Non-Viral Vectors for Liver Gene Transfer

描述(由申请人提供)： 肝脏是基因治疗的重要靶器官，因为它在血清蛋白的代谢和产生中起着核心作用。非病毒载体载体DNA被认为是将基因输送到肝脏最简单、最安全的方法。最近，人们已经证明，通过手动按摩肝脏(MML)，可以将质粒DNA导入肝脏。该方法是非侵入性的，在接受治疗的动物身上不会产生任何可测量的毒性。此外，基因表达水平足够高，足以在疾病肝脏模型中产生治疗效果。在这个提议中，通过回答特定的问题来阐明MML将DNA转移到肝细胞的机制：1)MML是否短暂地扩大了肝窦内皮细胞的窗孔，允许DNA到达肝细胞？2)MML是否导致肝内皮细胞回缩，开放肝内皮细胞间的紧密连接，暴露肝细胞？为了提高基因转移效率，将开发一种良好的非病毒载体用于MML基因转移，其中包括合成新的载体和构建新的载体。这种载体的设计是基于这样一个观察，即如果增加质粒DNA在肝脏中的保留时间和密度，使用MML的基因转移效率可以进一步提高。载体将是PNA-PEG-Gal，其中多肽核酸(PNA)将与聚乙二醇(PEG)偶联，并将目标配体半乳糖(Gal)附加到聚乙二醇远端。PNA-聚乙二醇半乳糖载体旨在通过受体介导的结合机制将质粒DNA携带到肝细胞中。这种策略使用半乳糖部分作为肝细胞的定位装置，并将PNA作为载体结合序列-特异性地与DNA结合。结果表明，质粒DNA在肝脏中的滞留时间和密度均有所增加。还将构建含有荧光素酶报告基因或羟基酶(PAH)基因的质粒，带有Epstein Barr病毒(EBV)元件/或PBS-HCRHPI-A(肝脏特异性基因表达盒)，以及具有与PNA杂交的特定序列的多个PNA结合元件。PAH基因转移后对苯丙酮尿症(PKU)小鼠代谢性疾病的治疗效果将使用本方案中的方法进行检测。

DESCRIPTION (provided by applicant): The liver is an important target organ for gene therapy because it plays a central role in the metabolism and production of serum proteins. Nonviral vector plasmid DNA has been considered as the simplest and safest method to delivery gene to the liver. More recently, it has been demonstrated that plasmid DNA could be transfected to the liver by manually massaging the liver (MML). The method is non-invasive and does not induce any measurable toxicity in the treated animal. Furthermore, the level of gene expression is high enough to produce therapeutic effects in a diseased liver model. In this proposal, the mechanisms underlying the DNA transfer into hepatocytes by MML will be elucidated by answering specific questions: 1) Does MML transiently enlarge the fenestrae in the sinusoid endothelial cells of the liver allowing DNA to reach the hepatocytes? And 2) Does MML induce the retraction of liver endothelial cells and open the tight junctions between the liver endothelial cell, exposing the hepatocytes? To increase gene transfer efficiency, a favorable non-viral vector for MML gene transfer will be developed, which will include the synthesis of a new carrier and construction of new plasmids. The design of this vector is based on an observation that gene transfer efficiency using MML could be further enhanced if the retention time and density of plasmid DNA in the liver are increased. The carrier will be PNA-PEG-Gal, in which peptide nucleic acid (PNA) will be conjugated with polyethylene glycol (PEG), and a target ligand, galactose (Gal), appended to the distal end of PEG. The PNA-PEG-galactose carrier is designed to carry plasmid DNA to hepatocytes through receptor-mediated binding mechanism. This strategy employs galactose moieties as homing devices for hepatocytes, and a PNA as a carrier to bind sequence-specifically to DNA. As a result, the retention time and density of plasmid DNA in the liver could be increased. The plasmids, containing either a luciferase reporter or hydroxylase (PAH) genes, with an Epstein Barr virus (EBV) element/or a pBS-HCRHPI-A (liver-specific gene expression cassette, and multiple PNA-binding elements that have specific sequences for hybridization with PNA will also be constructed. The therapeutic effects on the metabolic disease on phenylketonuria (PKU) mice, will be examined after the PAH gene transfer using the approach in this proposal.