FUNCTIONAL BIOMATERIALS FOR ANTISENSE DELIVERY TO ISLET

用于向胰岛输送反义酶的功能性生物材料

• 批准号: 6703536
• 负责人: SUNG WAN KIM
• 金额: $ 31.62万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-15 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6703536
• 关键词: NOD mouse; antigen antibody reaction; antisense nucleic acid; bioengineering /biomedical engineering; biomaterial development /preparation; biomaterial evaluation; biomaterials; biotechnology; chemoprevention; complementary DNA; diabetes mellitus therapy; gene delivery system; gene expression; gene therapy; genetic promoter element; glutamate decarboxylase; insulin dependent diabetes mellitus; laboratory mouse; oligonucleotides; pancreatic islets; pharmacokinetics; plasmids; polyethylene glycols; sulfonylurea; tissue /cell culture; transfection; transfection /expression vector

DESCRIPTION (provided by applicant): The goal of this application is to design functional biomaterials to carry antisense plasmids and antisense oligonucleotides to islet beta cell based on results from successful preliminary studies. The designed nontoxic and cationic polymeric carriers are conjugated with a targeting moiety of sulfonylurea (SU) or antiglutamic acid decarboxylase (antiGAD) antibody. These functional and biospecific biomaterials will carry the antisense GAD plasmid to islet beta cells, where they will suppress GAD antigen production and prevent autoimmune induced type I diabetes, which is triggered by GAD antigen. We will construct antisense GAD plasmids containing cDNAs of GAD-65 and GAD-67 with rat insulin promoters for site specific expression of antisense in RNAs. AntiGAD or SU conjugated polyethene glycol (PEG)/polyethylene imine (PEI) copolymer will be synthesized that will form stable complexes with the plasmids. These polymer complexes will target islet beta cells through antigen GAD or SU receptors present in the islet cells, which are expected to enhance transfection and expression. A second approach will also be investigated which includes direct chemical binding of synthesized antisense oligonucleotide (ODN) to SU grafted PEG or antiGAD grafted PEG using an acid labile linkage. A cationic fusogenic oligopeptide, WEAK-LAKA-LAKA-LAKH-LAKA-LKAC-EA (KALA) will be incorporated to form a stable micelle complex with the designed ODN conjugated polymer. This complex traffics to the cell via endocytosis and releases ODN in the acidic endosome by breakdown of the acid labile bond. The use of KALA significantly enhances trancription. After extensive characterization and analysis of in vitro cellular uptake, bioactivity and transfection with the above designed systems, systematic in vivo evaluation will be carried out in NOD mice. Biodistribution, efficacy, duration and optimal dosed delivery will be determined. The obtained results are anticipated to support the proposed application to treat early stage type I diabetes, which currently has no method for treatment.

描述（由申请人提供）： 本申请的目标是根据成功的初步研究结果，设计功能性生物材料，将反义质粒和反义寡核苷酸携带至胰岛β细胞。设计的无毒阳离子聚合物载体与磺酰脲（SU）或抗谷氨酸脱羧酶（抗GAD）抗体的靶向部分缀合。这些功能性和生物特异性生物材料将携带反义GAD质粒到胰岛β细胞，在那里它们将抑制GAD抗原的产生并预防由GAD抗原引发的自身免疫诱发的I型糖尿病。我们将构建含有 GAD-65 和 GAD-67 cDNA 的反义 GAD 质粒以及大鼠胰岛素启动子，用于 RNA 中反义的位点特异性表达。将合成抗GAD或SU缀合的聚乙二醇(PEG)/聚乙烯亚胺(PEI)共聚物，其将与质粒形成稳定的复合物。这些聚合物复合物将通过胰岛细胞中存在的抗原 GAD 或 SU 受体靶向胰岛 β 细胞，预计将增强转染和表达。还将研究第二种方法，包括使用酸不稳定连接将合成的反义寡核苷酸（ODN）直接化学结合到SU接枝的PEG或抗GAD接枝的PEG上。阳离子融合寡肽 WEAK-LAKA-LAKA-LAKH-LAKA-LKAC-EA (KALA) 将与设计的 ODN 共轭聚合物形成稳定的胶束复合物。该复合物通过内吞作用运输至细胞，并通过酸不稳定键的分解在酸性内体中释放 ODN。 KALA 的使用显着增强了转录。通过上述设计的系统对体外细胞摄取、生物活性和转染进行广泛的表征和分析后，将在 NOD 小鼠中进行系统的体内评估。将确定生物分布、功效、持续时间和最佳剂量递送。所获得的结果预计将支持治疗早期 I 型糖尿病的拟议应用，目前尚无治疗方法。