and then selected in G418. From 10-50 million transduced selected T cells were 
injected into the peritoneum of normal or athymic mice which were then 
periodically immunized with soluble myoglobin antigen. Blood, spleen and bone 
marrow cells were tested at 5, 8, and 15 weeks for the presence of the gene 
marked T cells. Bone marrow, blood, and spleen samples from the recipients were 
found to contain vector DNA by PCR analysis at 5,8, and 15 weeks after cell 
transfer. Spleen cells from recipient nude mice were stimulated with PHA and II- 
2 and then tested for resistance to G418 (Fig. 1). The control untreated nude 
spleen cells proliferated modestly to the PHA+I1-2 and this proliferation was 
inhibited by 0 . 3 mg/ml G418 . By contrast, the nude mice given the 14.1 SAX 
transduced T cells showed a brisk proliferative response to PHA+I1-2 in the 
presence of G418. These results indicate that the spleen of these mice contained 
T cells which continued to express the NeoR gene for at least 15 weeks after 
introduction. Therefore, we have clearly shown that cultured T cells can be 
successfully transduced with N2 based vectors and selected for NeoR expression 
with G418 . These selected gene marked cells can be given to animals and their 
marker genes can be detected in the lymphoid tissues of the recipient animals by 
PCR analysis several weeks after cell transfer. Finally, these NeoR expressing 
cells can be regrovn from the lymphoid tissues of the recipient animals and can 
be shown to continue to express the genes originally introduced with the N2 based 
vectors . 
b) The Subcommittee has recommended looking for tumorigenesis and 
other undesired effects following retroviral-mediated gene transfer using the TIL 
mouse model. Unfortunately, this is a difficult model to use for questions of 
tumorigenicity since the mice are pretreated with cytoxan (an ocogenic agent) and 
injected with tumor cells as well. Therefore long-term evaluation of these mice 
would be very complicated. We feel a better indication of tumorigenicity is 
obtained by reconstitution of the hematopoietic system with N2 infected 
progenitors cells. Since this permits reconstitution of the lymphoid system at 
all stages of development, it serves as a more stringent test of tumorigenicity 
and is not complicated by the injection of tumor cells. Our laboratory has 
followed mice initially described in Eglitis et. al . ( Science . 230 : 1395-1398 . 
1985) which underwent bone marrow transplantation with N2 infected bone marrow 
progenitors. Fifty mice were maintained and autopsies were performed at the time 
of death. These animals had a normal life-span and no tumors were noted. In 
addition, in the murine 14.1 T cell model, there has been no apparent pathology 
in any of the recipient mice. 
The results from our bone marrow transplantation/gene transfer experiments 
in primates, referred to in the principal proposal, are perhaps more relevant to 
this point than are murine experiments. Bone marrow cells containing N2 based 
vectors, given to immunosuppressed monkeys, have not been associated with any 
detectable abnormalities in the recipient monkeys followed for up to 3 years. 
Additionally, despite the extensive experience with N2 based vectors in many 
laboratories and in many species (including mice, dogs, cats, sheep and non-human 
primates) , there has not be a reported case of malignancy secondary to retroviral 
vectors . 
In addition to our Ln vivo experience and literature review, tumorigenesis 
is very unlikely on theoretical grounds. This is discussed in detail in the Human 
Gene Therapy Preclinical Data Document (page 56-60) and is addressed in our 
proposal. Since we will be using helper virus free supernate and the cells will 
be shown to be helper virus free prior to patient administration, the scenario 
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