Human Gene Therapy Subcommittee - 4/5/91 
either palliate their liver failure, or possibly, reconstitute the liver. He noted that in 
looking at numerous tandem repeats, which represent the polymorphisms that Dr. 
Epstein referred to, he has been unable to find evidence that one can detect these 
against a very high background of other positive signals. It is desirable to detect 
polymorphisms at 5 or 10 percent frequency, which is done in the bone marrow field all 
the time. Finding a polymorphism which occurs in less than 1 percent of the background 
signals from PCR is not possible. However, the technology is very attractive. 
Dr. Ledley presented data showing his group's ability to transduce the hepatocytes with 
the vector, LNL6. It is estimated that the investigators will transplant 5% of the 
hepatocytes of the donor liver; they expect that 2-3% of the liver cells in the recipient 
will be derived from the transplanted cells. With the lowest estimate for the number of 
cells transplanted, which is the less than 1%, it will still be possible to detect the neo 
gene by PCR. With 50% survival, between 5 in 10,000 and 1 in 1,000 cells should have 
the neo gene and should be easily detected by PCR. 
Dr. Ledley emphasized the therapeutic intent of this protocol. The use of marker genes 
is believed to be the best available technology. The risk-benefit ratio is tremendously 
enhanced by the effort taken to enroll patients early, to select patients, to think about 
the informed consent and to work on follow-up. He commented on the use of PCR 
versus in situ hybridization. In situ hybridization is sufficient to know whether the 
treatment is successful. He believes that it is within the realm of sensitivity of PCR to 
get the needed information. 
Dr. Parkman asked for a quantitative estimate of the transduction efficiency in the 
hepatocytes. Dr. Ledley replied that he did not have quantitative data, but would supply 
it at a later date. 
Dr. Woo said that his group has done 10 experiments using dogs in which they transfused 
1 billion hepatocytes into each one of the animals, either into the spleen, the splenic 
vein, or the splenic artery. The animals survived without any adverse effects. 
Dr. Mclvor asked for an estimate of the transduction frequency. Dr. Woo replied that 
using the dog hepatocytes and the beta-gal gene as with the amphotropic packaging cell 
as a control, 20% of the cells were transduced. Dr. Mclvor asked how many cells were 
actually engrafted into these animals. Dr. Woo said it was very difficult to calculate with 
only 4 observation days and no steady level. However, in the mouse experiment, 50% of 
the cells that were transplanted were in the liver. 
Dr. Epstein again raised the issue of using endogenous markers that are present in all 
cells. Dr. Ledley replied that there would be tremendous difficulty using these types of 
markers because of the low efficiency of doing restriction enzyme digest. 
Recombinant DNA Research, Volume 14 
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