ANIONIC LIPOSOMES FOR GENE DELIVERY TO APCS

用于将基因递送至 APCS 的阴离子脂质体

• 批准号: 6074206
• 负责人: FRANK TAGLIAFERRI
• 金额: $ 10.7万
• 依托单位: VALENTIS, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6074206
• 关键词: CD14 molecule; antigen presenting cell; biotechnology; dendritic cells; flow cytometry; gene targeting; human tissue; laboratory mouse; liposomes; plasmids; protamines; reporter genes; technology /technique development; transfection

The goal of this proposal is to develop plasmid DNA delivery technology that will efficiently deliver antigen-encoding DNA to antigen presenting cells (APCs) in vivo. This should produce improved transfection of the targeted APCs in vivo and induction of an immune response to the encoded antigen. Plasmid DNA encoding reporter genes or antigens will be condensed, encapsulated, and analyzed for physicochemical properties (e.g. size, mass/charge ratio). The resulting complexes will be mixed with mouse splenocytes, peritoneal macrophages, isolated dendritic cells, and human PBMC preparations in vitro and analyzed for uptake and expression. Flow cytometry will be used to identify the cells that preferentially internalize and express the complexes. If preferential expression is noted in APCs, the ability of anti-CD14 antibodies, LPS, and apoptotic bodies to interfere with this uptake will be tested. Complexes encoding GFP, luciferase or ovalbumin will be injected into mice and analyzed for induction of an antigen specific immune response. If successful, this vehicle could be applied to the formulation of vaccines for infectious disease and cancer. PROPOSED COMMERCIAL APPLICATIONS: Packaging the plasmid DNA with protamine sulfate and anionic lipids is expected to generate a complex that is more resistant to degradation than naked DNA, more stable in storage than attenuated virus, and preferentially internalized by APCs through apoptosis receptors. Such complexes, encoding antigen alone or in combination with genes encoding costimulatory molecules or chemokines, have many potential applications in tumor and infectious disease vaccines. In vivo loading of APCs would be more practical than the ex vivo peptide pulsing of dendritic cells currently in clinical trial.

该提案的目标是开发质粒DNA递送技术，该技术将有效地将编码抗原的DNA递送到体内的抗原呈递细胞（APC）。这将产生体内靶向APC的改善的转染和对编码抗原的免疫应答的诱导。对编码报告基因或抗原的质粒DNA进行浓缩、包封，并分析理化性质（例如大小、质荷比）。将所得复合物与小鼠脾细胞、腹膜巨噬细胞、分离的树突状细胞和人PBMC制剂在体外混合，并分析其吸收和表达。流式细胞术将用于鉴定优先内化和表达复合物的细胞。如果在APC中观察到优先表达，则将检测抗CD14抗体、LPS和凋亡小体干扰这种摄取的能力。将编码GFP、荧光素酶或卵清蛋白的复合物注射到小鼠中并分析抗原特异性免疫应答的诱导。如果成功的话，这种载体可以应用于传染病和癌症疫苗的配制。拟议的商业应用：预期用硫酸鱼精蛋白和阴离子脂质包装质粒DNA会产生复合物，该复合物比裸DNA更耐降解，比减毒病毒更稳定储存，并且优先通过凋亡受体被APC内化。 这种复合物单独编码抗原或与编码共刺激分子或趋化因子的基因组合编码抗原，在肿瘤和感染性疾病疫苗中具有许多潜在的应用。APC的体内负载将比目前在临床试验中的树突状细胞的离体肽脉冲更实用。