Interaction of cationic lipids with dendritic cells

描述（申请人提供）：我们最近发现，最初作为转染试剂开发的阳离子脂质DOTAP和DOEPC可以将肽抗原E7递送至树突状细胞（DC），并对表达该抗原的TC-1肿瘤细胞产生强效CTL活性。原代骨髓源性 DC (BMDC) 中的信号转导研究表明，DOTAP 会刺激活性氧 (ROS)，从而导致两种 MAP 激酶 (ERK 和 p38) 的磷酸化和激活。 pERK 激活导致 BMDC 产生和分泌强效趋化因子 CCL2 和 CCL4。 P38 激活导致 IL-12 的产生。 Mek1/2（磷酸化 ERK 的上游激酶）的特异性抑制剂对 pERK 激活的抑制，抑制了 CCL2 的产生，更重要的是，抑制了 DOTAP/E7 的抗肿瘤活性。我们假设 ERK 和 p38 激活在 DOTAP（或许还有 DOEPC）的佐剂活性中发挥关键作用。该项目将在目标 1 中研究 ROS 信号传导及其导致 CCL2 和 IL-12 产生的下游事件。在目标 2 中，我们将对阳离子脂质进行广泛的结构-活性关系研究，以确定适合疫苗配方的更多活性物种。将合成各种 DOTAP 类似物并测试 ROS 激活和疫苗活性。最后，在目标3中，我们将尝试使用TC-1肿瘤作为人类宫颈癌模型来提高抗肿瘤疫苗活性。许多配方改进策略将在模型中进行测试。此外，我们将测试靶向纳米颗粒制剂递送的 siRNA 针对 VEGF 和其他关键癌基因的活性，以增强 DOTAP/E7 疫苗的抗肿瘤活性。该研究的目标是开发化学性质明确且有效的阳离子脂质，用于治疗癌症的疫苗。 公共卫生相关性：该项目的目标是开发一种有效的宫颈癌治疗疫苗。该疫苗仅包含两种分子，即抗原和脂质，是一种非常简单但在小鼠模型中具有高活性的疫苗。项目将研究脂质佐剂的活性和作用机制，以寻找活性更高的脂质和更有效的疫苗。

DESCRIPTION (provided by applicant): We have recently discovered that the cationic lipids DOTAP and DOEPC, originally developed as transfection reagents, can deliver a peptide antigen E7 to the dendritic cells (DC) and illicit a potent CTL activity against TC-1 tumor cells expressing the antigen. Signal transduction studies in the primary bone marrow-derived DC (BMDC) indicate that reactive oxygen species (ROS) is stimulated by DOTAP which leads to the phosphorylation and activation of two MAP kinases, ERK and p38,. pERK activation leads to the production and the secretion of potent chemokines, CCL2 and CCL4, by BMDC. P38 activation leads to the production of IL-12. Inhibition of pERK activation by specific inhibitors of Mek1/2, the upstream kinase which phosphorylates ERK, inhibited CCL2 production and, more importantly, the antitumor activity of DOTAP/E7. We hypothesize that ERK and p38 activation play a key role in the adjuvant activity of DOTAP, perhaps also DOEPC. The project will study in aim 1 the signaling of ROS and its downstream events leading to the production of CCL2 and IL-12. In aim 2, we will perform an extensive structure-activity relationship study of the cationic lipid to identify more active species that are suitable for vaccine formulation. Various DOTAP analogs will be synthesized and tested for ROS activation and vaccine activity. Finally in aim 3, we will attempt to improve the anti-tumor vaccine activity using the TC-1 tumor as a model for human cervical cancer. A number of formulation improvement strategies will be tested in the model. Furthermore, we will test the activity of siRNA against VEGF and other key oncogenes delivered by a targeted nanoparticle formulation to enhance the anti- tumor activity of the DOTAP/E7 vaccine. The goal of the study is to develop chemically well defined and potent cationic lipids for therapeutic vaccines against cancer. PUBLIC HEALTH RELEVANCE: The goal of the project is to develop an effective therapeutic vaccine for cervical cancer. The vaccine contains only two molecules, the antigen and a lipid, and is a very simple but highly active vaccine in a mouse model. Project will study the activity and the mechanism of the lipid adjuvant, in order to find more active lipid and more efficacious vaccine.