Recombinant DMA Advisory Committee - ’hjyAl'H 
analysis to distinguish endogenous versus transgene expression. 
Investigator Response-Dr. Brigham 
Dr. Brigham responded to the questions concerning transduction efficiency and in vivo 
expression of the transgene in preclinical studies. He presented data demonstrating 
histochemical staining of the entire airway epithelium of rabbits with an AAT antibody 
following aerosol delivery of the DNA/liposome complex. Dr. Parkman commented that 
although transgene expression is demonstrated in cross sections obtained from airway 
tissue, a three-dimensional analysis is preferable. Dr. Brigham expressed his reservations 
about using the (5-galactosidase reporter gene to demonstrate transgene expression in the 
surface of bronchial epithelium, citing the possibility of obtaining false positive results. 
Serial cross sections will reveal expression in a three-dimensional sense. Dr. Brigham 
noted that in vitro and in vivo experiments have been published indicating that 10 to 15% 
of cells express the AAT protein. Dr. Parkman noted that the RAC has not had the 
opportunity to review this data and recommended that such data should be submitted as 
a stipulation for approval. Dr. Brigham agreed to submit the requested data with the 
reporter gene. Dr. Brigham presented in vitro data demonstrating protection against 
protease digestion of bronchial epithelial cells by the AAT transgene. Detection of 
transgene expression in normal individuals is complicated by the fact that normal lungs 
have high levels of endogenous AAT activity. Since the lungs are not the normal source 
of AAT synthesis (the protein is made in the liver and transported to the lungs), it is 
possible to demonstrate de novo synthesis of AAT by the transgene in organ cultures of 
the lung. The DNA/liposome complex will be instilled via bronchoscope to a distal 
wedge of the lung. Following resection, the tissue will be examined by serial cross 
sections to detect in vivo transgene expression. Histological toxicity data will be obtained 
regarding inflammatory responses. 
Dr. Parkman reviewed the published protein expression data described previously by Dr. 
Brigham. Following bronchial administration, approximately one-third of the rabbit 
epithelial cells express the human AAT protein as demonstrated by fluorescence 
labelling. The transgene was detected in the liver of rabbits 90 minutes following aerosol 
administration of DNA/liposome complex to the lungs. Such data raises questions about 
the possibility of germ-line integration and the necessity of long-term follow-up since 
some of the AAT deficient patients will have long life spans. 
Dr. Brigham said that systemic distribution of vector DNA is a legitimate concern; 
however, the rabbit experiments involved aerosol delivery to the entire lungs and not the 
localized delivery proposed for the human protocol. Vector DNA was not detected in 
either the ovaries or testes of animals via intravenous delivery of DNA/liposome 
complexes; therefore, germ-line transmission is not a concern. Dr. Parkman suggested 
inclusion of a statement in the Informed Consent document about the possibility of low 
level systemic absorption. Dr. Brigham agreed to include such a statement. 
In response to the issues raised by Dr. Zallen, Dr. Brigham stated that: (1) the 
experimental period for the human study will be reduced from 72 to 48 hours, (2) the 
Recombinant DNA Research, Volume 19 
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