Non-Viral Gene Targeting to the Brain

描述（申请人提供）：许多脑部慢性疾病可以用基因疗法治疗。然而，病毒载体不能穿过脑毛细血管壁，从而在体内形成血脑屏障（BBS）。该研究提出了一种新的、非病毒的、跨血管的脑基因治疗方法，其中编码治疗基因的表达质粒被封装在聚乙二醇化的免疫脂质体或pil中。PILs是一种纳米容器，具有针对转铁蛋白受体（TfR）的单克隆抗体（MAb），可以穿过血脑屏障和脑细胞膜（BCM）。针对TfR的单抗就像一个分子特洛伊木马，通过进入血脑屏障和脑屏障内内源性TfR介导的运输系统，将基因传递到血脑屏障和脑屏障。因为基因可以穿过血脑屏障，给药途径是非侵入性的，只需要静脉给药。这种跨血管的传递途径使基因能够分布到整个大脑。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。质粒DNA作为一个片段起作用，外源基因的持续表达是核DNA酶降解质粒的一个功能。在先前对帕金森病（PD）实验模型的研究中，通过单次静脉注射携带TH表达质粒的pil，可以使纹状体酪氨酸羟化酶（TH）活性完全正常化。这种方法的限制因素是基因表达持续时间有限。TH表达质粒传递后，单次静脉注射后脑组织TH酶活性衰减半衰期为6天。有证据表明，更长时间的基因表达可能与大脑传递的染色体衍生形式的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蛋白形成微染色质结构，这些结构对外源质粒的dna酶降解不太敏感。本研究将把PIL脑基因靶向与大鼠TH基因的染色体衍生形式结合起来。在克隆大鼠TH基因后，新的染色体衍生的TH表达质粒将被纳入tfrmab靶向的pil中，在静脉注射非病毒制剂后，将其传递给实验性PD大鼠的大脑。

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.