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 最近出现了许多最持久的医学问题的有效解决方案 有问题。这些治疗药物和反义寡核苷酸(ODN) 特别是，它具有巨大潜力来补充或取代常规 传统上基于小分子药物的药物疗法。 有一种神奇的能力选择性地下调一种基因的表达水平 特异性蛋白质，反义ODN可以高度调控疾病状态 专业的态度。最近加强网上解决稳定性的努力和在以下方面的进展 产生ODN的有效ODN序列的选择方法和设计 一旦被送到它们的作用部位，分子就会变得越来越强大 在胞浆或细胞核内。尽管有杰出的药理作用 特征，它们的全部潜力等待关键问题的解决 药物方面的挑战，主要是由于药物的膜渗透性低 ODN分子。与基因治疗普遍面临的困难类似， ODN药物疗法的成功依赖于将药物输送到特定类型的细胞 足够的浓度，然后是有效的细胞摄取和转运 细胞的细胞质。这项提议主要采取两种方法来解决 这些送货问题。首先，一种含有李斯特溶素O(LLO)的脂质体 将利用输送系统来克服细胞质的膜屏障 送货。LLO是单核细胞增多性李斯特氏菌的溶血素，可作用于 脂体制剂李斯特氏菌用来逃避 胞内体/溶酶体降解途径，进入胞浆。第二， 携带ICAM1或B7-1/2特异性反义ODN的LIO脂质体将成为靶向 通过结合靶向基序CTLA4Ig， 放到脂质体上。CTLA4Ig，B7的高亲和力、竞争性配体 APC表面的分子，已被证明能阻止必要的， CD28/B7介导的T细胞活化共刺激信号其有效性 靶向胞浆递送的B7特异性反义ODN CTLA4Ig偶联LLO脂质体载体将被评估为有效 用混合淋巴细胞反应抑制同种异体免疫反应 化验。将采取几种方法来优化各种参数 实现长期流通性与针对性的特殊平衡 CTLA4Ig-脂质体在体内。然后将结果扩展到活体小鼠身上 移植的模式。有效阻断T细胞共刺激作用 由ODN交付系统提供的多个级别的信号 在这个项目中表现和开发的，是一种理想的 移植受者的免疫抑制治疗。开发这样的一种 ODN的递送策略可以进一步推广到免疫应答之外 通过特定的蛋白质对多种疾病进行调节。

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.