Clinical Protocol 
Protocol 
Gene Therapy for the Treatment of Metastatic Breast Cancer by In Vivo Infection with Breast-Targeted 
Retroviral Vectors Expressing Antisense c-fos or Antisense c-myc RNA 
Co-Pnncipal Investigators: Jeffrey T. Holt MD, Department of Cell Biology, 
Vanderbilt University Medical School 
Carlos B. Arteaga MD, Division of Oncology, 
Department of Medicine, Vanderbilt University Medical School 
Associate Investigators: David Robertson MD, Director, Clinical Research Center, 
Department of Medicine, Vanderbilt University Medical School 
Harold L. Moses MD, Director, Vanderbilt Cancer Center 
Department of Cell Biology, Vanderbilt University Medical School 
Subject of Study 
Patients 
Number Sex Age Range 
10 Female >18 
1. Specific Aims 
To apply gene therapy to metastatic breast cancer we propose a method for in vivo gene transfer of 
antisense sequences directed against either c-fos or c-myc using retroviral vectors with expression regulated 
by the Mouse Mammary Tumor Virus (MMTV) long terminal repeat (LTR). We have demonstrated that 
infection of the human breast cancer cell line MCF-7 with an antisense retroviral vector producing either 
antisense c-fos or antisense c-myc RNA produces a marked decrease in tumor formation and tumor growth 
rate in nude mice. A control retroviral vector which differs only in the orientation of the gene insert did not 
inhibit tumor formation, providing evidence that the antitumor effect is mediated through production of 
antisense RNA. This effect is apparently mediated by the production of antisense RNA which binds to the 
target oncogene mRNA, resulting in decreased mRNA and protein levels. The production of antisense 
RNA is regulated by the breast-selective MMTV promoter which is designed to limit expression of the 
transferred gene to breast cells (which should generally be tumor cells in the therapeutic sites). The breast- 
selectivity of the MMTV promoter may contribute to the limited toxicity which we have observed in 
toxicity studies in nuce mice. Infection with these breast-targeted, antisense viral vectors can result in 
marked tumor inhibition or even cure of some experimental animals with no clearcut toxicity. Therefore we 
propose to apply this method for the treatment of human metastatic breast cancer. 
The clinical trial will focus on maximizing the delivery of retroviral vector to the tumor cells within the 
malignant fluid space, along with repeated administration in an attempt to maximize the antitumor effect. 
Patients will undergo a fluid examination prior to infusion of retroviral vector (lumbar puncture for 
carcinomatous meningitis, paracentesis for malignant ascites, thoracentesis for malignant pleuritis). 
Cytologic, biochemical, and molecular studies will be performed on aspirations of fluid to follow the 
extent of viral vector uptake by tumor cells and determine the stability of the viral vector within the fluid. 
The clinical extent of tumor spread will be measured before and after retroviral vector infusion by clinical 
exam , measurement of fluid accumulation and adbominal girth. After the initial infusion of retroviral 
vector, fluid will be removed and examined by methods cited above, followed by a secondand third 
injection of the retroviral vector. The initial studies and infusions will be performed as an in-patient 
procedure within the Clinical Research Center of Vanderbilt University Medical School. Following the 
third infusion, the patient may be discharged and then return at two weeks and four weeks for followup. 
In the event of death: Post-mortem examination will quantify tumor spread by careful disection, 
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