2 . 
PRECLINICAL STUDIES INCLUDING RISK-ASSESSMENT STUDIES 
a. LABORATORY STUDIES OF THE GENE TRANSFER DELIVERY SYSTEM: 
The delivery system is identical to that approved in previous 
studies (Rosenberg-TIL; Brenner-AML) and approved by this 
committee. 
b. LABORATORY STUDIES OF GENE TRANSFER AND EXPRESSION: 
bl. What animal and cultured cell models were used in 
laboratory studies to assess the in vivo and in vitro efficacy of 
the gene transfer system; In what ways are these models similar to 
and different from the proposed human treatment ? 
Incubation of autologous cells with the LNL6 vector at 5 x 10^ 
particles/cc was used to introduce the vector into hematopoietic 
cells. Producer cell-free supernatants, produced by GTI and 
approved by the FDA for this application, were used as in the 
previous N2-TIL experiments. At the present time, preliminary 
experiments described below with marrow cells from 5 chronic phase 
CML patients have been conducted in our laboratory. 
Autologous cells (marrow) were incubated in a 1 to 10 ratio 
(cells/viral particles) with the GTI LNL6 vector supernatant at 
37°C for 6 hours. Following centrifugation and rinsing, 1 x 10^ of 
the vector-exposed cells were plated in 1 cc of methylcellulose by 
standard methods, and grown in G418 for 14 days. A commercial 
preparation of methylcellulose was used for this purpose which was 
obtained from the Terry Fox Cancer Center, Vancouver, British 
Columbia. Individual colonies were picked and the DNA extracted by 
a single step procedure. The presence of the vector NEC sequences 
was assayed by PCR as described above. This vector is identical to 
that used in the N2-TIL clinical protocol of Rosenberg and the AML 
protocol of Malcolm Brenner. 
Cells of five chronic phase patients were evaluable for 
analysis following exposure to virus and plating for 14 days in 
methylcellulose. The percent of colonies grown in the presence of 
1 mg/ml of G418 from marrow cells either transduced by LNL6 or not 
exposed to LNL6 is shown in Figure 3, where a plating density of 5 
X 10^ cells/cc was used. The same data for cells exposed before or 
not exposed to the vector and grown in 1 mg/ml G418 is shown in 
Figure 4. Similar data is presented in Table II. The data in 
Table II is also the average of five patients. BFU-E and CFUGEMM 
were not plentiful, which may be due to the selective expansion of 
late myeloid progenitor which occurs in CML. The percent of CFU-GM, 
CFU-G, and CFU-M growing after vector exposure in 1.2 mg/ml G418 is 
at least 10%, which agrees closely with data presented by Brenner 
in his November 29, 1990 data on LNL6 transduction in AML. 
Recombinant DNA Research, Volume 14 
[749] 
