M.J. Welsh and A.E. Smith, RAC Application 
4.3. Design of the Present Generation Vector 
The vector to be utilized in the present protocol is an El -deficient Ad2 (Fig. 1). It is named 
Ad2/CF1R-1 and includes the coding sequence for CFTR in place of El. Removal of El 
Major Late Transcription ^ 
ITR El a Elb 
V/////A NLS-B-qalactosidase V////A Ad2/BGal-1 
\r* 
v pix > 
Figure 1. Ad2/CFTR-1 and Ad2/BGal-l 
has several advantages: a) it impairs lytic viral replication in human cells; b) it deletes the 
region of the genome associated with the in vitro transforming activity of the virus; and c) 
it creates space in the genome for the insertion of exogenous DNA. We also constructed a 
related vector encoding 8-galactosidase (Ad2/8Gal-l). 
Unlike many El deleted recombinant adenovirus vectors (10,11,80), Ad2/CFTR-1 retains 
the E3 region. This region is dispensable, at least for growth in tissue culture, and it is 
often deleted to allow space for the newly introduced DNA sequences. Since this was not 
done with Ad2/CFTR-1, its DNA is approximately 104.5% of the length of the wild-type 
adenovirus DNA. This means that the viral DNA is at the upper limit of size able to 
package into virions (88,89). For this reason, the Ad2/CFTR-1 virus grows less readily 
than normal, typically producing 5-10% of the yield obtained in 293 cells with vectors of 
wild-type size. 
The E3 region of the Ad2/CFTR-1 encodes a variety of proteins. One of these proteins, 
gpl9, is believed to interact with and prevent presentation of class I proteins of the major 
histocompatability complex (MHC) (reviewed in 90). This property prevents recognition 
of the infected cells and thus may allow viral latency. The presence of E3 sequences, 
therefore, has two useful attributes; first, the large size of the viral DNA renders it doubly 
defective for replication (i.e., it lacks early functions and is packaged poorly) and second, 
the absence of MHC presentation could be useful in later applications of Ad2/CFTR-1 in 
gene therapy involving multiple administrations, because it may avoid an immune response 
to recombinant virus-containing cells. 
Recombinant DNA Research, Volume 16 
[865] 
