Protein-DNA Drug Carriers for Tumor Targeting

描述（由申请人提供）：我们将测试一个假设，即来自细胞穿透病毒衣壳的独特重组融合蛋白可以通过微小的核酸载体运输DNA嵌入药物，并将这些药物靶向特定的肿瘤细胞，导致肿瘤特异性递送和细胞死亡。为了测试这种新型治疗方法的可行性，我们选择了HER2+乳腺癌作为我们的肿瘤靶点。HER2+乳腺肿瘤，过度表达人表皮生长因子受体（HER）亚基2，构成乳腺癌的一个重要亚群，对标准治疗方法无效，并且预测死亡率很高。我们将利用天然配体-受体相互作用诱导的快速内吞吸收和病毒蛋白的穿膜活性，将药物输送到癌细胞中，并从内部诱导细胞毒性。这个项目的具体目的是测试假设：1。关键分子基序增强细胞靶向和穿透修饰衣壳蛋白在体外和体内。heregulin靶向蛋白HerPBK10的一系列变体将被生成，并在一组表达不同HER亚基水平的HER2+和HER2-乳腺癌细胞系和原代细胞以及等基因细胞系上进行靶向细胞结合、摄取、细胞内渗透和运输测试。免疫荧光和共聚焦显微镜将用于分析摄取和运输，亚细胞分离将用于确认和量化结果。亚细胞标志物和运输抑制剂将有助于确定摄取途径。最后，这些变异将在体内进行肿瘤靶向性测试。产生最佳靶向和摄取的变体将被纳入随后目标中提出的生物偶联物中。2. 修饰的衣壳蛋白与双链寡核苷酸（ds-oligo）和DNA插入剂组装形成非共价生物偶联物，靶向HER2+乳腺癌细胞。从Aim 1中选择的最佳蛋白变体将与不同长度的ds-oligos和DNA插入剂结合形成非共价偶联物。我们将使用UV/Vis吸光度法和荧光法来评估组装参数及其在不同储存条件下和血清中的稳定性。我们将使用代谢试验在单独和混合的HER2+和HER2-细胞培养中测试每个组件的靶向毒性，并通过与自由配体的竞争性抑制来验证受体特异性。最后，我们将研究靶向毒性的机制，并测试我们提出的药物摄取和细胞内释放模型。这些结果将决定在Aim 3中测试的最佳组装参数和生物偶联物的剂量。3. 一种由修饰的衣壳蛋白、ds-oligo和DNA插入物组成的优化生物偶联物靶向HER2+肿瘤细胞，并在体内赋予治疗效果。我们将利用HER2+乳腺癌裸鼠模型建立体内剂量曲线，以确定影响最大肿瘤消融的最小瘤内剂量，并确定生长抑制和消退所需的时间。生物分布和药代动力学将分别通过体内成像和测量治疗小鼠血液中的偶联物水平来确定。我们将确定结合物全身递送的治疗效果，并测量肿瘤体积以确定生长抑制。最后，免疫原性将通过对免疫能力小鼠的血液进行吸光度测定来确定。公共卫生相关性：该研究项目与公共卫生相关，因为它将导致一种新的治疗方法的发展，可以专门针对HER2+乳腺癌。这种靶向治疗应该是对传统治疗方法的改进，因为正常细胞不应该受到影响。由于HER2+乳腺癌对常规疗法的反应不佳，这种替代疗法可以为乳腺癌治疗提供重大贡献，并且可以修改为针对其他类型癌症的靶向治疗。

DESCRIPTION (provided by applicant): We will test the hypothesis that a unique recombinant fusion protein derived from a cell-penetrating viral capsid shell can transport DNA intercalating drugs through a tiny nucleic acid carrier, and target such drugs to specific tumor cells causing tumor-specific delivery and cell death. To test the feasibility of this new type of therapeutic, we have chosen HER2+ breast cancer as our tumor target. HER2+ breast tumors, which overexpress subunit 2 of the human epidermal growth factor receptor (HER), comprise a significant subset of breast cancers that are recalcitrant to standard methods of treatment, and predict a high mortality. We will take advantage of the rapid endocytic uptake induced by natural ligand-receptor interactions and the membrane penetrating activity of a viral protein to deliver a drug into the cancer cell and induce cytotoxicity from within. The Specific Aims of this project are to test the hypotheses that: 1. Key molecular motifs enhance cell targeting and penetration of a modified capsid protein in vitro and in vivo. A series of variants of the heregulin-targeted protein, HerPBK10, will be generated and tested for targeted cell binding, uptake, intracellular penetration and trafficking on a panel of HER2+ and HER2- breast cancer cell lines and primary cells, and isogenic cell lines expressing different HER subunit levels. Immunofluorescence and confocal microscopy will be used to analyze uptake and trafficking, and subcellular fractionation will be used to confirm and quantify the results. Subcellular markers and trafficking inhibitors will help determine uptake pathways. Finally, the variants will be tested for tumor targeting in vivo. Variants that yield optimal targeting and uptake will be incorporated into the bioconjugate proposed in the subsequent aims. 2. A modified capsid protein assembles with a double-stranded oligonucleotide (ds-oligo) and DNA intercalating agent to form noncovalent bioconjugates that target HER2+ breast cancer cells in vitro. The optimal protein variant selected from Aim 1 will be combined with ds-oligos of varied lengths and DNA intercalating agent to form noncovalent conjugates. We will use UV/Vis absorbance and fluorometry to assess assembly parameters and stability under different storage conditions, and in serum. We will test each assembly for targeted toxicity in separate and mixed HER2+ and HER2- cell cultures using metabolic assay, and receptor specificity will be verified by competitive inhibition with free ligand. Finally, we will investigate the mechanism of targeted toxicity and test our proposed model of drug uptake and intracellular release. These results will dictate the optimal assembly parameters and dosage of bioconjugate to test in Aim 3. 3. An optimized bioconjugate comprised of a modified capsid protein, ds-oligo, and DNA intercalator targets HER2+ tumor cells and imparts therapeutic efficacy in vivo. We will utilize a nude mouse model of HER2+ breast cancer to establish an in vivo dose curve for determining the minimal intratumoral dosage effecting maximal tumor ablation and determine the timeline required for growth inhibition and regression. Biodistribution and pharmacokinetics will be determined by in vivo imaging and measuring conjugate levels in blood, respectively, in treated mice. We will determine the therapeutic efficacy of conjugate delivered systemically and tumor volumes will be measured to determine growth inhibition. Finally, immunogenicity will be determined using absorbance assays of blood taken from treated immune competent mice. PUBLIC HEALTH RELEVANCE: This research project is relevant to public health because it will result in the development of a novel therapeutic that can specifically target HER2+ breast cancer. This targeted therapy should be an improvement over conventional treatment methods because normal cells should not be affected. As HER2+ breast cancer does not respond well to conventional therapies, this alternative therapy could provide a significant contribution to breast cancer treatment, and could be modified to target therapy to other types of cancer.