Infections with retroviral vector supernatant (RVS) instead of the 
cocultivation method, complete and rapid hematopoietic reconstitution of four 
animals occurred. Five animals could be evaluated for gene transfer, including 
one that did not fully reconstitute its bone marrow and eventually died. 
Four of the five evaluable monkeys had low levels of human ADA activity 
equivalent to <0.1 to 0.5% endogenous monkey ADA activity. The peak of enzyme 
activity was present 60 to 80 days after transplantation and declined before 
disappearing altogether by day 170. Blood cells from three of the five animals 
contained NPT activity which declined over time and eventually could no longer 
be measured. 
Only one of the monkeys with detectable enzyme activity had vector DNA 
demonstratabl e by Southern analysis of bone marrow and peripheral blood cells. 
In this case the presence of a 4.9 kb band indicated that the vector had 
probably not suffered any major deletions or rearrangements. On the basis of 
the sensitivity of this assay, vectors could be present in no greater than 1 in 
10 cells of the "DNA-negative", ADA-positive, animals. In situ RNA 
hybridization of peripheral blood from one animal determined that 0.8% of the 
D 
cells expressed RNA transcripts from the Neo gene. Finally, clonable 
G418-resistant T lymphocytes were present in the peripheral blood of two 
animals at day 181 and 230, respectively. 
In summary, a scaling up of the bone marrow infection and transplantation 
protocol allowed the complete long term reconstitution of cynomolgus monkeys. 
The results indicate that, in contrast to the highly efficient gene transfer in 
mice, only a very few monkey bone marrow progenitor cells (between 1 in 100 and 
1 in 200 cells) could be infected with the retroviral vector. The limited 
time course over which detection of activity was observed may have resulted 
from the loss of vector DNA from the infected cells or. alternatively, from the 
sequential activation over time of different hematopoietic stem or other early 
Recombinant DNA Research, Volume 12 
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