7.1.2 
Re gulatory Elements of the tgAAVCF Vector 
The left-hand and right-hand inverted terminal repeats (ITRs) are derived from the AAV2 virus. 
These 145 base sequences contain internal palindromes that are thought to exist in a T-shaped 
hairpin structure*. The ITR sequences are required in cis for viral DNA replication, rescue, 
integration, and encapsidation^. The ITR also contains several regions that correspond to SP1 
binding sites^-S, as well as a sequence that is highly similar to the Inr sequence found at 
eukaryotic transcriptional initiation sites^-S. These sequences are likely to be responsible for 
the finding that the AAV ITR can function as a transcriptional promoter^. The tgAAVCF 
vector utilizes the promoter activity of the ITR to drive expression of the CFTR cDNA. The 
polyadenylation site is provided by a synthetic sequence based on the mouse 6-globin 
polyadenylation sequence *0. 
7.1.3 Construction of the tgAAVCF Vector 
The tgAAVCF vector is based on SA313, an AAV vector driving CFTR expression from the 
AAV p5 promoter^. The sequence of the CFTR cDNA in this construct has several differences 
from that described in Genbank (Accession number M28668* *). First, there are 3 clustered 
silent mutations that were introduced to allow propagation in a bacterial host *2 (T->C at 930; 
A->G at 933; T->C at 936). Second, there is an A->C change at nucleotide 1990 leading to a 
N->H amino acid change. This was found to be an inadvertent error in the original sequence 
(L.-C. Tsui, personal communication). Finally, there is a G->A change at position 4555, 
resulting in a V->M amino acid change *3. This change is a cloning artifact, but has been 
shown not to effect CFTR function as judged by electrophysiologic assays. 
The AAV sequences in this construct also were found to have changes relative to the accepted 
AAV2 sequence*. First, there is a T->G change at nucleotide number 2 in the left-hand ITR. 
Second, there was a deletion of the terminal 8 bases from the right-hand ITR. These changes 
do not appear to grossly effect replication, encapsidation, or transduction with this vector. 
A cloning strategy was devised to 1) correct the V->M mutation in the CFTR cDNA; 2) correct 
the mutation and deletion found in the AAV ITR sequences; and 3) remove the AAV p5 
promoter to allow transcription from the ITR and reduce the size of the vector to slightly larger 
than wild-type AAV. 
All constructions were performed using standard molecular biology techniques. Cloning steps 
were verified by restriction digest analysis, and where appropriate, DNA sequencing. 
Oligonucleotides were provided by Immunex Corporation. 
Correction of the V->M mutation in the CFTR cDNA was accomplished as illustrated in figure 
7.1.2. pSA313 was digested with Nco I and Stu I to release an approximately 450 bp fragment 
of 3' CFTR sequence containing the mutation. The plasmid BA-CFTR Afl III stop (kindly 
provided by M. Drumm), containing the correct 3' CFTR cDNA, was digested with Nco I and 
Hinc II and a 419 bp fragment isolated. These were ligated together to yield the plasmid p5CF. 
Removal of the AAV p5 promoter was accomplished in 3 steps (figures 7. 1.3-7. 1.5). A 934 
bp Aat II - Xba I fragment encompassing the left-hand ITR, p5 promoter, and 5' CFTR 
sequence was isolated and cloned into Aat II/Xba I digested pUC 19 to yield pUCAX. A 523 
bp Ava I/Xba I fragment containing the 5' end of the CFTR cDNA, including the initiator 
codon was isolated from 6ACFTRBQ (kindly provided by M. Drumm), and cloning into Ava 
I/Xba I digested pUC19 to yield pAVX. 
Recombinant DNA Research, Volume 20 
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