Human Gene Therapy Subcommittee - 11/21-22/91 
which they are injected? Dr. Nabel explained that the liposomes have been sized, and 
that this was defined in a paper by Dr. Stewart, et al. The liposomes traffic primarily to 
the liver and sometimes to the spleen; however, they are degraded rapidly. Dr. 
Leventhal asked if post mortem liver biopsies will be performed. Dr. Nabel said that 
liver biopsies will be requested in the informed consent document. 
Mr. Capron asked why the gene sequence of HLA-B7 was reported as two different 
lengths in the protocol. Dr. Miller explained the discrepancy is related to the addition of 
a linker. Dr. Miller asked if the entire gene was sequenced. Dr. Nabel responded that 
the entire HLA-B7 gene has been sequenced, including all of the junctions and 
restriction sites. 
Dr. Miller stated that he was concerned about whether the animal model chosen for the 
in vivo experiments is the most appropriate system to support the human experiment. 
Dr. Parkman explained that there is a basic problem with all animal model systems in 
that they all use transplantable tumors. This scenario is very different from human 
disease, particularly for solid tumors where the cells are nondividing. In murine models, 
the animals harbor numerous viruses that may elicit an immune response. Therefore, 
the immune responses in animal systems may not be to a specific tumor antigen and may 
not result in protective immunity. Currently, there are no in vivo systems in which 
significant regression of preexisting tumor has been observed in response to 
immunotherapeutic treatment. Dr. Parkman said that he supports the animal model data 
because it demonstrates that the introduction of a histocompatibility antigen appears to 
invoke an immunologic response against nontransfected tumor. 
Dr. Miller asked if the DNA used for the liposome preparation will be exposed to 
ethidium bromide. Dr. Nabel responded that ethidium bromide will not be used for the 
preparation and quantitation of DNA because of safety concerns. A column method of 
separation will be used and plasmid DNA quantitation will be compared to E. coli DNA. 
Dr. Miller asked about the upper limit of bacterial DNA that will be allowed in the 
preparation. Dr. Nabel said that he is currently discussing the acceptable limits of 
bacterial DNA with the FDA. 
Dr. Mclvor asked if the DNA will be stably integrated into the cells or will the 
expression be transient. What form is the DNA in? Dr. Nabel responded that the 
animal experiments have been performed using nonlinearized DNA. In these 
experiments, transient expression has been observed. It is preferable to use uncut 
plasmid DNA for transfection because long term over-expression will be avoided. Dr. 
Mclvor asked if the plasmid DNA will be analyzed for evidence of supercoiling. Dr. 
Nabel answered that aliquots from all DNA preparations will be analyzed on ethidium 
bromide agarose gels and probed with E. coli DNA in order to quantitate the amount of 
open circular versus supercoiled DNA. DNA analyses have demonstrated that there is 
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