TARGETED CYTOSOLIC DELIVERY OF ANTISENSE OLIGONUCLEOTIDE

反义寡核苷酸的靶向胞质递送

• 批准号: 6732065
• 负责人: KYUNG-DALL LEE
• 金额: $ 25.97万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2006-02-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6732065
• 关键词: CD28 molecule; animal genetic material tag; antigen presenting cell; antisense nucleic acid; biochemistry; bioengineering /biomedical engineering; biotechnology; biotherapeutic agent; cell adhesion molecules; cytoplasm; fluorescence microscopy; gene delivery system; gene expression; gene targeting; hemolysin; immunoconjugates; immunosuppression; intracellular transport; laboratory mouse; liposomes; mixed lymphocyte reaction test; oligonucleotides; surface antigens; tissue /cell culture; transplantation

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.

描述（逐字研究来自申请人的摘要）：基于核酸的药物具有 最近出现了许多医学最持久的强大解决方案 问题。这些治疗剂和反义寡脱氧核苷酸（ODN） 特别是，具有补充或替代常规的巨大潜力 传统上基于小分子量药物的药物疗法。 具有选择性下调A的表达水平的不可思议的能力 特异性蛋白质，反义ODN可以在高度调节疾病状态 专业方式。最近为增强ODN稳定性和进步的努力 有效ODN序列的选择方法和设计产生了ODN 一旦传递到其作用部位，越来越有效的分子 在细胞质或细胞核中。尽管有出色的药理 特征，他们的全部潜力等待着解决关键的解决 药物挑战，主要是由于低膜通透性的 ODN分子。类似于基因疗法所面临的困难， ODN药物疗法的成功依赖于在高处的特定细胞类型的递送 足够的浓度，然后有效地进行蜂窝摄取和运输到 细胞的细胞质。该建议采用两种主要方法来解决 这些交付问题。首先，含有含脂质体的李斯特氏蛋白酶蛋白O（LLO） 递送系统将用于克服胞质的膜屏障 送货。李斯特菌单核细胞增生李斯特菌的血解素LLO，同意 Lipsome配方李斯特菌利用的机制逃脱 内体/溶酶体降解途径并进入细胞质。第二， 携带ICAM1或B7-1/2特异性反义ODN的LLO-脂质体将被靶向 通过结合靶标基序Ctla4ig，以抗原呈递细胞（APC） 到脂质体上。 Ctla4ig，高亲和力，B7的竞争配体 APC表面上的分子已被证明可以阻止必需的， 用于T细胞激活的CD28/B7介导的共刺激信号。有效性 该靶向胞质输送提供的B7特异性反义ODN 将评估车辆，CTLA4IG偶联的LLO-脂质体，以提高效率 使用混合淋巴细胞反应抑制同种异体免疫反应 测定。将采用几种方法来优化各种参数 实现长流通和靶向的特殊平衡 ctla4ig脂质体在体内。然后将结果扩展到体内鼠标 移植模型。 T细胞共刺激性的有效封锁 由ODN输送系统呈现的多个级别的信号 在这个项目中表征和开发，是 移植受者的免疫抑制治疗。这样的发展 ODN的输送策略可以进一步推广到免疫反应之外 调节特定蛋白质介导的多种疾病。