July 30, 1990, Human Gene Therapy Subcommittee 
patient monthly. He noted that serum ADA level of the patient remained constant 
throughout the study and was efficacious in detoxifying the environment of the patient. 
Dr. Bordignon said that total lymphocytes and all the subsets of T cells were normal in 
the patient. The patient maintained a normal CD4/CD8 ratio with normal T cell 
responses after PEG-ADA treatment. The patient's immune function was analyzed by 
response to several different antigens both in vitro and in vivo and showed the patient's T 
cells were functional. He said that immunoglobulin (Ig) response to vaccination went 
from nothing before treatment to the normal range and then declined. After a second 
boost the level went back up and remains normal. 
Dr. Bordignon said that the vector used was produced by Petros Hantzopoulos in the 
laboratory of Eli Gilboa. It is the only vector used in the mice; therefore, he could not 
make any comparison to other vectors in this model. This vector proved to be most 
suitable by its frequency of infection and level of expression at the single cell level. He 
said that the model was analyzed by whether or not human IgG was present in the PBLs 
of mice and that the results were clear and reproducible. If the animals were untreated, 
they had no human IgG in peripheral blood. If they were treated with ADA-deficient 
human PBLs, there was very little IgG found, and after 2-3 weeks it was not detectable 
at all. However if normal human PBLs were used, there was human IgG in the PBLs of 
the mice. However, there was individual variation in levels of IgG expression. 
Dr. Bordignon said that Southern gel analysis was run for the presence of human ADA 
in the spleens of the mice, and once again the mice treated with ADA-deficient human 
PBLs showed no ADA, whereas mice treated with ADA-positive PBLs showed the ADA 
band. In order to demonstrate the human ADA band, it was necessary to increase the 
concentration of the protein lysates by 4-10 times. Utilizing PCR analysis, he was able to 
see CD4^ T cell clones in the spleen. He said that his conclusion was these cells 
represent one single clone with one single integration event. He noted that the controls 
were IL-2-stimulated PBLs which showed significantly more activity than can be expected 
when using the transduced cells. All clones were tested in parallel with PCR and were 
positive with the exception of one which was negative. 
Dr. Walters thanked Dr. Bordignon for making the long trip from Milan and for 
presenting the data. He then called for a brief recess before asking questions of Dr. 
Bordignon. 
Dr. Walters reconvened the subcommittee at 10:50 a.m., and asked Dr. Parkman to 
respond to Dr. Bordignon's presentation regarding issues that had been clarified and to 
allow him to ask Dr. Bordignon specific questions. 
Dr. Parkman said he found the presentation clarifying in several regards: 
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