Recombinant DMA Advisory Committee - 5/30-31/91 
He noted that they were not proposing any modem or sophisticated methods of dealing with 
this large number of cells, but noted that this procedure would be the same one used during 
the experiment. 
On the issue of alternative methods, he noted that there were two clinical models which he 
had discussed at the HGTS to look at the question of detecting a small population of cells 
among a larger population of unrelated cells. One is the use of PCR analysis, although the 
limit of sensitivity is never pushed to the limit needed for use in such a protocol. The other 
is fetal sexing, or Y chromosome analysis, which he said is the more appropriate model to be 
used in such experiments, and then to perform PCR or in situ hybridization techniques to look 
for mutations and do linkage analysis. He noted that this technique is very difficult and as 
yet is unproven. 
Dr. Ledley noted that the team intends to look at a very small number of subjects (6) to see 
if the marker gene is necessary to detect successful engraftment. He said that if it proved 
successful the plan was to turn the surgeons loose to perform the engraftments without 
markers. However, if better markers are needed or discovered, they would switch to those 
better markers. He noted that retroviruses at this point appear to be the only markers 
available to do the experiment. He noted that he had tried the in situ hybridization assay and 
the results are not yet available to confirm it, but that he felt that this coupled with confocal 
microscopy could be the best method for detecting successful engraftment. 
He said that as far as the dil was concerned there was some question as to its toxicity in 
humans and would require FDA approval. 
He noted that as far as the consent form went, the questionnaire had been tested on their 
nursing staff and that it will continue to be studied in the patient population to study its 
validity in this population. 
Dr. Ledley said that there were no hepatocyte cell lines available and that they had done 
extensive work in human hepatoma cell lines and those models would be used for many of 
the proposed studies, especially work on transduction efficiency required for gene therapy. 
He said the dosage had been worked up through the preclinical experiments and that there 
is an elaborate calculation in the original proposal of how many cells this will amount to 
relative to total liver mass and many variables. He noted that future animal experiments will 
continue to push this dose up, but that what is being contemplated here is a dose which has 
been administered without complication in preclinical experiments. 
He noted that they had also experimented with different infusion media and that after 
experimentation with various media this has ceased to be an issue. 
Dr. Ledley noted that patients were made aware of many of the risks of liver transplant via 
a publication outlining the procedure as well as undergoing counseling with a nurse 
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Recombinant DNA Research, Volume 14 
