Fairly extensive studies have also been performed on an AAV-CFTR transduced EB3-1 cell 
line, A35. These studies indicate that vector DNA integration is present in approximately 
5% of cells as judged by fluorescent in situ hybridization (FISH), while additional copies 
of the vector genome are detectable in the low molecular DNA fraction from these cells, 
indicating that both integrated and unintegrated forms may be present. The identifiable 
integration sites appear to be different from the described chromosome 19 integration site 
(AAVS 1). These studies taken in total indicate that while the use of AAV recombinant 
vectors may introduce some risk of insertional mutagenesis, this risk may be lessened by 
the fact that vector DNA may frequently be present in extra-chromosomal sites. 
3.4 Preliminary Animal Experiments 
3.4.1 Rabbit studies 
The same AAV-CFTR vectors used for the in vitro studies were evaluated for biological 
efficacy in vivo after delivery to the airway surface of New Zealand white rabbits^. A 
flexible fiberoptic bronchoscope was used to deliver 10^ particles of AAV-CFTR vector 
to the surface of the right lower lobe bronchus. The presence of AAV-CFTR vector 
genomes appeared to be limited to the right lower lobe of each of the vector-treated animals 
as assessed by in situ DNA PCR. RNA expression was detectable by PCR in lung 
homogenates from rabbits sacrificed 3 days, 10 days, 3 months, and 6 months after vector 
instillation, while none was observed in vehicle-treated controls. CFTR protein expression 
was detectable by immunohistochemistry and immunoblotting at each of these time points 
as well. There were no histopathologic changes seen in the lung, liver, kidney, or heart 
samples from any of these animals. These findings demonstrate both biologic activity and 
safety of AAV-CFTR vector administration to the airway surface, when given in a manner 
analogous to that which would be used for clinical gene therapy trials in CF patients. 
Another aspect of in vivo delivery which warrants consideration is the possibility that, in 
the course of vector delivery through a fiberoptic bronchoscope, the mucosal barrier may 
be breached resulting in significant systemic spread of recombinant virus. This issue has 
been studied by directly injecting a high dose (10^ particles) of AAV-CFTR into the 
auricular veins of New Zealand white rabbits. Animals were sacrificed at 1 week after 
vector injection and the spread of vector DNA was studied by standard PCR and in situ 
PCR of the following organs: RLL lung, LUL lung, trachea, thymus, bronchial lymph 
node, heart, liver, spleen, pancreas, kidney, jejunum, mesenteric lymph node, gonad, and 
brain. No vector DNA was detectable in a vehicle-injected control animal, but vector DNA 
was present in thymus of both of the vector-injected animals, and in the bronchial lymph 
node of one of two vector-injected animals. No vector DNA was detectable in the gonads 
or any other organs of either of the vector-injected animals. This study, indicates that while 
hematogenous dissemination of AAV-CFTR may occur after direct intravenous injection, 
this is not likely to result in germ-line alteration, even in the unlikely event that an entire 
bolus of the highest vector dose were to enter the bloodstream. 
3.4.2 Primate studies 
Based on the characteristics of the AAV vector system and the experimental preclinical 
biology described above, one might expect pneumonitis or other pulmonary toxicity to 
occur much less frequently with AAV-CFTR than with adeno-CFTR vectors. Rhesus 
monkeys were chosen for the final preclinical phase of AAV-CFTR vector testing since 
these primates provide a suitable host for AAV and adenoviruses. They are particularly 
relevant in the context of identifying potential problems related to recombinant virus 
f 120] 
Recombinant DNA Research, Volume 20 
