The DNA construct was made by modifying the basic N2 retroviral vector to produce unique cloning 
sites and then systemically clone in desired DNA sequences and regulatory elements. First, the Hind III 
and Bam HI sites within PBR sequences present in the vector were destroyed by blunting/religation so that 
these could be used as unique cloning sites in later constructs. Next, human beta globin sequences were 
cloned into the unique Xho I site to provide globin polyadenylation sequences for future constructs.The 
retroviral vector was now similar to the MMTV-antisense-globin vectors which we have previously used 
in transfection studies (22,24,25), allowing subcloning of Xhol-Bam HI MMTV-antisense sequences for 
either c-fos or c-myc. The antisense c-fos sequences is 196 base pairs which flank the translation initiation 
site and the antisense c-myc sequence is 750 base pairs which flank the translation initiation site. A control 
retroviral vector termed "sense" c-fos is identical to the XM6:antifos retroviral vector except the antisense 
sequence has been cloned in the reverse orientiation (thus it does not produce complementary antisense 
RNA). The recombinant DNA is purified by alkaline lysis and then twice- banded by ultracentrifugation 
with CsCl. 
The viruses were prepared by transfecting PA3 17 cells with either the XM6:anti-fos or XM6:antimyc 
retroviral vector DNAs which were purified as described in IBla(3). Following transfection, the PA317 
cells were split and then treated with G418 until individual clones could be identified and expanded. Each 
clone was then screened for its titer by analyzing its ability to transfer G418 resistance (since the retroviral 
vector contains a Neomycin resistance gene). The clones which had the highest titer were then frozen in 
numerous aliquots and tested for sterility, presence of replication-competent retrovirus, and presence of 
mycoplasm. The cells are grown in Dulbecco's modified essential medium with added fetal calf serum. 
The amount of fetal calf serum employed in the pre-clinical studies ranged from 2.5% (for studies of 
MCF-7 breast cancer cells whose growth is inhibited by serum) to 10% (for studies infecting fibroblast 
cells). We plan to collect virus from producer cells by adding serum-free media to the producer cells for 8 
hours so that patients are not exposed to bovine serum proteins. If the presence of serum proteins is 
shown to be important for efficient infectivity in future studies, then we may consider using appropriately 
tested Human AB serum for collection of retroviral stocks. We would make this change only with the 
approval of RAC and the FDA. We will only use media from retroviral producer clones which pass the 
following tests: S+L- assay, 3T3 amplification followed by S+L- assay, bacterial and fungal sterility 
testing, mycoplasma testing. Southern blot analysis of the retroviral producer clone, ability to transfer 
vector DNA to other cells (analyzed by transfer of G418 resistance and by Southern blotting of target cells 
to demonstrate efficient gene transfer), and additional testing as required or requested by RAC or die 
FDA. 
C. In vitro studies with antisense retroviral vectors 
1. Evidence for breast selectivity: 
Several different cell types have been infected with the amphotropic XM6:antifos and 
XM6:antimyc retroviral vector stocks including: mouse NIH 3T3 cells, human HL-60 leukemia cells, and 
MCF-7 human breast cancer cells. We have not observed expressed of the MMTV-regulated antisense 
sequences in 3T3 cells or HL60 cells unless exogenous steroid was added to the culture media. 
2. Effects of retroviral vectors in vitro: 
We have constructed a number of retroviral vectors which are designed for breast-targeted 
expression. These vectors can be divided into two basic groups: 1. Antisense dominant oncogene vectors; 
and 2. Tumor suppressor gene transfer vectors. The basic structure of these retroviral vectors has been 
reported [24] and is shown in Appendix C: Figure 1. Stable producer clones were produced by 
transfection of twice-cesium banded DNAs into the amphotropic producer line PA3 17. At least 10 
producer clones for each plasmid were titered by quantitating the transfer of neo resistance to NIH 3T3 
target cells. The producer clone which produced the highest titer from each plasmid DNA was identified 
Recombinant DNA Research, Volume 20 
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