Non-Viral Gene Targeting to the Brain

描述（申请人提供）：许多慢性脑疾病可以用基因疗法治疗。然而，病毒载体不穿过脑毛细血管壁，其在体内形成血脑屏障（BBS）。这项研究提出了一种新的非病毒的经血管脑基因治疗方法，其中编码治疗基因的表达质粒被封装在聚乙二醇化免疫脂质体或PIL中。PIL是纳米容器，其用针对转铁蛋白受体（TfR）的单克隆抗体（MAb）靶向穿过BBB和穿过脑细胞膜（BBB）。针对TfR的单克隆抗体充当分子特洛伊木马，其通过进入BBB和丘脑内的内源性TfR介导的转运系统，将基因运送穿过BBB和丘脑。由于该基因可以穿过BBB，因此给药途径是非侵入性的，仅需要静脉内给药。经血管的传递途径使基因能够分布到大脑的整个体积。质粒DNA不整合到宿主基因组中，这被认为是有利的，因为没有插入诱变的风险。质粒DNA作为附加体发挥功能，外源基因表达的持续性是质粒被核DNA酶降解的功能。在先前的工作中，在帕金森氏病（PD）的实验模型中，有可能完全正常化纹状体酪氨酸羟化酶（TH）活性与单次静脉注射携带TH表达质粒的PIL。这种方法的限制因素是基因表达持续时间有限。在递送TH表达质粒后，脑TH酶活性在单次静脉注射后6天的半衰期内衰减。有证据表明，较长的基因表达周期是可能的与脑递送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形成微型染色质结构的核蛋白，所述微型染色质结构对外源质粒的DNA酶降解较不敏感。本研究将联合收割机PIL脑基因打靶与大鼠TH基因的染色体衍生形式相结合。在克隆大鼠TH基因后，将新的染色体来源的TH表达质粒掺入TfRMAb靶向PIL中，用于在静脉内施用非病毒制剂后递送至具有实验性PD的大鼠的脑。

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.