Heregulin-Targeted Protein Uptake for Breast Cancer

描述（由申请人提供）： HER2阳性乳腺癌具有高度侵袭性，通常对化疗具有耐药性，并且与高死亡率相关。因此，通过替代手段的治疗，例如靶向递送治疗基因，可能证明对这些类型的癌症比标准治疗方法更有效。本文所述的基因递送系统在利用重组腺病毒组分方面是新颖的，所述重组腺病毒组分已被修饰以实现乳腺癌细胞特异性结合和缀合DNA的递送，同时保留病毒衣壳的高效细胞摄取特征。该系统将仅需要细胞表面结合和内化所必需的最少腺病毒蛋白，并且缺乏所有其他病毒蛋白和基因。因此，该系统避免了与使用病毒相关的问题，例如突变和病毒重组。具体目的是检验以下假设：1.重组可溶性五邻体和纤维衣壳蛋白易位需要在衣壳蛋白中发现的关键分子基序与细胞骨架和细胞内运输机制的组分的相互作用。这项研究将揭示有关乳腺癌细胞进入的决定因素的有用信息，因为对这些细胞中的病毒运输途径知之甚少。免疫荧光和共聚焦显微镜将用于观察野生型和突变蛋白的运输。增强免疫力的突变将被纳入载体设计中，并在Aims2&3中进行测试。 2.工程化衣壳蛋白介导非病毒基因递送至HER2+乳腺癌细胞，并且在体外比完整重组Ad毒性更低。将在非病毒复合物和Ad之间进行细胞毒性和基因转移的比较。我们将评估单个衣壳蛋白对Ad感染的细胞应答的贡献。 3.工程化衣壳蛋白比完整重组Ad毒性和免疫原性更低，并且可以在体内介导靶向非病毒基因递送至HER2+乳腺癌细胞。由于HER2+细胞过度表达调蛋白受体，我们检查调蛋白直接将非病毒基因递送到该组织的能力。我们将评估单个衣壳蛋白对Ad免疫应答的贡献，比较Ad的免疫原性，并测试免疫逃避的方法。

DESCRIPTION (provided by applicant): HER2-positive breast cancers are highly aggressive, frequently resistant to chemotherapy, and associated with a high incidence of mortality. Therefore, therapy by alternative means, such as targeted delivery of therapeutic genes, may prove much more effective on these types of cancers than standard methods of treatment. The gene delivery system described here is novel in its utilization of recombinant adenoviral components which have been modified to achieve breast cancer cell-specific binding and delivery of conjugated DNA, while retaining the high efficiency cellular uptake features of the viral capsid. This system would require only the minimal adenoviral proteins necessary for cell surface binding and internalization, and lacks all other viral proteins and genes. Thus, this system avoids the concerns associated with using viruses such as mutation and viral recombination. The Specific Aims are to test the hypotheses that: 1. Recombinant soluble penton and fiber capsid protein translocation requires interactions of key molecular motifs found in capsid proteins with components of the cytoskeletal and intracellular trafficking machinery. This study will uncover useful information about the determinants governing breast cancer cell entry, as little is known about viral trafficking pathways in these cells. Immunofluorescence and confocal microscopy will be used here to visualize trafficking of wild-type and mutant proteins. Traffic-enhancing mutations will be incorporated into vector design and tested in Aims2&3. 2. Engineered capsid proteins mediate non-viral gene delivery to HER2+ breast cancer cells and are less toxic than whole recombinant Ad in vitro. Comparisons of cytotoxicity and gene transfer will be made between non-viral complexes and Ad. We will assess the contribution individual capsid proteins make toward the cellular response to Ad infection. 3. Engineered capsid proteins are less toxic and immunogenic than whole recombinant Ad, and can mediate targeted non-viral gene delivery to HER2+ breast cancer cells in vivo. As HER2+ cells overexpress the heregulin receptor, we examine of the capacity for heregulin to direct non-viral gene delivery to this tissue. We will assess the contribution of individual capsid proteins to the immune response to Ad, compare immunogenicity to Ad, and test a method of immune evasion.