Recombinant DMA Advisory Committee- 9/12-13/94 
Investigator Response-Drs. Holt and Arteaga 
Dr. Holt said that marker rescue assays are used to detect RCR in their vector 
preparations. More stringent assays will be used to conform with the requirement of FDA 
including feline PG-4 S T/ assay, extended S T/ assay, and co-cultivation of test cells with 
Mus dunni cells with detection by the PG-4 S *L' assay. Dr. Miller said the criteria should 
be less than one RCR per 100 ml of the patient dose. Dr. Holt said that the RCR assay 
has been performed for 10 ml of supernatant, and it will be scaled up to one patient dose. 
Dr. D. Ginsburg said that since the LXSN vector constructs are being developed for 
antisense expression, it would be preferable to wait and use the better vectors for the 
human study. Dr. Miller said that if RCR is not detected by the stringent tests, there is 
no reason not to use the present vectors. Dr. Holt explained that the reason not to 
change the vector is that MMTV promoter may not function as well in the LXSN vector. 
He agreed with Dr. D. Ginsburg that it is a reasonable point to try the new vector in the 
future. 
Dr. Miller raised another question about the open reading frame of the gag gene in the 
N2 vector. Expression of gag proteins could complicate the interpretation of the results of 
myc and fos antisense expression due to potential immunogenicity. Dr. Holt explained 
that this complication is avoided by using the same vector with gag expression in the 
control studies. 
Dr. Miller said that in the new data, a diagram is presented to show the vector construct. 
A neo R gene is driven by the long terminal repeat (LTR) of the vector. The MMTV 
promoter drives the expression of the anti-/as in the opposite direction and the RNA is 
terminated with a polyadenylation signal from the globin gene. Dr. Holt said that a 
complete vector sequence is provided. 
Regarding the experiment of vector stability in pleural fluid, Dr. Holt said one explanation 
for the vector stability is that the level of complement, which inactivates the virus, may be 
lower in pleural fluid than in blood. As to the nude mouse experiment on established 
tumors, Dr. Holt said antitumor effects have been observed in a preliminary experiment 
with 6 mice, but additional studies with 20 nude mice are ongoing. 
Regarding the statement of alterative therapy in the Informed Consent document. Dr. 
Holt said that there is no alternative therapy for breast cancer metastasis in pleural or 
peritoneal effusions. Dr. D. Ginsburg commented that sclerosing treatment is quite 
effective for the symptom of pleural effusion by closing the pleural space, although it is 
not directed toward breast cancer itself. This treatment, however, would affect the vector 
access to the tumor cells, and Dr. D. Ginsburg asked if it should be considered as one of 
the exclusion criteria. 
Dr. Arteaga said that the sclerosing treatment will be useful for patients with serious 
symptoms of pleural effusion. The majority of patients to be enrolled in the study are not 
expected to have such severe symptoms. Patients will spend 4 days in the Clinical 
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Recombinant DNA Research, Volume 20 
