cells, 
the seed virus used for initial infection, the virus prior to purification 
and the final purified virus for adventitious agents, structure, and func- 
tion. All studies will be carried out under the U.S. Good Laboratory Prac- 
tices (GLP) and Regulations as promulgated in Title 21 CFR Section 5.8. 
The 293 cell bank will be assessed for bacteria and fungus, sterility, 
mycoplasma, karyology, (isozyme and cytogenetic analysis), in vitro evalua- 
tion for non-specific virus contaminants by evaluating indicator cells for 
cytopathic effect, in vivo evaluation for non-specific virus using adult 
mice, suckling mice, guinea pigs and embryonated hens egg for clinical 
signs of viral infection, Epstein-Barr virus, cytomegalovirus, bovine virus 
(including bovine viral diarrhea virus, infectious bovine rhinotracheitis 
virus, parainfluenza 3 virus, replication competent adenoviruses, and 
bovine parvovirus), hepatitis B virus, human immunodeficiency virus, por- 
cine parvovirus, adenovirus, and electron microscopy (as a general virus 
screen) . 
The genome of the AdCFTR seed virus will be evaluated for structural integ- 
rity by Southern analysis, DNA sequence of the left end of the vector 
through the region at the beginning of the large Clal fragment of Addl324, 
titer, expression of exogenous CFTR cDNA at the mRNA and protein levels in 
vitro and in vivo (see sections 3.1, 3.2), expression of viral genes 
(hexon) at the mRNA and protein levels (see section 4.2), DNA replication 
in cultured human epithelial cells (HeLa) and in freshly isolated human 
respiratory epithelial cells (see Section 4.1), for the presence of contam- 
inating adenovirus type 5 Ela sequences (e.g., from contamination in the 
laboratory), sterility, and adventitious agents. 
The final purified AdCFTR preparation will be evaluated for sterility, 
limulus amebocyte lysate (for gram negative bacterial endotoxin) and gener- 
al safety (inoculation into guinea pigs and mice and observation for overt 
signs of ill-health or unusual response). 
The evaluations listed above will be carried out using standard GLP condi- 
tions. The evaluation for the presence of contaminating Ad5 will be carried 
out by assessing the preparation for the presence of Ad5 Ela sequences. 
This is accomplished using a PCR-based amplification system with oligonu- 
cleotide primers located within the structural portion of the Ela 
gene (evaluating for the presence of Ad5 sequences 562 to 899 bp; (GenBank 
accession No. 73260). As a positive control, and to define the sensitivity 
of the assay, each assay includes a standard curve consisting of mixtures 
of the recombinant vector and an Ela + Ad5 virus. As a control for the 
presence of DNA, each sample is evaluated by PCR using oligonucleotide 
primers within the E2b region (Ad2 base numbers 6671 to 7072; Ad5 and the 
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