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中心作用。非病毒载体质粒DNA被认为是最简单、最安全的基因转移方法。最近，已经证明可以通过手动按摩肝脏（MML）将质粒DNA转染到肝脏。该方法是非侵入性的，并且在治疗的动物中不会引起任何可测量的毒性。此外，基因表达的水平足够高以在患病的肝脏模型中产生治疗效果。在这个提议中，MML将DNA转移到肝细胞的机制将通过回答特定的问题来阐明：1）MML是否瞬时扩大肝窦内皮细胞中的窗孔，允许DNA到达肝细胞？2）MML是否诱导肝内皮细胞回缩，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，将开发用于MML基因转移的有利的非病毒载体，这将包括新载体的合成和新质粒的构建。该载体的设计是基于这样的观察，即如果增加肝脏中质粒DNA的保留时间和密度，则可以进一步增强使用MML的基因转移效率。载体将是PNA-PEG-Gal，其中肽核酸（PNA）将与聚乙二醇（PEG）缀合，并且靶配体半乳糖（Gal）附加到PEG的远端。PNA-PEG-半乳糖载体设计用于通过受体介导的结合机制将质粒DNA携带至肝细胞。该策略采用半乳糖部分作为肝细胞的归巢装置，并使用PNA作为载体以序列特异性地结合DNA。结果，质粒DNA在肝脏中的保留时间和密度可以增加。还将构建含有荧光素酶报告基因或羟化酶（PAH）基因、具有爱泼斯坦巴尔病毒（EBV）元件/或pBS-HCRHPI-A（肝特异性基因表达盒）和具有与PNA杂交的特异性序列的多个PNA结合元件的质粒。将PAH基因导入苯丙酮尿症（PKU）小鼠体内，观察其对代谢性疾病的治疗效果。

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.