Optical Antisense Breast Tumor Targeting

描述（由申请方提供）：本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作用。我们已经成功地解决了大多数关于这种成像方式的先前问题，证明放射性标记的反义寡聚体通过反义机制在体外和体内癌细胞中积累，mRNA靶点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中获得令人信服的肿瘤图像。然而，在静脉内给予放射性反义寡聚体后，在此处或根据我们的判断，在其他地方尚未获得类似的成功图像，这可以通过正常组织中的高背景放射性水平来解释。虽然仅次于放射性方法，光学成像可能是最敏感的非侵入性体内成像方式，至少在涉及表面组织如乳腺癌时，光学成像方法相对于放射性方法的一个主要优点是通过明智地使用荧光共振能量转移（FRET）来打开和关闭信号的可能性。使用FRET来抑制和增强基于DNA的分子信标中的荧光是常见的，但主要是在体外和使用发夹DNA。据我们所知，线性荧光团缀合的寡聚物双链体的使用以前没有被考虑用于反义或其他成像应用。我们已经表明，Cy5.5发射体缀合的反义DNA的荧光在与较短的BHQ 3标记物缀合的互补DNA杂交时在细胞培养物中和在肿瘤动物中都可以被抑制，但是当双链体解离释放发射体时，在肿瘤中靶mRNA存在时荧光被表达（附带地，我们还观察到当寡聚体作为双链体施用时，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。我们建议研究针对生存素mRNA的反义寡聚体，该反义寡聚体在大多数（如果不是所有）癌症中以14磷酸二酯DNA、硫代磷酸酯DNA和磷酸二酰胺吗啉代MORFs双链体的形式过表达。将在不存在和存在存活素碱基序列的情况下评价每种双链体的稳定性，并且将在培养物中的MCF 7乳腺癌细胞中评价其中最有希望的五种双链体沿着对照，并且将在携带MCF 7肿瘤的小鼠中研究剩余的两种候选双链体。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，需要改进早期检测方法。我们的多学科团队在调查的各个方面都有经验。对于公众健康，我们在肿瘤动物中的初步结果表明，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可接近的癌组织的检测，例如减积后的原发性和残留乳腺癌，其上级于核反义成像和其他成像方式所提供的检测。如果进一步的研究证实了这些结果，将确定一种全新的和潜在的重要癌症检测方法，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.