2. 4. 4. 4 Master Virus Bank: The MVB will be evaluated using the procedures outlined below. 
If any test is not satisfactorily passed, the MVB will be discarded. In addition, the MVB will 
be extensively evaluated for the presence of wild type or replicative adenovirus in addition to 
other infectious agents. 
Sterility Test; M.A. Inc, Protocol No. 1514.510: See above (2. 4. 4. 3) for description. 
Mycoplasma; M.A. Inc, Protocol No. 1514.102003: See above (2. 4. 4. 3) for description. 
In Vitro Vims Assay; M.A. Inc, Protocol No. 1514.003: See above (2. 4.4. 3) for descrip- 
tion. 
Recombinant Adenoviral Vector-Derived Expression of Functional Human CFTR: The 
MVB will be evaluated for the presence and function of AvVlCF2 virus by several assays. The 
total amount of AE1 virus will be quantified by the plaque assay (see below) on 293 cells. The 
efficiency of gene transfer and expression of human CFTR and correction of the epithelial cell 
Cl* secretory defect of cystic fibrosis will be evaluated in CFPAC-1 cells by the chloride efflux 
assay as previously described (Trapnell et. al., 1991b; appendix 1; see below and Figure 21), 
and by measurement of forskolin-induced potential difference measurements (see below and 
Figure 22). 
Measurement of Forskolin-induced CFTR-Specific Changes in Electrical Potential: 
CFPAC-1 cells are cultured on millipore filter supports until confluent in DMEM, 10% fetal 
bovine serum (FBS) at 37°C in a humidified, 5% atmosphere C0 2 . When the electrical 
resistance across the membrane is high demonstrating confluence, the cells are infected with 
AvlCF2 or an irrelevant control adenovirus at several MOIs. After 48 hr, the filter is placed 
into a Ussing chamber and forskolin - stimulated electrical potential changes are evaluated (see 
below and Figure 22). 
Measurement of ^Cl* Efflux: The chloride efflux assay used has been previously described 
(Trapnell et.al., 1991b). Briefly, CFPAC-1 cells are seeded on 35 mm plates and cultured in 
DMEM, 10% fetal bovine serum (FBS) at 37°C in a humidified, 5% atmosphere C0 2 . The cells 
are 95% confluent within 24 hr and are infected with AvlCF2 or an irrelevant control 
adenovirus at several MOIs. After 48 hr, the cells are washed twice with bicarbonate-free 
lactated Ringer’s solution, and^oaded with 36 C1' at 3 /zCi/ml for 2 hr. After extensive washing 
(x6), cells are evaluated for secretion of 36 C1" into the media by sequentially removing and 
replacing the Ringer’s solution. The aliquots of removed Ringer’s lactate solution and finally 
the cell layer itself are counted by liquid scintillation counting. The data are compiled and 
represented as the amount (percent) of 36 C1" remaining within cells as a function of time after 
washing. Forskolin (13 ^M) is included in the Ringer’s lactate (except the wash) when 
evaluating cAMP-stimulated 36 C1' efflux (forskolin raises the intracellular cAMP concentration 
in CFPAC-1 cells). 
Replication Competent Adenovirus: The MVB will be evaluated extensively for the 
presence of contaminating wild type or parental (Ad-d]327) virus using a PCR detection system 
(Figures 14, 15, and see below section 2. 4. 4. 7). 
2. 4. 4. 5 Cell Harvest (Crude Viral Lysate): Aliquots of cells from the Master Cell Bank 
(MCB) are infected with AvlCF2 virus from the Master Virus Bank (MVB). A resulting 
"Batch" of Crude Viral Lysate (CVL) will then be evaluated by the assays listed below. Batches 
of CVL which fail any test will be discarded. Those that pass will be further purified for 
clinical certification. 
Sterility Test; M.A. Inc, Protocol No. 1514.510: See above (2.4.4. 3) for description. 
Mycoplasma; M.A. Inc, Protocol No. 1514.102003: See above (2.4.4. 3) for description. 
Recombinant DNA Research, Volume 17 
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