M.J. Welsh and A.E. Smith, RAC Application 
APPENDIX 5 
Scientific Abstract of the Protocol 
The goal of this protocol is to assess the safety and biochemical efficacy of a recombinant 
adenovirus for the treatment of cystic fibrosis (CF). Although the eventual aim of such an 
approach is to treat the airway epithelial cells in the lungs of CF patients, the initial test will be 
done in the nasal epithelia. We will use nasal epithelium because it resembles that of the 
intrapulmonary airways, because its use allows us to apply minimal amounts of virus, and 
because the accessibility of the tissue allows us to measure directly the transepithelial electrical 
potential difference, a property that is defective in CF patients. Safety can also be assessed by 
removing cells and biopsy tissue for examination. 
The adenovirus vector Ad2/CFTR-1 has the cDNA for CFTR inserted in place of the early region 
1 genes. This renders the virus impaired for replication because the El genes are req uired for the 
first stages of viral infection and impaired for packaging DNA into virions because CFTR cDNA 
is larger than the El DNA it replaces. The virus can be produced in a human cell line called 293 
because the cells constitutively expresses El proteins. Studies on the life cycle of Ad2/CFTR-1 
indicate that it is severely impaired for early and late gene transcription and DNA synthesis and 
no Ad2/CFTR-1 virus replication has been detected in tissue culture or animals. Safety and 
efficacy studies in cells in culture and in animals including nonhuman primates show: 1) that 
Ad2/CFTR- 1 can complement the chloride transport defect in human CF nasal epithelial 
monolayers in culture and 2) that other than a mild transient inflammatory response, Ad2/CFTR- 
1 has no adverse effects in hamsters and monkeys. 
The protocol involves production of Ad2/CFTR-1 virus in 293 cells that have been extensively 
tested for adventious agents, using a viral seed stock that has been similarly tested. Following 
purification and further testing, the Ad2/CFTR-1 stock will be diluted to 50-200 )J.l and applied 
directly to the nasal epithelium Virus will be applied to an area of about 0.5 cm 2 in each nostril 
using a small plastic applicator. After 30 minutes, the virus will be removed and the area 
washed. Three patients will be treated sequentially with doses of 1 x 10 6 , 1 x 10 7 and 5 x 10 7 
plaque forming units of virus. 
Participants will be patients with CF who are at least 18 years old and have only mild to 
moderate disease. We prefer patients homozygous for the common AF508 mutation. We will 
require patients to be seropositive for adenovirus 2 antibody and to have no evidence of Ad2 or 
Ad5 El DNA sequences in their nasal epithelium. Following treatment, patients will be 
followed in the hospital until the virus is no longer present. Samples of nasal cells will be 
brushed from the treated area and tested for the presence of CFTR mRNA and protein and for the 
presence of replicated Ad2/CFTR-1. Measurements of the transepithelial electrical potential 
difference will be made of the treated and surrounding areas. Precautions to prevent spread of 
the virus to health care workers and the environment are described. 
The successful outcome of this protocol will establish the safety and efficacy of Ad2/CFTR-1 
and will be invaluable in the design of subsequent protocols and of future generations of 
adenovirus vector. 
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Recombinant DNA Research, Volume 16 
