Development of a Gene and Oligonucleotide Delivery System

 描述（由申请人提供）：通过反义寡核苷酸（ODNS）和遗传药物（统称为G-MEDS）敲低靶基因有望用于多种治疗。然而，将有效量的ODNS递送到特定细胞已被证明是具有挑战性的。我们在这里提出了一种新的方法，涉及包膜病毒样颗粒（EVLPs）的ODN交付。这些递送剂通过纯G-MEDS和纯病毒衣壳蛋白（CP）体外自组装成病毒样纳米颗粒（VLP）来制备。正如我们已经证明的那样，衣壳保护内容物，但能够在哺乳动物细胞的细胞质中放弃其内容物。然后，高度单分散的颗粒被脂质双层包封，所述脂质双层可以抑制VLP的免疫原性并且能够被官能化以供目标哺乳动物细胞靶向和摄取。 在初步实验中，我们已经证明了我们使用豇豆褪绿斑驳病毒的CP和血管内皮生长因子（VEGF）的模型反义ODN制备EVLP的能力，VEGF是一种在许多癌细胞中过表达的蛋白质，以刺激血管生成，促进低氧条件下的肿瘤存活。我们建议优化组装并用表皮生长因子（EGF）使脂质双层功能化，表皮生长因子（EGF）结合在癌细胞，特别是乳腺癌细胞上过度表达的EGF受体。EVLP的有效性将通过测定培养的乳腺癌细胞中VEGF分泌的减少来证明。

 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.