Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
Adeno-associated virus (AAV) vectors might have some advantages over adenovirus. 
AAV constructs can be used to deliver functioning transgenes to the lungs in vivo . Flotte 
reported expression of the CFTR gene in airway epithelial cells of rabbits for several weeks 
following airway delivery of the gene in an AAV vector with no evidence of inflammation (6). 
Problems with this approach include difficulties in producing quantities of the AAV constructs 
which would be required clinically and at least theoretical problems resulting from 
incorporation of the vector into the host genome. 
Liposome mediated DNA delivery in vivo In 1 989, we initially demonstrated expression 
of a reporter gene in the lungs of animals following either intravenous or intra-airway delivery 
of plasmid/cationic liposome complexes (7). Since then several investigators have used this 
approach in experimental animals and, more recently, in phase I studies in humans. 
In both rabbits and cotton rats, delivery of reporter genes and of human CFTR to the 
lungs has been demonstrated following airway instillation or aerosol delivery of cationic 
plasmid/liposome complexes (8-1 1). Very high transfection efficiencies have been reported in 
airway epithelial cells (from 40% to 70% (8)) in the absence of demonstrable toxicity. In the 
transgenic mouse model of cystic fibrosis, delivery of a CFTR containing plasmid in this 
manner produced expression of the CFTR in the lungs and partial correction of the chloride 
transport defect (10). Our earlier observation that intravenous delivery of plasmid/liposome 
complexes was not toxic and resulted in transgene expression in the lungs has been confirmed 
by others (11). 
Delivery of plasmid/eationic liposome complexes to the lungs, at least over short 
periods, does not alter lung structure or funetion. Following aerosol delivery of a plasmid 
using cationic liposomes. Debs saw prolonged expression of a reporter gene in the lungs of mice 
without toxicity (8). Other kinds of liposomes have been delivered to humans by aerosol with 
no alteration in lung function (12). We have delivered plasmid/liposome complexes to rabbits 
repeatedly for four weeks by both aerosol and intravenous injection and have seen no effects 
on lung mechanics, arterial blood gases or lung strueture (13). 
Some phase I studies using cationic liposomes to deliver DNA to humans are now 
underway in this country and in England. Nabel and coworkers have used this technology with 
direct injection into melanomas in humans (14,15). More recently, the same group has injected 
the plasmid/cationic liposome complexes into the pulmonary artery of patient with lung cancer 
and observed no adverse effects (16). Initial studies using either intranasal instillation or 
aerosol administration of plasmid containing the CFTR gene eomplexed to cationic liposomes 
are now underway in England. 
Several different cationic liposome preparations are available which vary some in their 
ability to deliver DNA and, potentially, in their ijn vivo effects. The preparation which is 
being used in the human studies currently approved in this country are DC-chol liposomes, 
originally synthesized by Dr. Leaf Huang. Dr. Huang is a co-investigator in the studies we 
propose here and he will produce and supply DC-chol cationic liposomes identical to those 
being used in human studies by Nabel. 
DNA constructs for in vivo use Investigations of in vivo aene transfer to the lungs have 
used either viral vectors or liposome associated plasmids. As discussed above, replication 
deficient adenovirus vectors have received much attention and arc currently being used in 
preliminary studies in humans. Adeno-associated virus vectors have also been shown capable 
of delivering DNA to respiratory epithelium and may have less toxicity than adenovirus 
vectors ( 1 7). 
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Recombinant DNA Research, Volume 19 
