All adverse reactions should also be reported to the IRB. Data will be submitted 
to CTMS at least once every two weeks. The NCI/DCT case report or ACAS will be 
used to report to CTMS. 
10.0 POTENTIAL RISKS OF RETROVIRAL -MEDIATED GENE MODIFICATION 
10.1 Insertional Mutagenesis: 
The possibility of causing malignancy in cells secondary to the random insertion 
of the retroviral vectors in the genome exists, though the actual risk of this 
occurring is thought to be low. Nevertheless, transduced fibroblasts will be 
irradiated with 5,000 rads prior to vaccination. Tests of the viral supernatant 
as well as of the fibroblasts used for vaccination will be conducted to assure 
that no replication competent virus is present in either preparation. Since no 
other cells will be exposed to retroviral vector insertion, no other cells will 
be at risk for insertional mutagenesis. 
10.2 Risk from Murine Retrovirus: 
Exposure of the cancer patient to retrovirus could theoretically pose a risk of 
insertional mutagenesis. It should be emphasized, however, that careful tests 
will be conducted to assure that the patient is not exposed to replication 
competent virus. The retrovirus derived from the Moloney murine leukemia virus 
has been modified so that it no longer contains any intact viral genes and thus 
cannot produce the proteins necessary to package its RNA into an intact 
infectious virus (14, 15). To assemble the retrovirus, a retrovirus packaging 
cell line will be used that contains coding sequences that express the viral 
structural proteins. This packaging cell line does not produce replication 
competent retrovirus because of multiple modifications made to the gag, pol, and 
env coding sequences that prevent its replication. These modifications include 
removal of signals required for RNA encapsidation, priming of reverse 
transcription, and integration. Multiple assays will be performed on the 
packaging cell line, the retroviral vector supernatant as well as on the 
fibroblasts prior to vaccination to insure that no replication competent virus 
is present. These tests will include S+/L- assays and 3T3 amplification. Any 
supernatants or fibroblasts 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. These 
studies will initially be done at GTI and then subsequently in the IMDL/CT. 
Prior safety studies have shown that exposure of primates to large infusions of 
infectious murine amphotrophic virus produce no acute pathologic effects (16). 
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 for as long as 4 years after infusion (17). 
Patients in the proposed protocol will not be exposed to the vector supernatant. 
Fibroblasts will be transduced with the retroviral vector supernatant and then 
washed extensively. They will be grown for several weeks in the absence of 
supernatant. The fibroblasts will then be washed extensively again prior to 
reinfusion into the patient. 
Recombinant DNA Research, Volume 16 
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