M.J. Welsh and A.E. Smith, RAC Application 
B.2.a.(l) What cells are the intended target ce lls of recombinant DN A? If target cells are 
to be treated ex vivo and returned to the patien t, how will the cells be characterized 
before and after treatment? What is the theoretical and practic al basis for assuming 
that only the target cells will incorporate the DNA? 
The nasal respiratory epithelium is the intended target. Cells will not be treated ex vivo. 
The theoretical basis for assuming that only the target cells will incorporate DNA is: a) the 
adenovirus has a tropism for airway epithelium; b) application is to a localized area of nasal 
epithelium; c) the epithelium provides a barrier to virus movement from the airway lumen 
to the interstitial space; d) the total number of viruses applied to the surface epithelium will 
be very small. The practical basis for assuming that only the target cells will incorporate 
the DNA is that, in ongoing animal experiments, we have observed no transfer of DNA to 
cells other than the respiratory epithelium (Point B.2.c.(2) 3.c). However, adenovirus also 
has tropism for other cells such as the gut, and despite the precautions taken, we cannot be 
certain that no recombinant adenovirus will infect a cell other than a respiratory epithelial 
cell. 
B.2.a.(2) Is the delivery system efficient? What percentage of the target cells contain the 
added DNA? 
Because adenovirus DNA is predominantly not integrated and because the percentage of 
target cells that contain DNA and the copy number in those cells is dependent on the 
multiplicity of infection, we have not examined in detail the number of target cells that 
contain added DNA. Instead, we have measured the expression of the DNA; these 
experiments are described in response to point B.2.b.(3). 
B.2.a.(3) How is the structure of the added DNA sequences monitored and what is the 
sensitivity of the analysis? Is the added DNA extrachromosomal or integrated? Is the 
added DNA unrearranged? 
We examined the state and the copy number of Ad2/CFTR-1 DNA using Southern blot 
analysis. Rather than extract only soluble low molecular weight DNA by the Hirt 
procedure, total cellular DNA was prepared and the full complement of cellular DNA was 
digested with BstBl, electrophoresed and probed by Southern blotting. Figure 7 shows the 
state of Ad2 DNA in confluent monolayers of human primary nasal polyps cells at different 
times after infection with different multiplicities of Ad2/CFTR-1. The results indicate that 
Ad2 DNA can be detected as the predicted size BstB 1 fragment from the left hand end of 
the genome with no evidence of high molecular weight chromosomal integrated DNA. The 
sensitivity of the experiment shown in Fig. 7 is not certain, but longer exposures of this and 
similar experiments indicate that at the 0.1-1% detection level, no integrated DNA is 
detected. In similar experiments we found no evidence for Ad2 DNA integration in 
monkey, hamster or HeLa cells at this level of detection. However, based on the literature, 
we would expect perhaps some integration, but at a level far below our sensitivity of 
detection (81,84-86). 
The experiment in Fig. 7 also indicates that at high multiplicities of infection, there is time- 
dependent viral DNA synthesis in human nasal polyp cells. In other experiments, we have 
detected limited Ad2/CFTR-1 or Ad2/BGal-l DNA synthesis in HeLa, monkey bronchiolar 
and hamster primary tracheal cells. We have shown that synthesis is dependent on 
multiplicity of infection, and found that in human cells viral DNA accumulation peaks at 
about 2-4 days post infection. This result was not unexpected since earlier reports of Ela- 
or Elb-deleted vectors reported some DNA synthesis (123-126). 
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Recombinant DNA Research, Volume 16 
