Recombinant DNA Advisory Committee- 9/12-13/94 
expressed in the forward direction. The use of the MMTV promoter is to express the 
antisense sequences specifically in breast cells, and the retroviral vectors target specifically 
to the actively dividing cancer cells in the injected body spaces. Dr. Miller raised several 
specific questions: (1) Vector design and production. The vector sequence submitted on 
the computer disk does not match the description in the text or in the diagrams. The 
overall structure of the vector is fine but it is based on the old N2 retroviral vector. There 
was concern that the old N2 vector, when produced in the PA317 cells, has a high 
probability of generating replication competent retrovirus (RCR) due to recombination 
with other viral sequences in those cells. Dr. Miller asked why the newer LXSN vector 
was not used. The investigators responded in writing that they are concerned that the 
MMTV promoter may not be appropriately expressed in this new vector. Dr. Miller 
stated that the investigators should provide quantitative data regarding the lack of RCR 
production or the criteria to be used for testing clinical grade vector preparations. Dr. 
Miller asked what culture medium will be used to produce the clinical grade vector 
preparations. The standard culture media contain bovine serum which is not acceptable 
for human use. The investigators indicated in writing that human serum will be used or 
alternatively the vector will be produced in serum-free media. (2) The IBC has 
recommended a Biosafety Level (BL) 2 + physical containment for the present 
experiments. Dr. Miller recommended a BL1 containment level based on lack of 
oncogenic sequences in the vectors. (3) The IRB initially classified this study as high risk. 
The investigators have explained that the IRB means the protocol deals with high risk 
cancer patients, rather than the vector or the experiment being of high risk. (4) Have the 
investigators used the antisense vectors to transduce primary breast cancer cells? The 
investigators provided data to show that antisense RNAs were made, and they could 
inhibit fos and myc oncogene expression and could reduce cell growth. Dr. Miller was 
satisfied with these in vitro data. (5) Will body fluids of the vector injection sites directly 
inactivate the retroviral vectors? Human complement in blood can directly inactivate 
retroviruses. The investigators have provided data to show that at 37° C pleural effusions 
have little effect on virus infectivity, a twofold decrease after 24 hours. Overall, Dr. Miller 
was satisfied with the written response provided by the investigators. He asked to see the 
correct vector sequence and quantitative RCR data. 
Review-Dr. Haselkom (presented by Dr. Miller) 
Dr. Haselkom stated in his written review that since this new protocol is substantially 
different from others already approved by the RAC, it requires a thorough review. The 
protocol involves the administration of retroviral constructs expressing antisense RNAs of 
oncogenes to treat metastatic breast cancer. The vector has a MMTV promoter that 
requires estrogen for expression, so the transgenes should be transcribed only in cells such 
as breast cells that contain the estrogen receptor. Dr. Haselkom raised several specific 
questions: (1) Are the vectors targeted to breast cancer cells from the prospective 
recipients? (2) Are the antisense RNAs expressed in those cells? (3) If expressed, do 
they prevent expression of the fos and myc gene products? and (4) Is growth of the tumor 
cells affected? Dr. Miller said that these questions have been affirmatively answered by 
the investigators in their written response. The investigators noticed a "bystander effect" 
in that tumor growth was affected more than the number of cells transduced. Dr. Miller 
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Recombinant DNA Research, Volume 20 
