4.0 nRSTGN OF CLINICAL PROCEDURES 
4.1 Obtaining Fibroblasts for Use in this Study: 
In order to obtain large numbers of proliferating cells that can be transduced 
and available for use in a short period of time, we propose to use cultured, 
autologous fibroblasts grown from the skin resected at the time of 
metastectomy or as an outpatient procedure alone (19.2). An elipse of skin 
measuring 10cm by 2cm will be resected from a convenient location such as the 
anterior abdominal wall. This will be performed in sterile fashion using local 
anesthesia. All wounds will be closed primarily with either sutures or 
staples. The skin will be transferred in sterile fashion to the laboratory 
where it will be cleared of fat, divided into lmm-2mm diameter pieces 
incubated with tryple enzyme solution (Detailed protocol; Appendix 19.1) and 
plated in T25 flasks containing sterile culture medium and 10% human AB serum. 
Cultures will be split 1 to 3 when confluent monolayers develop. After the 
first passage, the fibroblasts will be transduced and replated as described 
below. 
4.2 Transduction and Growth of Fibroblasts: 
The procedures used here are the same as those used in our previous protocols 
at the NIH and the PCI, involving the infusion of TIL transduced with the Neo 
resistance gene (protocol 86-C-lo3c and 90-C-183c) (Rosenberg et al, 1990) and 
our IL-4 gene therapy protocol. These are detailed in the appendix (19.3). 
Cultured fibroblasts will be transduced during log phase of growth using a 
retroviral vector containing the genes for human IL-12 and neomycin 
phosphotransferase. Forty-eight hours after transduction, they will be exposed 
to tne selective pressure of 0.1 - 1.0 mg/ml of G418. This concentration may 
be increased to as much as 1 mg/ml depending on the health of the culture. 
Quality control of the retroviral supernatant and transfected fibroblasts will 
be conducted strictly observing FDA regulation. These regulations include 
detection of replication competent retrovirus using detection of env protein 
of retrovirus and other potential pathogens including mycoplasma or other 
bacteria. This testing and the growth of the fibroblasts will be conducted by 
the Immunologic Monitoring and Diagnostic Laboratory (IMDL) of the PCI. GLP 
will be followed. Fibroblasts will be cultured in the IMDL. 
4.3 Preparation of the IL-12 Vector-Containing Supernatant: 
Unlike our previously approved protocol of IL-4 gene therapy (P/I: Michael T. 
Lotze and Joshua T. Rubin), supernatants for this clinical trial will be 
prepared in the Human Gene Therapy Applications Laboratory (HGTAL) at the 
University of Pittsburgh HGTAL using the retroviral constructs developed by 
our group (Zitvogel et al, 1994). Thus, certification of the retroviral 
construct and helper- free viral supernatant for human clinical trials will be 
conducted based on our experience in Gene therapy for Gaucher Disease (P.I. 
John Barranger) . Testing for existence of helper virus in the viral 
supernatant and other adventitial agents will be carried out under contract 
with Mlcroblloglcal Associates (Appendix). The entire provlral construct, 
termed TFG-human IL-12-Neo, will be sequenced, and the packaging cell line, 
termed YCRIP, will be certified free of mycoplasma. This proviral construct 
will be tranfected into this certified packaging cell line using standard 
calcium phosphate methods to obtain retroviral supernatant. They will be 
tested and shown to be free of adventitious agents including RCR (replication 
competent retroviruses) replication-competent virus using the NIH 3T3 and 
S+/L- assays. 
Recombinant DNA Research, Volume 19 
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