FUNCTIONAL BIOMATERIALS FOR ANTISENSE DELIVERY TO ISLET

描述（由申请人提供）： 本申请的目的是基于成功的初步研究结果，设计功能性生物材料以将反义质粒和反义寡核苷酸携带到胰岛β细胞。所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（antiGAD）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抗原产生并预防由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GAD-65和GAD-67 cDNA的反义GAD质粒，用于RNA中反义核酸的位点特异性表达。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，预期其增强转染和表达。还将研究第二种方法，其包括使用酸不稳定键将合成的反义寡核苷酸（ODN）直接化学结合到SU接枝的PEG或antiGAD接枝的PEG。阳离子融合寡肽WEAK-LAKA-LAKA-LAKH-LAKA-LKAC-EA（KALA）将与所设计的ODN缀合聚合物结合以形成稳定的胶束复合物。该复合物通过内吞作用运输到细胞，并通过酸不稳定键的断裂在酸性内体中释放ODN。KALA的使用显著增强了转录。在对体外细胞摄取、生物活性和用上述设计的系统转染进行广泛表征和分析之后，将在NOD小鼠中进行系统的体内评价。将确定生物分布、疗效、持续时间和最佳剂量递送。预计所获得的结果将支持用于治疗早期I型糖尿病的拟议应用，目前还没有治疗方法。

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.