Engineered Outer Membrane Vesicles as DNA Vaccine Delivery Vehicles

工程化外膜囊泡作为 DNA 疫苗递送载体

• 批准号: 7015400
• 负责人: DAVID A PUTNAM
• 金额: $ 21.22万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7015400
• 关键词: DNA; bacteria; birth; cell membrane; gram negative bacteria; immune response; phagocytosis; plasmids; proteins; vector vaccine

DESCRIPTION (provided by applicant): BACKGROUND: The low immune response to DNA vaccines remains a challenge to their potential clinical use, and the development of new delivery strategies continues to be an area of significant effort. One clinically utilized strategy to enhance the potency of protein and polysaccharide vaccines is to deliver the antigens via bacterial "outer membrane vesicles", or OMVs, which are vesicles constitutively produced by gram-negative bacteria. OMVs are used by bacteria in nature as protein, and potentially DNA, delivery vehicles. The objective of this R21 exploratory research grant is to definitively determine if OMVs can be engineered to deliver plasmid-based DNA vaccines to mammalian antigen presenting cells. HYPOTHESIS: OMV characteristics can be engineered to enhance the delivery of plasmid-based DNA vaccines to mammalian antigen presenting cells. SPECIFIC AIMS: 1) To engineer, and quantitatively evaluate, the surface of plasmid-containing OMVs to maximize their non-specific binding to mammalian cell membranes, and 2) To engineer, and quantitatively evaluate, the surface of plasmid-containing OMVs to facilitate their release from acidic subcellular vesicles. The zeta potential of OMVs is approximately -40 mV which reduces their binding affinity to mammalian cell membranes. To increase the association of OMVs with mammalian cells, and increase the rate of internalization by non- specific endocytosis or phagocytosis, the surface of the OMVs will be engineered to possess sequences of polylysine to create OMVs with positive zeta potentials. Internalization of macromolecular structures by endocytosis/phagocytosis leads to their sequestration within acidic compartments and digestion by lysosomal enzymes. To facilitate the escape of OMVs from the lysosomal trafficking pathway, the surface of the OMVs will be engineered to possess sequences of polyhistidine to disrupt the membrane of acidic vesicles. Validation of the hypothesis will support the future engineering of OMVs with more intricate characteristics, such as cell specific ligands, fusogenic proteins and conformational protein adjuvants.

描述（由申请人提供）：背景：对DNA疫苗的免疫力低仍然是对其潜在临床使用的挑战，而新的分娩策略的发展仍然是巨大努力的领域。一种临床上利用的策略来增强蛋白质和多糖疫苗的效力，是通过细菌“外膜囊泡”或OMV传递抗原，或者是由革兰氏阴性细菌组成性产生的囊泡。细菌在自然界中用作蛋白质和潜在的DNA递送车。这项R21探索性研究赠款的目的是确定确定是否可以设计OMV以将基于质粒的DNA疫苗传递给哺乳动物抗原呈递细胞。假设：可以设计OMV特性以增强基于质粒的DNA疫苗向哺乳动物抗原呈递细胞的递送。具体目的：1）对含质粒的OMV的表面进行工程和定量评估，以最大化其非特异性与哺乳动物细胞膜的结合，以及2）对工程师进行工程，并进行定量评估，含有质粒的OMV表面以促进它们从酸性亚细胞囊泡中释放。 OMV的ZETA电位约为-40 mV，可降低其与哺乳动物细胞膜的结合亲和力。为了增加OMV与哺乳动物细胞的关联，并通过非特异性内吞和吞噬作用增加了内在化的速率，OMV的表面将被设计为具有多赖氨酸序列，以创建具有正Zeta电位的OMV。通过内吞和吞噬作用对大分子结构的内在化导致它们在酸性区室内隔离，并通过溶酶体酶消化。为了促进OMV从溶酶体运输途径中逃脱，OMV的表面将被设计为具有多组氨酸序列，以破坏酸性囊泡的膜。该假设的验证将支持具有更复杂特征的OMV的未来工程，例如细胞特异性配体，融合蛋白和构象蛋白辅助剂。