I 
the tumor is enhanced. Second, the brain is a partially immunologic privileged site, which 
should allow a somewhat longer survival of the xenogeneic murine cells in the brain and a 
greater transduction frequency of the growing tumor. This feature is further increased since 
human gliomas are known to further depress local immunity. This is thought to be secondary 
to a down regulation of IL-2 secretion and diminished expression of high affinity IL-2 
receptors on T-lymphocytes (1 1). The murine cells should survive sufficiently long in the 
brain to allow for the transduction of greater numbers of tumor cells. However, this period 
of survival will be limited since all cells that integrate and express HS-tk will be destroyed 
by the GCV or eventually destroyed by the immune system. 
II. Retroviral-mediated Gene Transfer (12,13). 
In contrast to chemical and physical cellular transfection methods, murine retroviral 
vectors have proven to be extremely efficient for gene transfer into mammalian cells, with 
efficiencies as high as 90% in cultured murine fibroblast cell lines. Murine retroviral vectors 
differ from the adenoviruses and herpes simplex viruses in that the retroviruses will only 
integrate and subsequently express their genes in proliferating tissues (e.g. tumors). This 
feature of the retroviral vectors may be particularly advantageous in the brain, where the 
tumor is the predominant mitotic cell type, maximizing specific transduction of tumor with 
minimal, or absent, transduction of normal brain. The Moloney murine leukemia virus- 
based (MoMLV) vectors have been designed to minimize the possibility of recombination 
resulting in regeneration of a replication-competent virus (14,15). 
In 1989, the first gene transfer experiment in humans was conducted at the NIH. This 
study involved the treatment of 10 patients with autologous T-cells (TIL) that have been 
transduced with a retroviral- vector. None of these patients have demonstrated any untoward 
effects secondary to receiving the genetically-altered cells (16). 
Human gene therapy for adenosine deaminase deficiency has been performed on 2 
children since the protocol opened in September, 1990. Twenty-three intravenous infusions 
of genetically altered autologous T-cells have been administered to date (17,18). Neither 
child has demonstrated any evidence of adverse effect due to the genetically altered cells. 
3. Preliminary Studies 
I. Pre-clinical Data 
A. In Vivo Transduction with NeoR and 0-galactosidase Vectors: 
1. In Vivo Transduction of the NeoR Vector (LNL6) into the MCA 205 Murine Fibrosarcoma 
(See Appendix A: Reprint 1)(10,27). Initial studies were performed in mice to determine if 
tumor cells could be transduced in vivo. The retroviral vector LNL6, which has a titer of 
1 .0x10^ cfu/ml, was utilized for these experiments. LNL6, which contains a NeoR gene 
promoted by the 5’-LTR, is free of replication-competent retrovirus. NeoR protects 
mammalian cells from the toxic effects of the neomycin analog G418. LNL6 VPC or 
control cells not producing vector but expressing the NeoR gene (LNL6 transduced 3T3 
cells) and MCA 205 tumor were mixed in vitro and injected subcutaneously in syngeneic 
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