Human Gene Therapy Subcommittee - 4/5/91 
what behavior the transplanted cells are likely to have, in terms of their migration from 
the point of entry. Even if in situ techniques could detect the presence of cells, one does 
not know how representative biopsy samples would be in characterizing the effectiveness 
of the transplantation process. He thought that these questions have to be answered 
before a definitive statement can be made as to whether the proposed gene marking of 
hepatocytes will add to the available approaches. A specific description of the types of 
experiments which need to be done, how they will be done, and whether they've been 
shown to be possible, is necessary. 
Dr. Epstein questioned whether it is necessary to do this experiment. He stated that one 
can find polymorphic markers which are detectable by PCR between any two individuals 
that one might select from the population. For PCR the introduction of another marker 
might be convenient, but not absolutely necessary. For in situ hybridization, one would 
need to have this kind of a marker available. This is the first protocol using 
heterologous transfer, and it allows another set of potentially available markers. The 
real issue is why marking with neo is preferable, and whether the neo marking will be 
used for something beyond PCR. He added that this committee and ultimately the RAC 
has to come to grips with some generic questions. Should the use of recombinant DNA 
markers be considered acceptable as long as it is believed that the procedure is safe? 
Should the validity and scientific merits of the marker experiment be unequivocally 
demonstrated prior to its approval? 
Dr. Mclvor stated that the ultimate aim of the overall protocol is one that is extremely 
important for improved therapy of liver diseases, given the lack of availability of donor 
organs and the ultimate aim of improving therapy through genetic engineering. The 
safety of LNL6 has not necessarily been established, but is in the process of being 
evaluated in terms of a number of different protocols. Since this protocol uses a 
different population of target cells that may differ in their response to insertion of a 
defective retrovirus, he requested a calculation of the rate of insertional mutagenesis. 
Dr. Mclvor expressed his concern that the infusion of these donor hepatocytes into a 
recipient may yield a maximum of 1 in 1,000 donor cells in the liver. If the transduction 
frequency of the cells that are infused falls to 1%, that's 1 in 100. Thus, the entire 
frequency of the cells that are actually transduced in the liver is 1 in 10^, and this is the 
limit of detection by PCR. He requested an indication that the frequency of donor liver 
cells might be more than 1 in 10^, or that the transduction frequency would be more than 
1 in 10^. He asked how the engraftment would be evaluated. If only a portion of the 
cells are going to be transduced, then there needs to be a way of looking for these 
allogeneic cells, whereby all of the donor cells would bear some sort of a distinguishable 
marker. He suggested immunohistology on these sections, using antibody staining for Y- 
specific protein or for different HLA polymorphisms, or any of a wide variety of 
monoclonal antibodies which will stain surface molecules on these cells. The advantage 
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Recombinant DNA Research, Volume 14 
