producing the virion proteins necessary to package its RNA into an intact 
infectious virus. In order to produce an infectious retroviral vector that is 
capable of transferring its RNA into a target cell, the vector construct is 
"enveloped" in a viral coat produced by a "packaging" call line containing a 
second defective retrovirus which produces the necessary gag, pol, and env 
proteins. The packaging cell line used for these studies does not produce intact 
replication competent retroviruses (helper viruses) because it has also been 
modified to remove the signal sequences in its genome which are required for its 
RNA to enter a virion. Thus, the patient's cells are never exposed to a 
competent retrovirus , but only to a viral coat within which is contained the 
recombinant retroviral vector that carries the marker gene. Specifically, our 
retroviral delivery system can transfer the NeoR gene into the TIL but then is 
metabolized and lost. An advantage of the TIL as a target for the retroviral 
vector is that the vector treated (transduced) cells can be grown in culture 
after treatment and then tested to be certain that the culture contains no intact 
infectious replication competent retroviruses . 
The Moloney murine leiikemia retrovirus parent of our vector can cause 
neoplastic disease in mice. Is this a threat to the patient given retrovirus 
marked TIL? There is no experience in man to answer this question directly so 
that it is necessary to extrapolate from observations in other species. We 
believe that the risk to the patient is very remote. First, as stated above, the 
viral genes have been removed from the vector and each TIL preparation will be 
tested to insure that no infectious replication competent retrovirus is present 
in the cells. Retroviruses can, however, induce neoplastic disease by a process 
of insertional mutagenesis. Since retroviruses integrate into the genome of the 
new host in a random manner, there is a remote but finite probability that an 
occasional insertion will be in a location that could activate a proto -oncogene. 
This mechanism is discussed in detail below (see Section VIII.A.). In an 
experimental mouse model which is exposed to intact replication competent 
retrovirus from birth, the development of malignancy occurs only after months of 
exposure and then the tumors are clonal suggesting that transformation is a very 
rare event despite this continuous exposure to the retrovirus. There are no 
published examples of the transformation of primate cells by a murine retrovirus 
either in vitro or In vivo . We have performed retroviral-mediated gene transfer 
into 21 primates in vivo using an autologous bone marrow transplantation protocol 
and to date no animal has shown any evidence of neoplasia after as long as 2 1/2 
years of observation. Finally, we will observe the growth of the retroviral 
vector treated TIL in culture and confirm that their growth has not become 
autonomous (i.e., independent of IL-2) before infusing these cells into the 
patients. We have not observed the development of IL-2 independence in any of 
our preliminary experiments of N2 vector treated TIL and believe that this test 
will rule out the possibility that transformation has occurred in these treated 
cell populations . 
Recombinant DNA Research, Volume 14 
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