Recombinant DNA Advisory Committee - 2/10-11/92 
not a primary cell line. Dr. Mclvor stated that while it would be very difficult to perform 
in vitro experiments demonstrating ADA gene expression in lymphocyte lineages, the 
investigators are capable of doing some experiments following differentiation into myeloid 
lineages. These myeloid cells could be expanded and assayed for enzyme activity. 
Dr. Dunbar responded that the investigators had selected granulocytes from the second 
patient that are 99% pure, and that these cells can be readily assayed for ADA activity. 
However, these granulocytes represent cells from a patient who already received the first 
vector. If the same cells are transduced with the second vector, it will not be possible to 
differentiate one ADA from the other ADA. Dr. Nienhuis stated that in his interpretation 
of the discussion, the investigators are being asked if they could take CD34 selected cells, 
infect them in vitro, culture them, harvest the myeloid colonies, and examine for ADA 
expression. This experiment would be feasible, and the investigators will plan to conduct 
such a study. Dr. Mclvor commented that it should be possible to fractionate granulocytes 
away from the lymphocytes. The granulocytes in the untransduced control should be 
negative; when transduced with the new ADA vector, there should be activity. 
Dr. Mclvor asked two questions concerning the safety of the protocol. First, since a new 
target cell population will be used, what would be the number of estimated events that 
would be necessary for insertional mutagenesis to occur? He acknowledged that the 
investigators would have to make some assumptions as to the number of insertional events 
necessary to produce tumor formation. Dr. Anderson stated that this estimate would 
probably have to be multiplied by the number of cell divisions. The risk of insertional 
mutagenesis with the CD34 selected cells should be lower because there are fewer 
numbers of transduced cells; however, the total risk will be greater because both 
procedures are being conducted. By taking the bone marrow assessment, the percentage 
of totipotent stem cells in the peripheral blood fraction could be compared with those in 
the bone marrow and make the reasonable assumption that the risk factors will be reduced 
by one hundred. 
Dr. Dunbar responded that the number of insertional events that are observed in the 
totipotent stem cells of mice using these growth factors is one to four. If the marked cells 
are considered and the colonies analyzed, the number of insertions is one to four. There 
is an increased efficiency in the percentage of inserted cells; however, there has been no 
increase observed in the number of insertions per cell. If helper-free virus is used, 
circulating totipotent cells can be given back to the host and no further insertional events 
will occur after the original one. Even though the cells can live longer and have a chance 
of a second event, once the first event has occurred, there is probably no excess risk of 
another insertional event. 
Dr. Mclvor asked what assays for replication-competent helper virus and infectious agents 
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Recombinant DNA Research, Volume 15 
