Recombinant DMA Advisory Committee - 5/30-31/91 
large bursts of the human marker protein in the blood during the first couple of weeks, which 
receded and dropped to zero after a month. The animals were then sacrificed and different 
sections of the livers were looked at via PCR analysis for the presence of the provirus. Four 
of 8 spots from the liver showed positive by ethidium staining and additional blots are being 
examined to date. He said that he wanted to know what percentage of the transplanted cells 
were actually recoverable from the liver and that until quantitative data is in hand he would 
be reluctant to make any estimates. However, the research is still ongoing and results are 
expected within a week. 
Dr. Ledley then turned to the question of quantitation of the transplant and its relationship 
to how many cells they can get into an animal. He said that he felt the most appropriate 
animal model for this was the baboon. He presented data on a 2 year old baboon that was 
being sacrificed because of a seizure disorder in which the investigators were able to perform 
a left lateral lobectomy. They were able to harvest hepatocytes and stain them with the 
fluorescent dye dil, which is an extremely hydrophobic molecule which binds to membranes 
and is extremely fluorescent as well as being absolutely stable in vivo. It does not transfer 
from cell to cell and it can be used to track single neurons and their projections. The cells 
were stained and transplanted into the spleen of the animal using 2 X 10® cells per kilogram, 
which is the same level proposed for the human experiments. Then the animal was sacrificed 
a week after the transplant and histology showed the animal to be normal. Donor cells could 
not be discriminated from recipient cells on H&E section. 
Dr. Ledley noted that one question which had been brought up was whether these cells could 
be found throughout the liver or whether you had to biopsy in the right place to find them. 
He said that they looked at 13 segments of liver from all different regions and no differences 
could be found. What was seen in 2 out of the 13 sections were small emboli in some of the 
small portal venules, but no evidence of infarction or damage to the liver resulting from these 
cells. In the spleen the cells could be found within the splenic pulp, therefore showing that 
clearly in the baboon this did engraft well. On counting, 5 percent of the total cells counted 
were found to be fluorescent, thus reflecting the exact prediction of what would be found in 
the animal by putting in 2 X 10® cells per Idlogram. 
Dr. Ledley said the question then was can the cell which takes up the provirus which is then 
implanted be detected. He said this depended on the following three variables: 
1. Transduction efficiency; 
2. What fraction of donor cells originate from the transplant; and, 
3. How sensitive are the methods of detection. 
Dr. Ledley said that he believed this experiment shows that they can detect cells to a 
sensitivity level down to 1 in 10^, which is consistent with the preclinical data presented. 
As far as the appropriateness of animal models. Dr. Ledley said that this varied depending 
upon the aspects which were being looked at. For surgical aspects of the experiment the dog 
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