2. Replication Deficient Recombinant Adenovirus Containing the Human CFTR 
cDNA 
The two vectors evaluated in this protocol, AdCFTR and AvlCFl , are identi- 
cal in general construction, differing only in the "expression cassette" 
relating to control of expression of the CFTR cDNA (see Figures 2.3-A, 
2.3-B and Table 2.3-B, in Section 2.3 below). AdCFTR was constructed by 
Transgene SA, Strasbourg, France and M. Perricaudet (CNRS) , Institut 
Gustave Roussy, Villejuif, France (see Appendix 1). AvlCFl was constructed 
by B. Trapnell, M. Rosenfeld and R. Crystal, in the Pulmonary Branch, 
NHLBI . Both are based on the genome of Ad5 , a member of the common subgroup 
C of adenoviruses (see Section 2.1, below). In both, all of the Ela region 
has been deleted, as have the 69.5% of the left hand portion of Elb and 66X 
of the middle part of the E3 region. Both vectors begin (starting from the 
left end) with the left inverted terminal repeat (ITR) and origin of repli- 
cation from Ad5 and the encapsidation signal and Ela promoter/enhancer 
element from Ad5 . In both vectors, this is followed by the major late 
promoter (MLP) and tripartite leader sequences; in AdCFTR these elements 
are from Ad2 (also a member of subgroup C, with 94.7% sequence homology to 
Ad5) , while in AvlCFl they are from Ad5 . Following the tripartite leader, 
both contain the normal human CFTR cDNA flanked by short 5' and 3' untrans- 
lated regions from the CFTR genome; there are minor differences in the two 
vectors in the length of these regions and the sequence of the CFTR cDNA 
(see Table 2.3-B). The major difference in the two vectors is following the 
CFTR cDNA 3' untranslated region. In AdCFTR there is a poly A signal (the 
SV40 early region poly A signal) whereas this element is not present in 
AvlCFl (AvlCFl uses a poly A signal in the Elb region, see Figure 2.3-A). 
Following the CFTR cDNA "expression cassette", AdCFTR and AvlCFl are iden- 
tical and are based on the sequences of Ad5 from 9.24 m.u. through the 
right ITR. Both have two deletions in this sequence (see Appendix 3) : (1) a 
2 bp deletion in the VA-I promoter that interrupts a minor alternative 
transcription start site for VA-I RNA; and (2) a deletion of the 66% of 
middle part of 3. AvlCFl and AdCFTR were derived from different base Ad5 
deletion mutants (Addl327 for AvlCFl, Addl324 for AdCFTR); this difference 
is irrelevant to the final construct, with the right end of AdCFTR and 
AvlCFl identified after the CFTR expression cassette. For a discussion of 
the derivation and final structure of AvlCFl, see Section 2.1, and appendix 
3. For a discussion of the derivation and final structure of AdCFTR, see 
appendix 1 and appendix 3. The recombinant genomes of both AdCFTR and 
AvlCFl are packaged into replication deficient recombinant adenovirus using 
293 cells (Graham et al., 1977), a human embryonic kidney cell line con- 
taining El sequences of Ad5 . 
Both AdCFTR and AvlCFl will infect human epithelial cells and express the 
human CFTR cDNA as evidenced at the mRNA level (Northern analysis) and 
protein level ( immunohis tochemis try , immunoprecipitation followed by phos- 
phorylation with kinase). For both vectors, the expression of the CFTR cDNA 
is in amounts sufficient to complement mutations of the CFTR gene, enabling 
the epithelial cells derived from individuals with CF to secrete Cl - in 
response to elevations in cAMP. In the sections dealing with efficacy 
(Section 3) and safety (Section 4) data with both vectors are presented; 
for all of the data presented, the specific vector used (AdCFTR or AvlCFl) 
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Recombinant DNA Research, Volume 16 
