Interaction of cationic lipids with dendritic cells

描述（由申请人提供）：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递送至树突细胞（DC），并产生针对表达该抗原的TC-1肿瘤细胞的有效CTL活性。在原代骨髓来源的DC（BMDC）中的信号转导研究表明，活性氧（ROS）被DOTAP刺激，这导致两种MAP激酶ERK和p38的磷酸化和激活。pERK激活导致BMDC产生和分泌有效的趋化因子CCL 2和CCL 4。P38激活导致IL-12的产生。通过Mek 1/2（磷酸化ERK的上游激酶）的特异性抑制剂抑制pERK活化，抑制CCL 2的产生，更重要的是，抑制DOTAP/E7的抗肿瘤活性。我们假设ERK和p38激活在DOTAP的佐剂活性中起关键作用，也许也是DOEPC。该项目将在目标1中研究ROS的信号传导及其导致CCL 2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，以确定适合疫苗制剂的更多活性物质。将合成各种DOTAP类似物并测试ROS活化和疫苗活性。最后，在目标3中，我们将尝试使用TC-1肿瘤作为人宫颈癌模型来提高抗肿瘤疫苗活性。一些配方改进策略将在模型中进行测试。此外，我们将测试通过靶向纳米颗粒制剂递送的针对VEGF和其他关键癌基因的siRNA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。该疫苗仅含有两种分子，抗原和脂质，并且是一种非常简单但在小鼠模型中具有高活性的疫苗。本项目将对脂质佐剂的活性及其作用机制进行研究，以寻找更有活性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.