Non-Viral Gene Targeting to the Brain

许多慢性脑部疾病可以用基因疗法来治疗。然而，病毒载体不会交叉 脑毛细血管壁，在体内形成血脑屏障(BBS)。建议的研究进展 一种新的、非病毒的、跨血管的脑基因治疗方法，其中编码 治疗基因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， 跨血脑屏障和跨脑细胞膜(Bcm)的单抗(Mab)靶向 转铁蛋白受体(TFR)。TfR的单抗充当分子特洛伊木马，它将 基因跨血脑屏障和血脑屏障，通过进入体内内源TFR介导的运输系统 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 只需静脉注射即可。通过血管传递的途径使基因能够 分布到整个大脑体积。质粒DNA没有整合到宿主基因组中，这是 被认为是有利的，因为没有插入突变的风险。该质粒DNA的功能是 外源基因的持续表达是外源基因降解的函数 用核糖核酸酶将其转化为质粒。在之前的帕金森氏病(PD)实验模型中， 一次静脉注射即可使纹状体酪氨酸羟化酶(TH)活性完全正常化 携带TH表达载体的PILs。这种方法的限制因素是 基因表达的持续性。在TH表达载体递送后，脑TH酶 单次静脉注射后，活动减弱，半衰期为6天。有证据表明 更长时间的基因表达是可能的，因为大脑递送的是染色体衍生形式的 TH基因。与基因的cdna形式相比，外源基因的基因组形式吸引核。 形成微小染色质结构的蛋白质，这些结构不太容易受到DNA酶降解的影响 外源质粒。本研究将PIL脑基因打靶与染色体衍生相结合 大鼠TH基因的形式。继大鼠TH基因克隆后，新的染色体衍生TH表达 TfRMAb靶向PILs将被整合到实验性帕金森病大鼠的脑内 在静脉注射非病毒制剂后。

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.