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，经常对化疗耐药，并与高死亡率相关。因此，通过其他方法进行治疗，例如靶向传递治疗基因，可能会证明对这些类型的癌症比标准治疗方法更有效。本文描述的基因递送系统的新颖之处在于它使用了重组腺病毒成分，这些重组腺病毒成分已经被改造，以实现乳腺癌细胞特异性结合和传递结合的DNA，同时保留了病毒衣壳的高效细胞摄取特征。这个系统只需要细胞表面结合和内化所需的最低限度的腺病毒蛋白，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，如突变和病毒重组。本研究的具体目的是验证以下假设：1.重组可溶五酮和纤维衣壳蛋白的转运需要衣壳蛋白中的关键分子基序与细胞骨架和细胞内运输机制的组件相互作用。这项研究将揭示有关控制乳腺癌细胞进入的决定因素的有用信息，因为人们对这些细胞中的病毒传播途径知之甚少。免疫荧光和共聚焦显微镜将在这里用来显示野生型和突变蛋白的运输。交通增强型突变将被纳入载体设计并在Aims2和3.2中进行测试。2.工程衣壳蛋白介导非病毒基因传递到HER2乳腺癌细胞，体外毒性低于完整的重组Ad。将比较非病毒复合体与Ad.的细胞毒性和基因转移。我们将评估单个衣壳蛋白对细胞对Ad感染的反应的贡献。3.基因工程衣壳蛋白的毒性和免疫原性均低于完整的重组腺病毒，并能在体内介导靶向性非病毒基因导入HER2乳腺癌细胞。由于HER2细胞过度表达Hereglin受体，我们检测了Hereglin将非病毒基因传递到该组织的能力。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，并测试一种免疫逃避方法。

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.