2 . 1.2 
uninfected cells expressing HLA class II was evaluated. Cytotoxicity 
assays employing a cell line that expresses very high levels of HLA class II 
as a target, namely the human B cell line Raji, demonstrated that CD4- 
UR+CD8+ T cells were unable to kill this HLA class Il-expressing target, 
even at effector: target ratios as high as 20: 1 . 
Since the genetically modified CD8+ lymphocytes will express the CD4- 
UR cell-surface protein, the theoretical possibility exists that these 
transduced cells will become susceptible to HIV infection, as the virus 
gains entry into cells using the CD4 glycoprotein as a receptor. 
Consequently, studies were designed to test the infectability of CD4- 
UR+CD8-I- T cells in vitro. Using two different strains of virus (HIV-1 
IIIB and JR-CSF) in 7-day cultures, preliminary results have detected no 
evidence of infection in the genetically modified cells based on absence of 
p24gag in the supernatant (data on file, Cell Genesys, Inc.). 
Assessment of the Safety of Retro viral-mediated Gene Transfer 
In the ADA-deficient SCID clinical protocol, a total of approximately 20 
infusions of up to 2 x cultured cells have been performed, and no 
significant side effects related to the cell infusions have been observed. In 
addition, there have been no side effects related to gene transfer. Similarly, 
in the N2/TEL protocol, there have been no side effects due to gene transfer 
and no evidence of infection by replication-competent virus. Since foreign ' 
DNA is inserted randomly into the genome of cells that are reinfused into 
the patient, however, there is some potential for the insertional event to 
result in an unfavorable outcome. If the insertion disrupts a gene essential 
for maintaining cell function, that particular cell might die. More 
threatening is the possibility that insertion may initiate oncogenic 
transformation of the cell. The magnitude of the risk that gene transfer 
poses to a patient cannot be accurately stated at present, but based on the 
accumulated experience the risk appears to be very low. 
Some of the potential hazards were illustrated in a primate model of 
retrovirally-mediated gene modification, in which lethally irradiated rhesus 
monkeys were exposed to massive amounts of replication-competent 
retrovirus. When autologous stem cells used to reconstitute the bone 
marrow were incubated in vitro in the presence of retroviral vector particles 
with accompanying replication-competent virus, retroviral vector genome 
was detected in circulating cells of 5 of 8 of the transplant recipients of 
CD34+ cells and in 2 recipients of unfractionated bone marrow cells (35). 
Three recipients of CD34 cells had a productive infection with replication- 
competent virus. Within 7 months of transplantation, each of these three 
animals developed a rapidly progressive T cell neoplasm; lymphoma cells 
contained 10 to 50 copies of the replication-competent virus but, 
importantly, lacked the retroviral vector. Thus, these tumors occurred in 
the setting of intentional contamination of the bone marrow transplant with 
replication-competent virus, were associated with continuous retroviremia, 
but did not involve the retroviral vector. This experience underscores the 
importance of carefully screening retroviral producer clones used in human 
trials to exclude contamination with replication-competent virus. 
In the more than 30 patients who have now received gene-modified cells, 
no evidence for the generation of replication-competent virus has been 
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