Targeted Double Stranded mRNA Nanoparticles

项目摘要和摘要 该方案旨在开发靶向纳米颗粒，在肝脏中表达人因子 小鼠肝细胞。安全有效的静脉注射。纳米粒给药系统对肝脏的转染性研究 肝细胞表达分泌型hFVIII对治疗血友病A具有变革性作用。 对于质粒DNA的流体动力剂量，所有其他纳米颗粒递送系统目前也是 在肝脏中达到治疗水平的蛋白质表达效率低下，主要是由于无法 质粒DNA穿过未分裂的肝细胞的核膜。初步数据 证实了利用双链mRNAs可以实现高效表达。纳米颗粒 是使用一种新型的含有Lys-ACR残基的聚乙二醇肽与ds mRNA结合而产生的。这个 双链信使核糖核酸纳米颗粒进入胞浆绕过限制表达的主要屏障 质粒DNA的表达。该提案的第一个目标是通过增加 循环稳定性和表达的持久性。多价聚乙二醇肽将被开发出来 以更高的亲和力结合ds RNA，进一步提高静脉注射后ds mRNA纳米粒的稳定性。 给药。持续表达将通过发展自我放大的信使核糖核酸结构实现 目的是在胞浆中复制信使核糖核酸并扩展其表达。高效的肝细胞靶向 将使用高亲和力的三天线N-糖链连接到聚乙二醇肽来介导 通过去唾液酸糖蛋白受体将纳米颗粒内吞入肝细胞。强效膜 裂解肽蜂毒素将可逆地结合到双链mRNA上，以提供触发的mRNA释放到 细胞质。优化的双链信使核糖核酸纳米递送系统将用于表达人 因子VIII，目的是实现对小鼠血友病A的功能性纠正。成功者 靶向双链信使核糖核酸纳米粒高效转染肝细胞的研究 通过建立范式转换战略来推进纳米医学领域，以实现 在静脉注射后的非分裂细胞中的表达。给药。

Project Summary and Abstract This proposal aims to develop targeted nanoparticles to express human factor VIII in liver hepatocytes of mice. A safe and efficient i.v. dosed nanoparticle delivery system to transfect liver hepatocytes to express secreted hFVIII would be transformative for treating hemophilia A. Compared to hydrodynamic dosing of plasmid DNA, all other nanoparticle delivery systems are currently too inefficient to achieve therapeutic levels of protein expression in liver primarily due to the inability of plasmid DNA to traverse the nuclear membrane of non-dividing hepatocytes. The preliminary data establishes that efficient expression can be achieved using double stranded mRNA. Nanoparticles are generated using a novel PEG-peptide containing Lys-Acr residues that binds to ds mRNA. The delivery of ds mRNA nanoparticles to the cytosol circumvents the major barrier that limits expression of plasmid DNA. The first aim of the proposal will advance ds mRNA nanoparticles by increasing circulatory stability and persistence of expression. Multivalent PEG-peptides will be developed that bind ds RNA with higher affinity to further increase ds mRNA nanoparticles stability following i.v. dosing. Persistent expression will be achieved by developing self-amplifying mRNA constructs designed to replicate mRNA in the cytosol and extend its expression. Efficient hepatocyte targeting will be attained using a high-affinity triantennary N-glycan attached to the PEG-peptide to mediate nanoparticle endocytosis into hepatocytes via the asialoglycoprotein receptor. The potent membrane lytic peptide melittin will be reversible attached to ds mRNA to afford triggered release of mRNA into the cytosol. The optimized ds mRNA nanoparticle delivery system will be used to express human factor VIII with a goal of achieving functional correction of hemophilia A in mice. The successful development of targeted ds mRNA nanoparticles for efficient transfection of liver hepatocytes will advance the field of nanomedicine by establishing a paradigm changing strategy to achieve expression in non-dividing cells following i.v. dosing.