Genzyme Corporation Ad2/CFTR-2 Gene Transfer Protocol: Single 
One Kendall Square, Cambridge, MA 02139 Aerosol Administration to the Lung of CF Patients 
instillation and not vector-related injury at the early time point, but that this resolved by Day 10. On Day 10 there was 
very mild lung inflammation associated with multiple injections of the adenovirus vector. However by Day 21 all 
parameters were resolving or resolved with only the high dose animals remaining affected by some indicators of injury. 
Others have reported adenovirus vector associated inflammation, however we had not seen significant inflammation in 
the cotton rats, rabbits or monkeys in previous experiments. Therefore, pilot studies were performed under conditions 
that would produce moderate to severe injury in the lung as a result of vector administration. The lungs of BALB/c mice 
were moderately injured only in response to a partially purified preparation of Ad2/CFTR-2 and not to high doses of 
purified virus. However, in the C57BL/6 mouse, severe inflammation was seen in the lungs at 7 days and longer after 
administration of a high dose of purified Ad2/CRR-2 (3 x 1 0 9 IU). Thus inflammation due to adenovirus may vary with 
different species or genetic background as well as with vector and dose. 
Additional studies in rodents are being conducted to measure adenovirus vector-induced inflammation in response to 
the variables of dose, time post treatment, virus purity, virus activity and genetic composition of the recipient rodent. 
Although we conclude that rodents represent good models in which we can explore the inflammatory response to 
adenovirus vectors, questions remain as to the mechanism and significance of inflammation seen only in a subset of 
animal models. 
Rhesus Monkey Studies: As part of our long-term program to develop adenovirus vectors for CF gene therapy we have 
conducted a series of experiments in Rhesus monkeys ( Macaca mulatta). Rhesus monkeys were studied because the 
physiology and anatomy of the airway epithelium closely resembles that of humans [23]. Initially we applied Ad2/CFTR- 
1 into the nasal cavity of 3 monkeys on 3 occasions. Then we applied Ad2/CFTR-2 to the nasal epithelium on 2 
additional occasions for a total of 5 administrations. Although all 3 monkeys had positive antibody titers to adenovirus, 
none of the animals developed any clinical signs of viral infection or inflammation, either systemically or in the nasal 
epithelia. There was no viral replication and biopsies and brushings of treated epithelia showed no evidence of 
inflammation. This repeated administration did not prevent expression of CFTR. 
In an ongoing study, to examine the effects of repeat administration of Ad2/CFTR-2 to the lung of Rhesus monkeys, 8 
monkeys are arranged in 3 groups for treatment; 2 animals are treated with vehicle alone, 3 animals are treated with 3 x 
10 9 IU (approximately 4.5 x 10 11 particles), and 3 animals are treated with 3 x 10 10 IU (approximately 4.5 x 10 12 
particles). To date these animals have been treated 11-16 times by instilling virus through a bronchoscope into the left 
caudal lobe at 3 week intervals. All treated animals have consistent dose-related elevations in anti-adenovirus 
antibodies. The animals have remained clinically normal at these doses throughout the study as assessed by body 
weight, respiratory rate, heart rate, clinical chemistry, CBCs and X-ray. Protein and cell content of bronchoalveolar 
lavage (BAL) is generally higher in treated versus control animals but the levels are not consistently altered in proportion 
with either the concentration of virus administered or number of treatments. One parameter that is consistently altered 
in treated monkeys is an increase in the percentage of lymphocytes (mostly CD8+) in the BAL. One animal from the low 
dose and one from the high dose groups were sacrificed after 1 1 doses and examined for evidence of histopathology in 
the lungs. There were some minimal changes involving <5% of the alveoli in the treated lobes of both animals. In 
addition, the treated lobe of the higher dose animal also had a focal fibrotic lesion that was characterized by an outside 
pathologist as moderate to severe. Several features of this lesion including its focal nature suggested that it may not 
have been directly related to the protocol but to a pre-existing condition. The relative contribution of the repeated 
instillation of vector is unknown. The doses have been increased from 3 x 10 9 IU to 6 x 10 10 IU and from 3 x 10 10 IU to 
3 x 10 11 IU in the remaining animals for further observations. Although no serious adverse events have been detected 
after 6 exposures to these increased doses, some lesions have been noted on the chest x-rays at the site of treatment. 
In our second study of repeat administration of Ad2/CFTR-2 to the lungs of Rhesus monkeys, 2 animals that had been 
exposed previously to 5 nasal exposures received administration of 1-2 x 10 11 IU (2.2 - 3 x 1 0 1 2 /kg body weight - 
approximately 1 00 times the proposed maximum human dose) of Ad2/CFTR-2 to the whole lung through a 
bronchoscope. No treatment-related adverse reactions were observed in either monkey, and the lungs of the animals 
were judged as normal by HRCT. 
Recombinant DNA Research, Volume 20 
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