AAV is not known to cause any disease. AAV is not a transforming or oncogenic virus. 
AAV integration into chromosomes of human cell lines does not cause any significant 
alteration in the growth properties or morphological characteristics of the cells. These 
properties of AAV recommend it as a potentially useful human gene therapy vector because 
most of the other viral vectors proposed for this application such as retroviruses, 
adenoviruses, herpes viruses, and poxviruses can cause disease. 
The general principles of AAV vector construction have been defined as reviewed 
recently 14,15 AAV vectors are constructed in AAV recombinant plasmids by substituting 
portions of the AAV coding sequence with foreign DNA to generate a vector plasmid. In 
the vector plasmid, the terminal (ITR) portions of the AAV sequence must be retained intact 
because these regions are required in cis for several functions, including excision from the 
plasmid after transfection, replication of the vector genome, and integration and rescue 
from the host cell genome. The vector plasmid is transfected into a producer cell line and 
packaged into an AAV particle to generate an AAV transducing virus by infection of the 
producer cells with an appropriate helper virus such as adenovirus. In order to achieve 
replication and encapsidation of the vector genome into AAV particles, the vector plasmid 
must be complemented for any AAV functions required in trans, namely rep and cap, that 
were deleted in construction of the vector plasmid. From lysates of such producer cells the 
AAV vector particles can be purified and concentrated. 
There are several desirable features of AAV vectors that are designed for use in human 
gene therapy. First, the transducing vector can be generated at sufficiently high titers that it 
is practical as a delivery system. This is especially important for gene therapy stratagems 
aimed at in vivo delivery of the vector. Secondly, the vector preparations can be generated 
free of wild type AAV virus. Third, the vector need not contain any coding sequences 
except the gene to be used as the therapeutic agent. 
3.3 In Vitro Characterization of the AAV vector system 
The construction and packaging of recombinant adeno-associated virus vectors suitable for 
transduction of the airway epithelium was first accomplished with the neomycin 
phosphotransferase reporter gene vector, pAAVp5neo^. This vector was used to 
transduce the CF bronchial epithelial cell line, D33-1, and up to 70% of the cells expressed 
neo, as assessed by G418 resistance. Similar vectors expressing the CFTR cDNA were 
constructed and packaged into infectious AAV virions 1 '. These vectors were used to 
transduce the CF bronchial epithelial cells in culture. Even in the absence of selection, 
CFTR protein expression was detectable by immunofluorescence, and CFTR-mediated 
cAMP-dependent chloride conductance was observed. 
Further delineation of the nature of the complementation seen in these cells was 
accomplished using excised patch-clamp recordings^. These studies revealed that the 
corrected cells demonstrated both (1) the appearance of small linear chloride conductances, 
previously associated with recombinant CFTR expression, and (2) the restoration of cAMP 
regulation of the outwardly rectifying chloride channel, previously shown to be defective in 
the airway epithelium in CF. 
The ability to transduce non-dividing cells is an additional consideration relevant to in vivo 
gene transfer to the airway epithelium, where most surface ciliated cells are in G 0 at any 
given point in time. Experiments in growth-arrested cells indicate that cell division is not 
absolutely required for AAV vector gene transfer and expression, although cell division 
appears to enhance the efficiency of gene transfer^. 
Recombinant DNA Research, Volume 20 
[ 119 ] 
