Protein-DNA Drug Carriers for Tumor Targeting

用于肿瘤靶向的蛋白质-DNA 药物载体

• 批准号: 7645497
• 负责人: LALI K MEDINA-KAUWE
• 金额: $ 27.23万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7645497
• 关键词: Ablation; Affect; Alternative Therapies; Base Pairing; Binding; Biodistribution; Biological Assay; Blocking Antibodies; Blood; Breast Cancer Cell; Breast Cancer Treatment; Cancer cell line; Capsid; Capsid Proteins; Cell Culture Techniques; Cell Death; Cell Fractionation; Cell Line; Cells; Chemicals; Chimeric Proteins; Confocal Microscopy; Cytoplasm; DNA; DNA Binding; Development; Diffuse; Dose; Doxorubicin; Drug Carriers; Drug Delivery Systems; Drug Kinetics; ERBB2 gene; Engineering; Epidermal Growth Factor Receptor; Figs - dietary; Fluorometry; Growth; Heregulin; Human; Image; Immune; Immunofluorescence Immunologic; In Vitro; Individual; Intercalating Agents; Length; Ligands; Link; Malignant Neoplasms; Mammary Neoplasms; Measures; Membrane; Metabolic; Methods; Modeling; Modification; Molecular; Mus; Normal Cell; Nuclear Pore; Nucleic Acids; Nude Mice; Oligonucleotides; Pathway interactions; Patients; Penetration; Pharmaceutical Preparations; Polylysine; Proteins; Public Health; Recombinant Fusion Proteins; Recombinant Proteins; Research Project Grants; Series; Serum; Signal Transduction; Specificity; System; Testing; Therapeutic; TimeLine; Toxic effect; Toxicity Tests; Treatment Efficacy; Tumor Volume; Ursidae Family; Variant; Viral; Viral Proteins; attenuation; base; cancer cell; cancer type; conventional therapy; cytotoxicity; design; dosage; gene therapy; immunogenicity; in vivo; inhibitor/antagonist; intercalation; malignant breast neoplasm; mortality; mouse model; neoplastic cell; novel therapeutics; overexpression; plasmid DNA; public health relevance; receptor; receptor binding; response; success; therapeutic target; trafficking; tumor; uptake

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.

描述（由申请人提供）：我们将测试这一假设，即源自细胞穿透性病毒衣壳壳的独特重组融合蛋白可以通过微小的核酸载体转运DNA嵌入药物，并将此类药物靶向特定的肿瘤细胞，从而导致肿瘤-特异性递送和细胞死亡。为了测试这种新型疗法的可行性，我们选择 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) 2 亚基，是乳腺癌的一个重要子集，这些乳腺癌难以接受标准治疗方法，并且死亡率很高。我们将利用天然配体-受体相互作用诱导的快速内吞摄取和病毒蛋白的膜穿透活性，将药物递送到癌细胞中并从内部诱导细胞毒性。该项目的具体目标是测试以下假设： 1. 关键分子基序增强修饰衣壳蛋白在体外和体内的细胞靶向和渗透。将生成调蛋白靶向蛋白 HerPBK10 的一系列变体，并在一组 HER2+ 和 HER2- 乳腺癌细胞系和原代细胞以及表达不同的 HER 亚基水平。免疫荧光和共聚焦显微镜将用于分析摄取和运输，亚细胞分级将用于确认和量化结果。亚细胞标记和运输抑制剂将有助于确定摄取途径。最后，将测试这些变体的体内肿瘤靶向性。产生最佳靶向和吸收的变体将被纳入后续目标中提出的生物结合物中。 2. 修饰的衣壳蛋白与双链寡核苷酸 (ds-oligo) 和 DNA 嵌入剂组装，形成在体外靶向 HER2+ 乳腺癌细胞的非共价生物缀合物。从目标 1 中选择的最佳蛋白质变体将与不同长度的双链寡核苷酸和 DNA 嵌入剂结合形成非共价缀合物。我们将使用紫外/可见吸光度和荧光测定法来评估不同储存条件下和血清中的组装参数和稳定性。我们将使用代谢测定在单独和混合的 HER2+ 和 HER2- 细胞培养物中测试每个组件的靶向毒性，并通过与游离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。这些结果将决定目标 3 中测试的生物结合物的最佳组装参数和剂量。 3. 由修饰的衣壳蛋白、ds-oligo 和 DNA 嵌入剂组成的优化生物结合物靶向 HER2+ 肿瘤细胞并赋予体内治疗功效。我们将利用HER2+乳腺癌裸鼠模型建立体内剂量曲线，以确定实现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线。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缀合物水平来确定。我们将确定全身递送的缀合物的治疗效果，并测量肿瘤体积以确定生长抑制。最后，将使用从经处理的免疫能力小鼠采集的血液的吸光度测定来确定免疫原性。公共健康相关性：该研究项目与公共健康相关，因为它将导致开发出一种专门针对 HER2+ 乳腺癌的新型疗法。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+ 乳腺癌对传统疗法的反应不佳，这种替代疗法可以为乳腺癌治疗做出重大贡献，并且可以进行修改以针对其他类型的癌症进行靶向治疗。