Development of a Gene and Oligonucleotide Delivery System

基因和寡核苷酸递送系统的开发

• 批准号: 8981573
• 负责人: Gary Fujii
• 金额: $ 32.29万
• 依托单位: MOLECULAR EXPRESS, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981573
• 关键词: Antisense Oligonucleotides; Binding; Biological Assay; Breast Cancer Cell; Capsid; Capsid Proteins; Cell Line; Cell membrane; Cells; Cellular Assay; Cytoplasm; DNA; Development; Effectiveness; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Fluorescence Microscopy; Gene Delivery; Gene Targeting; Genes; Genetic; Genetic Medicine; Healthcare; Housing; Hypoxia; Hypoxia Inducible Factor; In Vitro; Incubated; Ionic Strengths; Label; Length; Ligands; Lipid Bilayers; Lipids; Literature; MDA MB 231; Mammalian Cell; Measurement; Medical; Medicine; Methods; Modeling; Mono-S; Oligonucleotides; Organ; Oxygen; Phospholipids; Plant Viruses; Plants; Polymers; Production; Proteins; RNA; Relative (related person); Small Interfering RNA; System; Technology; Testing; Tissues; Toxic effect; Vascular Endothelial Growth Factors; Viral; Virus; Virus-like particle; Work; angiogenesis; base; cancer cell; cowpea chlorotic mottle virus; immunogenicity; interest; malignant breast neoplasm; nanoparticle; novel; novel strategies; particle; public health relevance; reconstitution; research study; self assembly; targeted delivery; tumor; uptake; vector

 DESCRIPTION (provided by applicant): The knockdown of targeted genes by anti-sense oligonucleotides (ODNS) and genetic medicines (collectively, G-MEDS) holds promise for a variety of therapies. The delivery of effective quantities of ODNS to specific cells, however, has proved to be challenging. We propose here a novel approach to ODN delivery that involves enveloped virus-like-particles (EVLPs). These delivery agents are prepared by the in vitro self- assembly of pure G-MEDS and pure viral capsid protein (CP) into virus-like nanoparticles particles (VLPs). The capsid protects the contents yet, as we have demonstrated, is capable of giving up its contents in the cytoplasm of mammalian cells. The particles, which are highly mono-disperse, are then enveloped by lipid bilayers that can suppress the immunogenicity of the VLPs and are capable of being functionalized for targeting and uptake by mammalian cells of interest. In preliminary experiments, we have demonstrated our ability to prepare EVLPs using the CP of the cowpea chlorotic mottle virus and a model antisense ODN for vascular endothelial growth factor (VEGF), a protein over-expressed in many cancer cells to stimulate angiogenesis a facilitate tumor survival under low- oxygen conditions. We propose to optimize the assembly and to functionalize the lipid bilayers with epidermal growth factor (EGF), which binds the EGF receptor that is over-expressed on cancer cells, especially those of breast cancer. The effectiveness of the EVLP will be demonstrated by assaying the reduction in the secretion of VEGF in cultured breast cancer cells.

 描述(申请人提供)：通过反义寡核苷酸(ODN)和基因药物(统称为G-meds)敲除靶基因有望实现多种治疗方法。然而，向特定细胞提供有效数量的ODN已被证明是具有挑战性的。我们在这里提出了一种新的ODN递送方法，它涉及到被包裹的病毒样颗粒(EVLP)。这些递送剂是通过将纯G-meds和纯病毒衣壳蛋白(CP)在体外自组装成病毒样纳米颗粒(VLP)而制备的。正如我们已经证明的那样，衣壳可以保护内容物，但它能够释放哺乳动物细胞细胞质中的内容物。这些颗粒是高度单分散的，然后被脂双层包裹，脂双层可以抑制VLP的免疫原性，并能够被靶向和被感兴趣的哺乳动物细胞摄取。在初步实验中，我们已经证明了我们能够利用豌豆褪绿斑驳病毒的CP和血管内皮生长因子(VEGF)的模型反义ODN来制备EVLP。血管内皮生长因子是一种在许多癌细胞中过表达的蛋白质，可以刺激血管生成，促进肿瘤在低氧条件下的生存。我们建议用表皮生长因子(EGF)来优化组装和功能化脂质双层，EGF与癌细胞，特别是乳腺癌细胞上过度表达的EGF受体结合。EVLP的有效性将通过检测培养的乳腺癌细胞分泌血管内皮生长因子的减少来证明。