Interaction of cationic lipids with dendritic cells

描述(申请人提供)：我们最近发现了阳离子脂类DOTAP和DOEPC，最初是作为转染剂开发的，可以将多肽抗原E7传递到树突状细胞(DC)，并对表达该抗原的TC-1肿瘤细胞产生强大的CTL活性。原代培养的骨髓来源DC(BMDC)的信号转导研究表明，DOTAP刺激细胞内的ROS，导致ERK和p38两个MAP激酶的磷酸化和激活。PERK激活导致BMDC产生和分泌强大的趋化因子CCL2和CCL4。P38的激活导致IL-12的产生。用MEK1/2的特异性抑制剂抑制PERK的激活，会抑制CCL2的产生，更重要的是，抑制DOTAP/E7的抗肿瘤活性。我们推测ERK和p38的激活在DOTAP，可能也包括DOEPC的佐剂活性中起关键作用。该项目将在目标1中研究ROS的信号及其下游导致CCL2和IL-12产生的事件。在目标2中，我们将对阳离子脂类进行广泛的构效关系研究，以确定更多适合疫苗配方的活性物种。各种DOTAP类似物将被合成并测试ROS激活和疫苗活性。最后，在目标3中，我们将尝试使用TC-1肿瘤作为人宫颈癌的模型来提高抗肿瘤疫苗的活性。一些配方改进策略将在模型中进行测试。此外，我们将测试针对血管内皮生长因子和其他关键癌基因的siRNA的活性，通过靶向纳米颗粒配方来增强DOTAP/E7疫苗的抗肿瘤活性。这项研究的目标是开发化学上定义明确和有效的阳离子脂类，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性疫苗。该疫苗只包含两个分子，即抗原和脂质，在小鼠模型中是一种非常简单但活性很高的疫苗。本项目将对脂质佐剂的活性和作用机制进行研究，以期找到更具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.