Optical Antisense Breast Tumor Targeting

描述(申请人提供)：该实验室继续研究放射性标记的反义DNA和其他寡聚体的肿瘤靶向。我们已经成功地解决了大多数先前对这种成像方式的担忧，证明了放射性标记的反义寡聚体通过反义机制在体外和体内的癌细胞中积累，mRNA靶的数量足以提供成功的核成像，并且在肿瘤内给药后通过反义机制在小鼠身上获得了令人信服的肿瘤图像。然而，根据我们的判断，静脉注射放射性反义寡聚体后，尚未在这里或其他地方获得类似的成功图像，这可能是因为正常组织中的高本底放射性水平。虽然光学成像可能是仅次于放射性方法的最敏感的非侵入性体内成像手段，但至少对于乳腺癌等表面组织而言，光学成像方法相对于放射方法的一大优势是可以通过明智地使用荧光共振能量转移(FRET)来打开和关闭信号。在基于DNA的分子信标中使用FRET来抑制和增强荧光是很常见的，但主要是在体外和使用发夹DNA。据我们所知，使用线性荧光团-偶联低聚物双链以前还没有被考虑用于反义或其他成像应用。我们已经证明，在细胞培养和肿瘤动物中，当与较短的BHQ3抑制剂结合的互补DNA杂交时，Cy5.5发射体结合的反义DNA的荧光可能被抑制，但当双重结构解离释放发射体时，荧光在肿瘤中的靶mRNA存在的情况下表达(顺便说一句，我们还观察到当寡聚体作为双重构体给药时，细胞递送得到改善)。因此，光学反义靶向的这些概念验证结果表明，进一步的研究，特别是那些旨在优化乳腺癌成像方法的研究，是合适的。我们建议为这一应用研究针对Survivin mRNA的反义寡聚体，Survivin在大多数癌症中过表达，其形式为14个磷酸二酯DNA、硫代磷酸DNA和磷二酸吗啉双链。将在没有Survivin碱基序列和存在Survivin碱基序列的情况下评估每个双链的稳定性，其中最有希望的五个将在培养的MCF7乳腺癌细胞中与对照一起评估，其余两个候选双链将在MCF7荷瘤小鼠中进行研究。这项调查的重点是乳腺癌的检测，部分原因是这种疾病是美国妇女的最大杀手之一，需要改进早期发现的方法。我们的多学科团队在这次调查的各个方面都有经验。为了公众健康，我们在肿瘤动物上的初步结果表明，使用荧光线性反义DNA寡聚体的光学成像可以提供对表面或以其他方式接近的癌症组织的检测，例如去茎后的原发和残留乳腺癌，优于核反义成像和其他成像方式。如果进一步的研究证实了这些结果，一种全新的、潜在意义重大的癌症检测方法将被确定，这可能对乳腺癌患者特别有益。

DESCRIPTION (provided by applicant): 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.