Recombinant DNA Advisory Committee - 06/1-2/92 
Dr. Dunbar presented an update on the rhesus primate data. The experiments 
performed over the last six months were performed with helper virus free retroviral 
vectors. One of these vectors contains the murine adenosine deaminase (ADA) gene 
and the other contains the human multi-drug resistance (MDR) gene. Both of these 
vectors have titers between 10 s and 10 6 . These vectors have been helper virus free when 
used with IL-3, IL-6, and SCF supernatant infection daily, following 3-4 days in culture. 
The mortality rate in primates is approximately 50%, due to the lack of cooperation of 
the animals and inadequate critical care services; patient mortality should be less than 
5%. 
Dr. Dunbar explained that four monkeys have received cells transduced with the MDR 
vector. One monkey has survived 75 days with this vector. Retroviral transfer in the 
peripheral blood and bone marrow of three of these monkeys has been detectible by 
PCR. Data was presented from one animal that demonstrated that approximately 80% 
of the circulating cells were marked at day 45. Although this persistence appears to be 
stable, more time will be required, as well as sorting these cells into T cell, B cell, and 
myeloid fractions. When cells are grown in methylcellulose, approximately 8-10% of the 
circulating cells are marked. 
Dr. D. Miller asked what dose of radiation these monkeys received? Dr. Dunbar 
responded that the animals received 1200 rads in two fractions. This response is in 
contrast to the 1000 rads used previously. 
Dr. D. Miller asked about the manner in which the marrow was prepared prior to 
harvest. Dr. Dunbar answered that the marrow is CD34( + ) selected. The monkeys 
were stimulated with 5-fluorouracil (5-FU) prior to marrow harvest. Melanoma patients 
will receive granulocyte colony stimulating factor (GCSF) and cyclophosphamide, and the 
CML patients will receive cytosine arabinoside and daunorubicin. 
Dr. Dunbar described the rhesus transplantation protocol. Autologous blood is collected 
and stored. 5-FU is administered on day minus 9 at a dose of 70 milligrams per 
kilogram. The bone marrow is harvested on day minus 4. These cells are CD34( + ) 
selected and irradiated with two doses of 600 rads. These irradiated, selected, bone 
marrow cells are cultured and reinfused on day 0. GCSF is administered on day 1. 
Dr. Dunbar described experiments using the murine ADA retroviral vector. Data was 
presented that showed a monkey that was PCR( + ) for the murine ADA gene at days 34 
and 48. Murine ADA expression was also observed in the peripheral blood mononuclear 
cells of this monkey. Five of the eight monkeys that received the helper contaminated 
retroviral producer line were positive for the neomycin resistance gene. PCR analysis 
demonstrated that expression of this gene was stable and persisted for a long period of 
time. Long-term stem cell gene transfer was probably established in this system. 
Recombinant DNA Research, Volume 15 
[723] 
