TARGETED CYTOSOLIC DELIVERY OF ANTISENSE OLIGONUCLEOTIDE

描述（来自申请人摘要的逐字）：基于核酸的药物具有 最近成为许多医学上最持久的 问题这些治疗剂和反义寡脱氧核苷酸（ODN） 特别是具有补充或取代常规 传统上基于小分子量药物的药物疗法。 它具有选择性下调一种基因表达水平的神奇能力， 特异性蛋白，反义ODN可以高度调节疾病状态， 专业的方式。增强ODN稳定性的最新努力和 有效ODN序列的选择方法和设计产生了ODN 这些分子一旦到达作用部位， 在细胞质或细胞核中。尽管有杰出的药理学 其全部潜力有待关键问题的解决。 药物的挑战，主要是由于低膜渗透性， ODN分子。与一般基因治疗所面临的困难类似， ODN药物疗法的成功依赖于以高浓度递送至特定细胞类型。 足够的浓度，然后有效的细胞摄取和运输到 细胞的细胞质。该建议采取两种主要方法来解决 这些交付问题。首先，使用含有鲎溶血素O（LLO）的脂质体， 传递系统将被用来克服细胞溶质的膜屏障， 交付. LLO是单核细胞增生李斯特菌的溶血素， 脂质体制剂是李斯特菌利用的机制，以逃避 内体/溶酶体降解途径并进入胞质溶胶。二是 携带ICAM 1或B7 - 1/2特异性反义ODN的LLO-脂质体将被靶向 通过将靶向基序，CTLA4Ig， 到脂质体上。CTLA4Ig，B7的高亲和力竞争性配体 APC表面上的分子，已被证明可以阻断必需的， CD28/B7介导的T细胞活化的共刺激信号。有效性 B7特异性反义ODN通过这种靶向胞质递送 将评估载体CTLA4Ig缀合的LLO-脂质体的有效性， 使用混合淋巴细胞反应抑制同种异体反应性免疫应答 比色法将采取几种方法来优化各种参数， 实现长循环和针对性的特殊平衡， 体内CTLA4Ig-脂质体。然后将结果扩展到体内小鼠 移植模型。T细胞共刺激因子的有效阻断 在多个级别上的信号，如由ODN递送系统所呈现的 本项目的特点和发展，是一个理想的模式， 免疫抑制治疗移植受体。发展这种 ODN的递送策略可以进一步推广到免疫应答之外 调节由特定蛋白质介导的多种疾病。

DESCRIPTION (Verbatim from Applicant's Abstract): Nucleic acid-based drugs have recently emerged as powerful solutions to many of medicine's most enduring problems. These therapeutic agents, and antisense oligodeoxynucleotides (ODN) in particular, possess enormous potential to complement or replace conventional pharmaceutical therapies based traditionally on small molecular weight drugs. With the uncanny ability to selectively downregulate the expression level of a specific protein, antisense ODN can modulate disease states in a highly specialized manner. Recent efforts to enhance ODN stability and advances in the selection method and design of effective ODN sequences have produced ODN molecules that are increasingly potent once delivered to their site of action in the cytosol or the nucleus. Despite the outstanding pharmacological characteristics, their full potential awaits resolution of critical pharmaceutical challenges, due primarily to the low membrane permeability of ODN molecules. Similar to the difficulties facing gene therapy in general, the success of ODN drug therapies relies on delivery to specific cell types at high enough concentrations, followed by efficient cellular uptake and transport into the cytoplasm of the cells. This proposal takes two main approaches to address these delivery problems. First, a listeriolysin O (LLO)-containing liposomal delivery system will be utilized to overcome the membrane barrier for cytosolic delivery. LLO, the hemolysin of Listeria monocytogenes, confers upon the lipsome formulation the mechanism that is utilized by Listeria to escape the endosomal/lysosomal degradation pathway and enter the cytosol. Second, the LLO-liposomes carrying ICAM1 or B7-1/2 specific antisense ODN will be targeted to antigen presenting cells (APC) by conjugating a targeting motif, CTLA4Ig, onto the liposomes. CTLA4Ig, a high affinity, competitive ligand for the B7 molecules on the surface of APC, has been shown to block the essential, CD28/B7-mediated co-stimulatory signal for T cell activation. The effectiveness of B7-specific antisense ODN delivered by this targeted cytosolic delivery vehicle, the CTLA4Ig-conjugated LLO-liposomes, will be assessed for efficient inhibition of alloreactive immune responses using the mixed lymphocyte reaction assay. Several approaches will be taken to optimize various parameters for achieving the special balance of long circulation and targeting of CTLA4Ig-liposomes in vivo. The results will then be extended to in vivo mouse models of transplantation. Efficient blockade of the T cell co-stimulatory signal at multiple levels, as rendered by the ODN delivery systsem characterized and developed in this project, is an ideal modality of immunosuppressive treatments for transplant recipients. Development of such a delivery strategy for ODN can be further generalized beyond immune response modulation to a wide range of diseases mediated by specific proteins.