Genzyme Corporation Ad2/CFTR-2 Gene Transfer Protocol: Single 
One Kendall Square, Cambridge, MA 02139 Aerosol Administration to the Lung of CF Patients 
A 6 x 10 11 IU dose of Ad2/CFTR-2 (approximately 4.8 x 10 13 particles; approximately 10 13 particles/kg body weight) 
was instilled into the lung of one of the monkeys through a bronchoscope. This dose is nearly 400 times higher than the 
proposed human dose range. This was the seventh dose of an Ad2-based vector (3 doses of Ad2/CFTR-1 and 4 
doses of Ad2/CFTR-2) administered to the animal. One day later, the animal had tachypnea and hypoxemia and high 
resolution chest CT revealed diffuse ground-glass infiltrates in the left lung with some air bronchograms suggestive of a 
diffuse pneumonitis. On the 5^ day after vector administration the monkey was lethargic, its mucosal membranes were 
dry, and it was hypothermic. Despite the administration of resuscitative fluids and O 2 , the animal died. 
At necropsy there were areas of discoloration noted in the left lung. On microscopic examination chronic inflammation 
was observed in large areas of the lung, while other regions had no apparent or minimal involvement. Importantly, the 
lungs showed evidence of healing in the damaged areas at the time of death. The independent pathologist doubted that 
the pathologically significant pulmonary disease was adequate to cause death. Congestion and edema were also 
observed in several organs suggesting heart failure and there was multifocal infarction of the adrenal gland. 
Another Rhesus monkey (that had previously received 1 intranasal and 1 whole lung exposure to Ad2/CMVB-Gal) was 
instilled with approximately 1 .4 x 10 11 IU (approximately 1.1 x 10 13 particles; approximately 1.1 x 10 12 /kg body weight) 
of the same preparation of Ad2/CFTR-2 to the whole lung. This monkey experienced no treatment-related adverse 
reactions, and the lungs of this animal were normal as judged by HRCT. 
Based on extensive studies in which primates and rodents have been treated with doses of adenovirus vectors several 
logs greater than our maximum proposed human dose, we conclude that very high doses of Ad2/CFTR-2 can induce 
lung inflammation. The monkey death may be an indication of the maximum tolerated dose of an adenovirus vector in 
Rhesus monkeys. 
Genzyme believes that our current, and proposed, Ad2/CFTR-2 clinical protocols will expose the participants to little 
risk. Specifically, the doses of Ad2/CFTR-2 in these clinical studies are much lower than those used in the animal 
studies. The first human nasal dose is approximately 5 logs lower than the dose given the monkey that died on a per kg 
body weight basis while the maximum human dose of 10 10 is approximately 400 fold less than the dose given the 
monkey on a per kg body weight basis. 
Preclinical Aerosol Studies: For our initial studies in the lung of humans, we will be evaluating the aerosol delivery of 
Ad2/CFTR-2. Therefore, we are testing the safety and efficacy of single and repeat aerosol administration of 
Ad2/CFTR-2 and Ad2/CMV(3-gal in the lungs of cotton rats, mice and Rhesus monkeys. The objective of the first 2 
studies was to determine the safety of a single aerosol dose of Ad2/CFTR-2 to the lungs of cotton rats and mice. An 
additional aerosol study was conducted to evaluate the amount and distribution of aerosolized Ad2/CMVB-gal virus to 
cotton rats and mice (C57 BL/6 mice and BALB/c mice). Together these studies demonstrate that vector can be 
aerosolized effectively into the lungs of rodents, that the levels of expression are equal to or greater than other routes of 
administration and that the aerosol route of administration may possibly be safer. 
Finally, a study was conducted to assess the safety of aerosolized Ad2/CFTR-2 in the lungs of Rhesus monkeys 
exposed to a dose of 5 x 10 10 IU which is twice the proposed human dose. As a preliminary experiment, we 
aerosolized 2 doses of Ad2/CMVB-gal virus. Each dose (5x1 0 9 IU and 5x1 0 10 IU) was aerosolized into the lungs of 1 
monkey which was analyzed 3 day post-exposure. In these monkey studies we demonstrated that vector could be 
effectively aerosolized into the lungs of a nonhuman primate. At the highest dose tested there was some mild 
inflammation produced in the lungs of these monkeys, but none was seen at doses comparable to those planned 
clinically in humans. 
Additional information on the in vivo studies which investigated the safety of Ad2/CFTR-2 and the efficacy of adenovirus 
vectors is presented in Appendix 1 of this application. 
Clinical Studies of Ad2/CFTR-2 (ORDA Protocol #9312-0671 : Adenovirus may be a good vector system for transfer of 
CFTR cDNA to correct the CF pulmonary defect. However, use of adenovirus vectors as a treatment for CF lung 
disease will require repeat administrations. Moreover, the use, and further development of adenoviral vectors requires 
some demonstration of clinical efficacy in CF airway epithelia involved by the disease. 
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Recombinant DNA Research, Volume 20 
