DELIVERY OF BIOPOLYMERS USING CATIONIC PEPTIDES

本建议的长期目标是使用短，阳离子 肽作为递送生物聚合物的有效方法， 自身抗原、免疫显性肽和反义寡核苷酸 进入抗原呈递细胞和T淋巴细胞的细胞质， 诱导抗原特异性无反应性和/或影响分泌的 细胞因子作为实验性变态反应性脑脊髓炎（EAE）的治疗。 我们已经证明，对应于九个肽的肽的缀合 从HIV达特蛋白的氨基酸，到蛋白抗原， 它们被各种细胞快速吸收，并允许分子 进入MHC I类和II类生物合成途径， 其抗原性和免疫原性急剧增加。在 此外，为了将抗原递送至MHC I类和II类分子，我们 建议使用达特肽将PNA反义试剂转运到 细胞来调节炎症特征性的细胞因子产生。的 该项目中概述的研究旨在探索治疗 特异性抑制促炎细胞因子表达的潜力 通过引入转录激活因子NF-κ B的反义PNA 转化成小鼠T细胞克隆。如果成功，这些方法可以用作 在小鼠体内减少CNS炎症的治疗策略 对小鼠该项目有三个具体目标，1。证明 - 将达特肽缀合至完整的髓鞘碱性蛋白， 蛋白脂质蛋白及其免疫显性决定簇， 改变的肽配体显著增加了它们诱导 抗原特异性耐受性和/或改变细胞的细胞因子谱。 自身抗原特异性克隆，2.证明了达特结合蛋白 和肽进入抗原呈递细胞，如树突细胞， 小的静息B细胞，并探讨是否过继转移， 负载抗原的细胞更有效地诱导免疫刺激， 或耐受性，以及3. 表明多种促炎蛋白的表达 可以用HIV-1达特缀合的反义PNA显著降低 针对NF-κ B的65千道尔顿亚基， 当直接注射反义PNA构建体时， 感染EAE的啮齿动物

The long term objective of this proposal is to use short, cationic peptides as a efficient method of delivering biopolymers, such as intact autoantigens, immunodominant peptides, and antisense oligonucleotides into the cytoplasm of antigen presenting cells and T lymphocytes to induce antigen specific anergy and/or affect the profile of secreted cytokines as a therapy for experimental allergic encephalomyelitis (EAE). We have demonstrated that conjugation of a peptide, corresponding to nine amino acids from the HIV tat protein, to protein antigens results in their rapid uptake by a variety of cells and permits the molecules to enter both the MHC class I and ll biosynthetic pathways, which results in a dramatic increase in their antigenicity and immunogenicity. In addition, to delivering antigens to the MHC class I and ll molecules, we propose to use the tat peptide to transport PNA antisense reagents into cells to modulate cytokine production characteristic of inflammation. The studies outlined in this project seek to explore the therapeutic potential of specifically suppressing proinflammatory cytokine expression by introducing antisense PNA for the transcriptional activator NF-kappaB into murine T cell clones. If successful, these methods could be used as therapeutic strategies in mice in vivo to reduce inflammation in the CNS of mice. This project has three specific aims, 1. demonstrate that conjugation of the tat peptide to intact myelin basic protein and proteolipid protein as well as their immunodominant determinants and altered peptide ligands dramatically increases their ability to induce antigen specific tolerance and/or modify the cytokine profile of the autoantigen specific clones, 2. demonstrate that tat conjugated proteins and peptides enter antigen presenting cells, such as dendritic cells and small resting B cells, and explore whether adoptive transfer of the antigen loaded cells more efficiently induces either immune stimulation or tolerance than immunization with the peptides or proteins, and 3. demonstrate that the expression of a variety of proinflammatory proteins can be dramatically reduced with HIV-1 tat conjugated antisense PNA against the 65 kilodalton subunit of NF-kappaB in vitro and that the antisense PNA constructs can reduce inflammation when directly injected into rodents with EAE.