Non-Viral Delivery of DNA Vaccines to the Buccal Mucosa

描述（由申请人提供）：我们的目标是利用新的生物有机化合物介导口服递送来增强DNA疫苗的功效。口服疫苗接种主要保护个人免受口腔病原体的侵害，其中包括艾滋病逆转录病毒和伴随艾滋病的机会性病原体。口服疫苗接种也对通过其他途径感染的病原体提供一定的保护。在临床试验中，口服活疫苗一直比DNA疫苗更有效，但最近允许DNA递送到特定细胞的技术为DNA疫苗提供了新的可能性。在这里，我们建议测试口服DNA疫苗靶向递送到树突状细胞（DC）。采用新型糖多胺作为递送载体。DC是主要的抗原提呈细胞，它们表达丰富的细胞表面碳水化合物受体，如甘露糖受体和DC- sign。在我们的研究中使用的基于环糊精的糖多胺包括两个功能域：与DNA结合并形成复合物的多个氨基，以及结合细胞表面凝集素并将DNA复合物靶向到DC的糖配体。增强DC对DNA的摄取将增强DNA疫苗的免疫原性。细胞培养研究已经证明，甘露糖基化多胺显著增强质粒DNA进入DC的摄取。在这里，我们建议在体内测试靶向递送的有效性。我们假设针对树突状细胞表面碳水化合物受体的DNA疫苗将引发增强的细胞介导反应。为了验证这一假设，我们将采用lcmv感染的小鼠模型系统，已知该模型系统可引发强烈的细胞介导免疫反应。我们将有两个具体目标。在目标1中，我们将合成新的糖基化环糊精多胺（CDPA），并测试报告基因在树突状细胞培养中的摄取和表达。将甘露糖和寡甘露糖配体引入CDPA，分别靶向树突状细胞上的甘露糖受体和DC-SIGN。在目标2中，我们将测试不同配方的DNA疫苗口服接种后细胞介导免疫的强度。编码LCMV NP基因的质粒DNA将以裸DNA或与CDPA或糖基化CDPA复合物的DNA形式口服给药给小鼠。一旦我们找到一种最适合引发细胞介导免疫的配方，我们将用编码与艾滋病保护性免疫相关抗原的DNA取代NP基因（p27 gag）。我们将确定，在小鼠系统中，p27 DNA与我们的化合物复合物是否仍然可以引起高细胞介导的免疫。这些研究将有助于改进预防艾滋病及其伴随的机会性感染的口服疫苗。一旦我们在小鼠模型系统中优化了疫苗配方，我们将把靶向递送系统应用于猴子模型和人类临床试验。

DESCRIPTION (provided by applicant): Our goal is to enhance the efficacy of DNA vaccines using new bio-organic compounds to mediate oral deliver. Oral vaccination primarily protects an individual from oral pathogens, which include the AIDS retroviruses and opportunistic agents that accompany AIDS. Oral vaccination also confers some protection against pathogens that infect via other routes. Live oral vaccines have always been more effective than DNA vaccines in clinical trials, but recent technology allowing DNA delivery to specific cells provides new possibilities for DNA vaccines. Here we propose to test oral DNA vaccines for targeted delivery to dendritic cells (DC). using novel glyco-polyamines as the delivery vehicle. DC are the primary antigen-presenting cells and they express abundant cell surface carbohydrate receptors, such as mannose receptor and DC-SIGN. The cyclodextrin-based glyco-polyamines used in our studies consist of two functional domains: the multiple amino groups that bind and form complexes with DNA, and the sugar ligands that bind cell surface lectins and target the DNA complexes to DC. Enhancing the uptake of DNA by DC will enhance the immunogenicity of DNA vaccines. Cell culture studies have already demonstrated that mannosylated polyamines significantly enhance the uptake of plasmid DNA into DC. Here we propose to test the efficacy of targeted delivery in vivo. We hypothesize that DNA vaccines targeted to the cell surface carbohydrate receptors of dendritic cells will elicit enhanced cell-mediated responses. To test this hypothesis, we will employ the LCMV-infected murine model system that is known to elicit strong cell-mediated immune responses. We will have two specific aims. In aim 1, we will synthesize novel glycosylated cyclodextrinbased polyamines (CDPA) and test the uptake and expression of reporter genes in dendritic cell culture. Mannose and oligomannose ligands will be introduced into CDPA for targeting to mannose receptors and DC-SIGN on dendritic cells, respectively. In aim 2, we will test the magnitude of cell mediated immunity after oral inoculation with DNA vaccines in various formulations. Plasmid DNA encoding the LCMV NP gene will be orally delivered to mice as naked DNA or as DNA complexed with CDPA or glycosylated CDPA. Once we find a formulation that is optimum for eliciting cell-mediated immunity, we will replace the NP gene with DNA encoding an antigen associated with protective immunity in AIDS (p27 gag). We will determine, in the murine system, whether p27 DNA complexed with our compound can still elicit high cell-mediated immunity. These studies will lead to improved oral vaccines against AIDS and its attendant opportunistic infections. Once we have optimized the vaccine formulation in the murine model system, we will apply the targeted delivery system to monkey models and human clinic trials.