2.2. Molecular Biology of AE1 Recombinant Adenovirus Vectors: The two best studied 
adenoviral genomes are from serotypes 2 and 5 and complete nucleotide sequences have been 
determined for each. Most recombinant adenoviral vector gene transfer systems are derived 
from one or parts of both of these viruses. It has been known for some time that removal of 
the El region will render the adenovirus unable to replicate in many cell types. This is true of 
all cells which do not produce an ElA-like factor. Such an Ela-like factor can act in trans to 
restore the replication of AE1 viruses back to wild type levels. Replication deficient 
recombinant adenovial vectors have been studied for several years with regard to developing 
their potential for use for in vivo human gene therapy. In this context, Perricaudet et.al., (1992) 
have developed a recombinant adenoviral vector system based on Ad5 carrying deletions in the 
viral early region 1 (AE1) and 3 (AE3). The E3 deletion creates space for insertion of an 
exogenous cDNA as does the El deletion. However, as noted above, the El deletion also 
largely inactivates the endogenous genetic program of the virus including its capacity to 
replicate. Several recombinant human adenoviral vectors have been constructed which are 
deficient in replication due to El region deletions and contain an expression cassette containing 
the normal human CFTR cDNA (Figure 4). These vectors have minor differences with regard 
to the structure of the expression cassette and are expected to function similarly. Multiple in 
vivo studies have now shown that these first generation adenoviral (Avl) vectors can 
efficaciously and safely be used in animals even when large amounts of virus are used (Wilson 
et.al., RAC Proposal, 1992; Crystal et.al., RAC Proposal, 1992). Studies by Levrero et.al., 
1991 have been conducted in non-human primates with an Ad5 based AE1, AE3 vector 
expressing the hepatitis surface antigen. Rosenfeld et.al., (1991a) has also carried out studies 
in cotton rats where similar vectors except expressing either human a 1 -antitrypsin or CFTR 
were successfully transferred to the respiratory airway epithelium in vivo with no reported 
toxicity. Finally, up to 3 x 10 11 plaque forming units of recombinant AE1, AE3 adenovirus has 
been administered into the lung of a non-human primate with no (Wilson et.al., RAC Proposal, 
1992). Thus, it appears that AE1, AE3 recombinant adenoviral vectors may provide a safe 
mechanism for in vivo gene delivery. 
2.3. Construction of AvlCF2: AvlCF2 (Figure 5) was constructed in several steps consisting 
of routine prokaryotic cloning procedures and a homologous recombination procedure in the cell 
line 293. First, an adenoviral construction shuttle plasmid (pAvS6) was assembled (see below 
for details). The human CFTR cDNA insert from the plasmid pBQ4.7 was then removed and 
inserted into pAvS6 to yield pAvS6-CFTR. The sequence of pAvS6-CFTR was confirmed by 
direct sequence analysis of both strands and was determined to have the sequence expected (see 
appendix 7). pAvS6 was then linearized and co-transfected into 293 cells and allowed to 
recombine with a fragment of the Ad5-derived virus Ad-dl327 to yield AvlCF2. Purified DNA 
from AvlCF2 was evaluated by Southern analysis to confirm structural integrity (not shown). 
2.3.1 Construction of The Shuttle Plasmid pAvS6: The adenoviral construction shuttle 
plasmid pAvS6 was constructed in several steps using standard cloning techniques and 
polymerase chain reaction based cloning techniques. First, the 2913 bp Bglll, Hindlll fragment 
was removed from Ad-d]327 and inserted as a blunt fragment into the Xhol site of pKSII 
(Stratagene, La Jolla, CA) (Figure 6). The orientation of this fragment was such that the Bglll 
site was nearest the T7 RNA polymerase site of pKSII and the Hindlll site was nearest the T3 
RNA polymerase site of pKSII'. This plasmid was designated pHR. 
Recombinant DNA Research, Volume 17 
[371] 
