IV. Rationale for Gene Therapy and Significance 
There is no standard therapy for metastatic breast cancer which has spread to peritoneum, pleura, or 
meningeal spaces. Some patients with large malignant pleural effusions are treating by removing the fluid 
with or without sclerosing the previous space which was filled with fluid. Alternative therapies include 
systemic chemotherapy and/or systemic hormonal therapy . These therapies do not presently provide 
effective treatment or cure to these patients.This treatment may be used in combination with other 
chemotherapeutic or hormonal approaches, but no combination of these therapies has demonstrated 
substantial efficacy or cure.This transcription factor-targeted antisense approach is specifically targeted to 
the MMTV -expressing breast tumor cells. Hence, theoretically it is a more rational approach than the use 
of cytotoxic nonselective chemotherapy agents. Unfortunately, most patients with metastatic breast cancer 
with malignant effusions also have disease at other clinical sites. Therefore this therapy is designed to halt 
progression or reverse disease manifestations, and is not intended to prevent all manifestations of disease. IJ 
The recombinant viral vector will be transferred into malignant tumor cells which are growing 
within mesothelial-lined fluid spaces. The use of recombinant DNA is necessary for these studies because 
the transfer of antisense sequences requires an efficient gene delivery mechanism such as a retroviral 
vector. The use of recombinant viral vectors in the proposed clinical protocol would also provide important 
data regarding the transfer for retroviral vectors into cells growing within peritoneal, pleural, or 
cerebrospinal fluid. These fluids may provide a special site for effective gene transfer. 
3. Preliminary Studies: 
I. Pre-clinical Data 
A. In vitro studies with plasmid-mediated and oligonucleotide-mediated antisense methods. 
We have previously employed antisense methods to demonstrate that antisense RNA 
complementary to c-fos results in inhibition of cell proliferation in cultured fibroblasts. This work is 
presented in several publications which are included in Appendix A. This work has also demonstrated that 
antisense inhibition of the c-myc gene results in decreased cell proliferation and an induction of 
differentiation [19]. In both cases: we demonstated that the antisense produces decreased expression of the 
target gene mRNA and protein, and that the effect could be rescued by sequence competition [19] or by 
antisense rescue [25]. 
B. Construction of Retroviral vectors and production of viral stock 
The specific retroviral vectors are XM6:antifos and XM6:antimyc which differ only in the 
antisense region between the MMTV promoter and the globin 3' sequence. These retroviral vectors are 
based on the N2 retroviral vector, have been previously published (25) and their structures were confirmed 
by restriction enzymes analysis and DNA sequencing across cloning and tinkering sites. Maps and 
computer-generated DNA sequences for both retroviral vectors are presented in Appendix B. 
The XM6 retroviral vectors used in these antisense retroviral vectors contain the breast-targeted Mouse 
Mammary T umor Virus Long Terminal Repeat (MMTV) which is selectivity expressed in breast cells in 
transgenic mice and in steroid-treated tissue culture cells. The specific cis-elements within the MMTV 
promoter which are responsible for breast-targeted expression have not been definitively identified, but 
studies in transgenic mice have shown that certain truncations of the MMTV promoter show promiscuous 
expression in diverse tissues. For these reasons, the breast- targeted retroviral vectors employed in this 
protocol employ the entire 5' MMTV LTR region. Because antisense sequences need to be fused to other 
regulatory elements, these retroviral vectors include the human beta globin polyadenylation site which 
provides stability to the antisense transcripts and marker sequences for ready identification of gene 
transfer. 
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