Non-Viral Gene Targeting to the Brain

描述(申请人提供)：许多慢性脑部疾病可以用基因疗法来治疗。然而，病毒载体不会穿过脑毛细血管壁，在体内形成血脑屏障(BBS)。这项拟议的研究提出了一种新的、非病毒的、跨血管的脑基因治疗方法，其中编码治疗性基因的表达质粒被包裹在聚乙二醇化的免疫脂质体或PILs中。PILs是一种纳米容器，靶向跨越血脑屏障和脑细胞膜(BCM)，带有针对转铁蛋白受体(TFR)的单抗(MAb)。TFR的单抗作为一个分子特洛伊木马，通过进入BBB和BCM内的内源性TFR介导的运输系统，将基因运送到BBB和BCM。因为基因可以穿过血脑屏障，所以给药途径是非侵入性的，只需要静脉给药。跨血管传递途径使基因能够分布到整个大脑体积。质粒DNA没有整合到宿主基因组中，这被认为是有利的，因为没有插入突变的风险。外源基因的持续表达是核DNA酶降解质粒的函数。在帕金森氏病(PD)的实验模型中，通过一次静脉注射携带TH表达质粒的PILS就有可能完全恢复纹状体酪氨酸羟化酶(TH)的活性。这种方法的限制因素是基因表达的持续时间有限。TH表达载体导入后，脑组织TH酶活性在单次静脉注射后的半衰期为6天。有证据表明，通过大脑传递TH基因的染色体衍生形式，可能会有更长时间的基因表达。与基因的cDNA形式相比，外源基因的基因组形式吸引形成微小染色质结构的核蛋白，这不太容易受到外源质粒DNA酶降解的影响。目前的研究将结合PIL脑基因打靶和大鼠TH基因的染色体衍生形式。在克隆大鼠TH基因后，新的染色体衍生TH表达载体将被整合到TfRMAb靶向PILs中，用于静脉注射非病毒制剂后，将其输送到实验性帕金森病大鼠的脑内。

DESCRIPTION (provided by applicant): Many chronic diseases of the brain could be treated with gene therapy. However, viral vectors do not cross the brain capillary wall, which forms the blood-brain barrier (BBS) in vivo. The proposed research advances a new, non-viral, trans-vascular approach to brain gene therapy, where an expression plasmid encoding the therapeutic gene is encapsulated in pegylated immunoliposomes or PILs. The PILs are nano-containers that are targeted across the BBB, and across the brain cell membrane (BCM), with a monoclonal antibody (MAb) to the transferrin receptor (TfR). The MAb to the TfR acts as a molecular Trojan horse, which ferries the gene across the BBB, and the BCM, by accessing the endogenous TfR-mediated transport systems within the BBB and the BCM. Because the gene can cross the BBB, the route of administration is non-invasive and requires only an intravenous administration. The trans-vascular route of delivery enables the gene to distribute to the entire volume of brain. The plasmid DNA is not integrated in the host genome, which is considered advantageous, since there is no risk of insertional mutagenesis. The plasmid DNA functions as an episome, and the persistence of expression of the exogenous gene is a function of the degradation of the plasmid by nuclear DNases. In prior work in an experimental model of Parkinson's disease (PD), it was possible to completely normalize striatal tyrosine hydroxylase (TH) activity with a single intravenous injection of PILs carrying a TH expression plasmid. The limiting factor in this approach is the limited duration of persistence of gene expression. Following the delivery of TH expression plasmid, the brain TH enzyme activity decays with a half-time of 6 days following the single intravenous injection. There is evidence that longer periods of gene expression are possible with the brain delivery of chromosomal derived forms of the TH gene. Genomic forms of the exogenous gene, as compared to cDNA forms of the gene, attract nuclear proteins forming mini-chromatin structures, which are less susceptible to DNase degradation of the exogenous plasmid. The present research will combine PIL brain gene targeting with chromosomal derived forms of the rat TH gene. Following cloning of the rat TH gene, novel chromosomal derived TH expression plasmids will be incorporated into TfRMAb-targeted PILs for delivery to brain of rats with experimental PD following intravenous administration of non-viral formulations.