M.J. Welsh and A.E. Smith, RAC Application 
Ad2 /CFTR-1 
24 hr 
48 hr 
72 hr 
96 hr 
MOI 
1 10 50 
1 10 
50 1 
10 50 
1 10 50 
23.1 kb- 
9.4 kb- 
6.6 kb — 
4.4 kb- 
Figure 7. Southern blot analysis of Ad2/CFTR-1 
The only evidence as to whether the Ad2/CFTR-1 is rearranged is the length of the viral 
DNA on Southern blots. Although the sensitivity of this assay is low, there is no evidence 
of altered molecular species indicating rearrangement. 
B.2.a.(4) How many copies are present per cell? How stable is the added DNA both in 
terms of its continued presence and its structural stability? 
Copy number per cell and DNA stability 
The number of copies of extrachromosomal Ad2/CFTR-DNA per cell will depend on the 
multiplicity of infection (MOI) used. In the proposed protocol, three different multiplicities 
will be tested. Because the intended area of application is about 1 cm 2 and the human nasal 
epithelium contains 2 x 10 6 cells/cm 2 , these multiplicities are 1, 10, and 25 PFU/cell. 
Assuming all cells take up viral DNA equally, quantitation of the Southern blot experiment 
using human primary nasal polyp cells grown in monolayer (Fig. 7) indicates that following 
infection with Ad2/CFTR-1 at an MOI of 50 the maximum number of DNA molecules 
detected per cell is approximately 550. In treated monkeys, we have detected 6- 
galactosidase expression 2 weeks following exposure to Ad2/6Gal-l and CFTR expression 
1 week after exposure to Ad2/CFTR-1. There are reports in the literature suggesting 
transcripts from a related virus can be detected in cotton rats 42 days after exposure, using 
PCR methods (11). Presumably, viral DNA persists for at least this period. We do not 
Recombinant DNA Research, Volume 16 
[885] 
