of AdCFTR and AvlCFl compared to the genome of Ad5 is shown in Figure 2.3-A 
and details of the "expression cassette" of both vectors is shown in Fig- 
ures 2.3-B. In both vectors, the El deletion encompasses the entire Ela 
coding region and 69. 5X of the left hand end of the Elb region. The E3 
deletion in both vectors comprises 66% of the E3 region (leaving 20% of the 
left end of E3 and 14% of the right end of E3) . AdCFTR and AV1CF1 have 
minor differences in regards to the "expression cassette" relating to the 
expression of the CFTR cDNA. These are detailed in Section 2., above and in 
Table 2.3-B. The sequence of AvlCFl (based on direct sequencing of the 
expression cassette and the published sequence of Ad5) is in appendix 3. 
The minor sequence differences of AdCFTR from this sequence are detailed in 
Table 2.3-B. These regions of minor differences of AdCFTR as compared to 
AvlCFl in the "expression cassette" [the left hand end through the begin- 
ning region of homology with the regions of minor differences of Ad5 (9.24 
mu)] are being sequenced and will be made available to the Recombinant DNA 
Advisory Committee and the FDA. 
The deletions of Ad5 used as the base for AdCFTR total 4754 bp and AvlCFl 
total 4743 bp. To make AdCFTR amd AvlCFl, the expression cassettes detailed 
in Figures 2.3-B and Table 2.3-A were combined by homologous recombination 
with the E1“E3 _ Ad5 base to yield a final genome of approximately 101 mu. 
The upper limit of genomic DNA that can be packaged into an infectious Ad 
virion is 105 mu. At 101 mu, AvlCFl and AdCFTR are approximately 3.8% less 
than the maximal packagable genome. 
From a functional point of view, AdCFTR and AvlCFl are replication defi- 
cient since both are missing El genes coding for Ela proteins [28K, 42-54K, 
and 48-58K proteins] and Elb proteins [15K, 22K and 55K proteins] that play 
a critical role in regulating replication. Given that these vectors are 
replication deficient and missing most of E3 , they should not have the 
ability of Ad5 to subvert the immune/inf lammatory system (See section 4.8). 
From the newly inserted information, AdCFTR and AvlCFl contain elements to 
direct the expression of the human CFTR cDNA [the Ela promoter/enhancer, 
the MLP and the tripartite leaders (sequences that increase translation 
efficiency)]. This is followed by the CFTR cDNA itself. The CFTR mRNA 
directed by AdCFTR likely uses the SV40 early region polyadenylation signal 
3' to the CFTR 3' untranslated region, while the CFTR mRNA directed by 
AvlCFl likely uses the polyadenylation signal within the Ad5 Elb region 
(4038 to 4043 bp of Ad5) . 
The packaged AdCFTR and AvlCFl virions have a CsCl density similar to Ad5 
(1.33-1.35 g/ml) . 
2.4 Fabrication and Production 
AdCFTR and AvlCFl were constructed by homologous recombination of an adeno- 
virus vector construction plasmid containing the human CFTR cDNA and Ad5 
sequences and the large Clal fragment of Addl327 (for AvlCFl, a replication 
competent adenovirus type 5 based E3~ mutant) or Addl324 (for AdCFTR, a 
replication deficient adenovirus type 5 based E1~E3 - mutant) . The regions 
of Addl327 and Addl324 that are in AvlCFl and AdCFTR, respectively, have 
identical sequences (Addl327 was derived from Addl324; T. Shenk, personal 
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