•Limulus Amebocyte Lysate (LAL) (Protocol SPGT332070 M.A., Inc.): 
The purpose of this study is to detect and quantitatively determine the gram negative 
bacterial endotoxin level of a test article or extract using the Limulus Amebocyte 
Lysate (LAL) gel-clot method for testing. 
•Replication Competent Ad5-please see description of assays in section IV.D.2.e 
•Presence and quantity of functional Ad.CB-CFTR-see description of assay in section 
IV.D.2.C 
IV.D.2.e Assays for functional CFTR virus and contaminating wild type Ad5 virus 
The final preparation of purified Ad.CB-CFTR virus will be evaluated for the presence and 
concentration of virions capable of transducing CF defect. The total particle number will be 
determined by measurement of the absorbance at 260nm. An aliquot of the virus preparation 
will be diluted serially and used to infect both 293 cells and the cell line CFPAC which was 
derived from a pancreatic adenocarcinoma of a patient with cystic fibrosis. The infected 293 
cells will be evaluated for El transcomplementing virus using the previously described plaque 
forming assay. The infected CFPAC cells will be evaluated for CFTR protein expression by 
immunocytochemistry and correction of cAMP mediated chloride transport using SPQ assay. 
Descriptions of these assays are provided in section II.B.3. 
Several experiments will be performed to evaluate the virus preparations for contamination 
with wild type adenoviruses. The presence of wild type adenoviruses in the 293 cell line will 
be evaluated by M.A., Inc. using a standard assay in which a variety of indicator cell lines are 
exposed to 293 supernatants, passaged, and inspected for cytopathic effects. 
Detection and quantitation of low level wild type adenovirus in high titer preparations of the 
recombinant Ad.CB-CFTR virus is confounded by the observation that El deleted viruses will 
cause cytopathic effects (CPE) in non El expressing cells, such as HELA cells infected at high 
MOI's. Mechanism(s) for this relate to the direct toxic effect of high concentrations of viral 
proteins such as fiber and the possibility of low level viral replication of El deleted virus at 
high MOI. We find it necessary to dilute the concentrated stock at least 100 to 1000 fold to 
avoid CPE in most indicator cell lines. Two types of assays will be performed. A typical virus 
preparation contains 5 x 10 12 particles/ml based on OD at 260 nm and ~10 11 functional 
virions/ml based on pfu or assays that measure CFTR protein expression 
(immunocytochemistry or SPQ functional analyses). The first assay is based on the ability of 
wild type Ad (but not El deleted Ad) to replicate in nonEI expressing cell lines. An aliquot of 
each preparation representing 5% of the total will be diluted and exposed to a variety of 
indicator cell lines. The cells are passaged for 2 weeks to allow spead of wild type virus and 
subsequently examined for CPE. Specifically, for each ml of stock, 5 pi is removed, diluted and 
exposed to 10 x 15 cm plates of confluent indicator cells. Preliminary results with purified 
preps of recombinant viruses have failed to demonstrate CPE under these conditions. The 
sensitivity of the assay is currently being defined in reconstitution experiments. 
Another assay has been developed to detect and quantify wild type Ad5 in the stocks of 
recombinant virus. The potential sources of wild type Ad5 are contamination from the initial 
transfection or recombination with Ad5 sequences originally transfected into the 293 cells. The 
assay is based on PCR detection of El sequences in DNA isolated from the stock of recombinant 
virus. Figure 20 shows an agarose gel of PCR products in which 125 ng of total cellular DNA 
was supplemented with varying amounts of wild type Ad viral DNA (from 100,000 to 0.1 copies 
of Ad5 per reaction). Amplification for 35 cycles afforded a sensitivity equal to 100 viral 
[832] 
Recombinant DNA Research, Volume 16 
