Protocol 
RAC Application 
Kenneth L. Brigham, M.D. 
For the studies proposed here, a large batch of plasmid ( 1.3- 1.4 kg cells) will be prepared 
at the University of Iowa College of Medicine, Department of Microbiology, Iowa City, Iowa 
52242 (Lacy Daniels) according to the following protocol. One 100 liter fermentor run is 
performed with E. colt strain NM522-pCMVj-A AT grown in medium composed of a mixture of 
Luria Broth Base and Terrific Broth. An inoculum is prepared from an isolated colony, scaled 
up by growth on plates and grown finally in Fernbach flasks for the inoculum. All media 
contain originally ca. 75-100 /ig ampicillin/ml. Cells are harvested with a Sharpies continuous 
centrifuge after absorbance reaches a near maximal point. Cells are frozen directly in liquid 
nitrogen and shipped on dry ice. Cell yields arc usually 1.3-1. 4. kg wet cells per fermentor run. 
Liposome production 
DC-Chol/DOPE liposomes arc prepared by one of the investigators. Dr. Leaf Huang, at 
the Department of Pharmacology, School of Medicine, at the University of Pittsburgh, 13th 
Floor, Biomedical Science Tower, Pittsburgh, PA 15261. DOPE is obtained from Avanti Polar 
Lipids, Inc., 700 Industrial Park Drive, Alabaster, AL 35007. DOPE has a Drug Master File 
Number with the U.S. FDA. The microf luidizer (model 110-S) was purchased from 
Microfluidics Corp., Newton, MA. 
DC-Chol and DOPE (in chloroform) are mixed in a 6:4 molar ratio in a 250 ml round 
bottom flask. The lipid mixture is evaporated to dryness in a stream of nitrogen and allowed 
to vacuum desiccate (for at least 2 hours) to remove any residual chloroform. The thin lipid 
film is hydrated overnight (at least 12 hours at 4"C) with sterile water. Upon sufficient 
hydration, the flask is briefly sonicated to remove the lipid form from the walls of the flask. 
The hydrated lipids arc shaken to disperse the lipids in the aqueous solution. 
The microfluidizer is powered by compressed nitrogen gas and is operated at a pressure 
of 80 psi. The liposomes arc cycled through the microfluidizer several times (5-15 passes) to 
reduce the mean diameter of the liposomes. The size of the liposomes is monitored by quasi- 
elastic light scattering (QLS). The liposome size is reduced by cavitation which occurs in the 
interaction chamber. This chamber is packed in ice to eliminate any heat which may be 
produced during operation of the microfluidizer. 
Once the liposome size is reduced below 200 nm, the liposomes are passed through a 0.22 
nm nylon filter to sterilize the liposomes. 
The liposomes are assayed for phosphate content by a calorimetric method according 
to Bartlett (25) and diluted to a final concentration of 2 /xmol of total lipids per ml. 
The liposomes are packaged in 4 ml Wheaton screw cap vials which have been 
autoclaved. The packaging occurs in a sterile hood and the headspace of the vials is filled with 
nitrogen to prevent oxidation of the DOPE. The vials are stored a 4°C until use. 
Liposomes will be sent by overnight express mail to one of the investigators at 
Vanderbilt, Dr. Hans Schreier, who will receive the shipment, document date, declaration 
(content, lot number) and amount of shipment received, and will store the material at 4°C in 
a refrigerator in the Liposome Laboratory of the CLR (Room B 1222 MCN). 
Generation of plasmid/liposomc complexes 
DNA and liposomes will be mixed together at 37X in a plasmid/liposome w:w ratio of 
1:3 in sterile, pyrogen free polystyrene tubes and incubated for 30 minutes prior to 
administration. 
Patient protocols 
Patients undergoing lung resection Wc have designed the protocol to insure, as 
much as possible, safety to the patients being studied. The purpose of this study is to determine 
whether the human alpha 1-antitrypsin gene in a plasmid construct delivered with a liposome 
vehicle via the airways into the lungs of humans will function in such a way that the human 
Recombinant DNA Research, Volume 19 [185] 
