Targeted Double Stranded mRNA Nanoparticles

项目概要和摘要 该提案旨在开发靶向纳米颗粒以在肝脏中表达人因子VIII 小鼠肝细胞。一种安全有效的静脉给药纳米粒给药系统用于肝脏切除 表达分泌的hFVIII的肝细胞对于治疗血友病A将是变革性的。相比 对于质粒DNA的流体动力学定量给药，所有其它纳米颗粒递送系统目前也是如此。 在肝脏中不能有效地达到治疗水平的蛋白质表达，主要是由于 质粒DNA穿过非分裂肝细胞的核膜。初步数据 确立了使用双链mRNA可以实现有效表达。纳米颗粒 使用一种新的含有Lys-Acr残基的PEG-肽与ds mRNA结合。的 将ds mRNA纳米颗粒递送到胞质溶胶绕过了限制表达的主要屏障 质粒DNA。该提案的第一个目标将通过增加 循环稳定性和表达持久性。将开发多价PEG肽， 以更高的亲和力结合ds RNA，以进一步增加静脉内给药后的ds mRNA纳米颗粒稳定性。 剂量。通过开发自扩增mRNA构建体， 设计用于在胞质溶胶中复制mRNA并延长其表达。高效肝细胞靶向 将使用连接到PEG-肽的高亲和力三触角N-聚糖来介导 纳米颗粒通过脱唾液酸糖蛋白受体内吞入肝细胞。有效的膜 裂解肽蜂毒肽将可逆地连接到dsmRNA上， 细胞质优化的ds mRNA纳米粒递送系统将用于表达人 因子VIII，目的是在小鼠中实现血友病A的功能矫正。成功 开发用于有效转染肝细胞的靶向dsmRNA纳米颗粒将 通过建立一个范式改变战略，以实现推进纳米医学领域 i. v.给药后在非分裂细胞中的表达。

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.