was used that contained a second defective retrovirus which expresses the viral 
structural proteins. This packaging cell line does not produce replication 
competent retrovirus because of multiple modifications made to the second 
retrovirus that prevent its replication, Including removal of signals required 
for RNA encapsidation , reverse transcription, and Integration (28). Multiple 
assays will be performed on the packaging cell line, the retroviral vector 
supernatant as well as on the TIL prior to infusion to Insure that no 
replication competent virus is present. These tests will include S+L- assays 
including 3T3 amplification, PCR assays for the envelope gene, and assays 
for reverse transcriptase. Any supernatants or TIL with evidence of any 
replication competent virus will not be utilized. The 3T3 amplification and 
S+L- assays are thought to be capable of detecting a single replication 
competent viral particle per ml. 
Prior safety studies have shown that exposure of primates to large 
infusions of infectious murine amphotrophic virus produce no acute pathologic 
effects (32). In a study of 21 primates receiving retroviral mediated gene- 
modified autologous bone marrow cells no animal showed evidence of toxicity 
related to the gene transfer as long as 4 years after infusion (33, unpublished 
data) . 
More recently we have seen cancer develop in a mouse that received large 
amounts of retroviral vector supernatant by the intraperitoneal route. We are 
currently conducting studies to see if this tumor is related to the retroviral 
transfer. It should be emphasized, however, that these mice were exposed to 
large amounts of retroviral vector and that the patients in the proposed 
protocol will not be exposed to the vector supernatant. TIL will be transduced 
with the retroviral vector supernatant and then the TIL will be washed 
extensively and then grown for several weeks in the absence of supernatant. 
The TIL will then be washed extensively again prior to reinfusion into the 
patient . 
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