FUNCTIONAL BIOMATERIALS FOR ANTISENSE DELIVERY TO ISLET

描述(由申请人提供)： 这项应用的目标是基于成功的初步研究结果，设计将反义质粒和反义寡核苷酸携带到胰岛β细胞的功能生物材料。设计的无毒阳离子聚合物载体与磺酰尿素(SU)或抗谷氨酸脱羧酶(AntiGAD)抗体的靶向部分偶联。这些具有功能和生物特异性的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，并防止由GAD抗原触发的自身免疫诱导的I型糖尿病。我们将用大鼠胰岛素启动子构建含有GAD-65和GAD-67基因的反义GAD真核表达载体，用于反义RNA的定点表达。合成抗GAD或SU共轭的聚乙二醇/聚乙烯亚胺(PEI)共聚物，并与其形成稳定的络合物。这些聚合物复合体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，这有望促进胰岛细胞的转染和表达。第二种方法也将被研究，包括利用酸不稳定的键将合成的反义寡核苷酸(ODN)直接化学结合到SU接枝的聚乙二醇或反义GAD接枝的聚乙二醇上。将阳离子融合寡肽-弱LAKA-LAKA-LAKH-LAKA-LKAC-EA(KALA)与所设计的ODN共轭聚合物形成稳定的胶束复合体。这种复合体通过内吞作用运输到细胞，并通过破坏酸性不稳定键在酸性内吞体内释放ODN。Kala的使用显著提高了转录水平。在利用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小鼠身上进行系统的体内评估。将确定生物分布、有效性、持续时间和最佳剂量给药。所获得的结果预计将支持拟议的应用于治疗早期I型糖尿病，目前尚无治疗方法。

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.