This laboratory continues to investigate tumor targeting with radiolabeled antisense DNAs and other oligomers. We have successfully addressed the majority of the prior concerns regarding this imaging modality by demonstrating that radiolabeled antisense oligomers accumulate in cancer cells in vitro and in vivo by an antisense mechanism, that the number of mRNA targets is sufficient to provide successful nuclear imaging and that a convincing tumor image in mice by an antisense mechanism was achieved following intratumor administration. However, that a similar successful image has not yet been achieved here or, in our judgment, elsewhere following intravenous administration of radioactive antisense oligomers may be explained by the high background radioactivity levels in normal tissues. While next to radioactivity methods, optical imaging may be the most sensitive of noninvasive in vivo imaging modalities, at least as concerns surface tissues such as breast cancers, one major advantage of optical imaging methods over radioactivity methods is the possibility of turning the signal on and off through the judicious use of fluorescence resonance energy transfer (FRET). The use of FRET to inhibit and enhance fluorescence in DNA-based molecular beacons is common but primarily in vitro and using hairpin DNAs. To our knowledge, the use of linear fluorophore-conjugated oligomer duplexes have not previously been considered for antisense or other imaging applications. We have shown that the fluorescence of a Cy5.5 emitter-conjugated antisense DNA may be inhibited both in cell culture and in tumored animals when hybridized with a shorter BHQ3 inhibitor-conjugated complementary DNA but that fluorescence is expressed in the presence of the target mRNA in tumor as the duplex dissociates freeing the emitter (parenthetically we have also observed that cellular delivery is improved when oligomers are administered as duplexes). These proof of concept results of optical antisense targeting therefore suggests that further studies, in particular those designed to optimize this approach for breast cancer imaging, are appropriate. We propose to investigate for this application antisense oligomers against the survivin mRNA, overexpressed in most if not all cancers, in the form of 14 phosphodiester DNAs, phosphorothioate DNAs and phosphodiamidate morpholino MORFs duplexes. The stability of each duplex will be evaluated in the absence and in the presence of the survivin base sequence and the most promising five of these will be evaluated along with controls in MCF7 breast cancer cells in culture and the remaining two candidate duplexes will be studied in MCF7 tumor-bearing mice. This investigation is focused on the detection of breast cancer in part because this disease is among the largest killer of US women and improved methods for early detection are needed. Our multidisciplinary team has experience in each aspect of this investigation. To public health, we have preliminary results in tumored animals suggesting that optical imaging with fluorescent linear antisense DNA oligomers may provide detection of surface or otherwise accessible cancer tissues, such as primary and residual breast cancer following debulking, superior to that provided by nuclear antisense imaging and other imaging modalities as well. If further studies confirm these results, an entirely novel and potentially significant method of cancer detection will have been identified that may be of particular benefit to patients with breast cancer. ? ? ?
