endogenous retroviral sequences and the N2 provirus. 
(2) In addition to studies in our laboratory, supemate for patient use will be 
tested in an independent laboratory which performs testing for amphotropic virus 
regularly and under the guidelines and surveillance of the U.S. Food and Drug 
Administration. We specifically will be using Microbiologic Associates, Bethesda 
MD . 
While the protocol we have submitted will utilize helper-virus free 
supemate and TIL will be tested for helper virus prior to patient exposure, 
other applications of retroviral-mediated gene transfer in man may not permit 
such detailed analysis before patient exposure. Since this could result in 
accidental exposure to helper virus, we chose to study non-human primates 
directly inoculated with murine helper virus to assess the risk, and to identify 
disorders, that might arise from such an exposure. Our preliminary findings are 
described in Appendix B. 
II. Response to the Comments of Howard Temin: 
1) "Should the optimal system as far as safety be used?" 
Yes. N2 and N2-based vectors have been studied in mice in vivo for 
over four years and in monkeys in vivo for over three years. We believe that the 
advantages of this wealth of safety experience far exceeds the potential safety 
advantages of new vectors or packaging systems which would first need to be 
evaluated in long-term animal studies before use. We are, of course, evaluating 
new systems for future protocols. It should be emphasized again that the unique 
characteristic of the present protocol is that the transduced TIL can (and will) 
be studied in vitro before re -insertion into the patient in order to be certain 
that no infectious virus is present. Since it can be shown with the present 
system that no infectious virus is present, a theoretically "safer" system is not 
necessary. Therefore, considering the years of animal in vivo experience with 
N2, this is the "optimal system as far as safety" at this time. 
2) The sensitivity of the tests: see I.B.3.b. 
3) Efficiency of initial infection: see I.B.3.a. 
4) Sensitivity of the test for neoplastic transformation: 
Dr. Temin is correct that two weeks is not sufficiently long to prove 
that there are no 11-2 independent TIL. We are carrying transduced human TIL in 
culture for several weeks in order to determine if any cell in the culture can 
continue to divide in the absence of 11-2. Thus far we have seen no growth in 
the absence of 11-2 for as long as 4 weeks. In addition, we have studied many 
vector transduced cultures for thymidine incorporation in the absence of 11-2 and 
have seen no incorporation signficiantly above background by 2 weeks after 11-2 
withdrawal . 
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Recombinant DNA Research, Volume 13 
