Imaging of gene delivery in the central nervous system

描述(申请人提供)：基因替代疗法具有极大的潜力来缓解许多无法治愈的神经退行性疾病的预后。在这种方法中，缺陷基因被其产物可以恢复正常功能的基因原位取代。到目前为止，还没有直接的方法来非侵入性地监测基因传递的准确性及其在长时间内的表达水平，并将其与大脑中的解剖结构共同配准。当前提议的主要目标是开发一种基于人工磁共振成像(MRI)报告基因的非侵入性成像方法，该方法基于人工磁共振成像(MRI)报告基因，例如我们先前开发的用于跟踪细胞的富赖氨酸蛋白(LRP)报告基因。该方法基于化学交换饱和转移(CEST)对比机制。射频脉冲被应用于饱和特定的酰胺质子，从而减少水信号。由于不同的可交换质子具有不同的共振频率，这允许创建一组报告基因，这些基因可以以频率选择性的方式相互区分。为了验证我们的假设，即可以通过CEST报告基因在大鼠大脑中直接监测转基因表达，将使用CEST MRI监测病毒传递和LRP的表达，并将通过使用荧光素酶的生物发光成像进行验证。接下来，将产生两个具有不同射频选择性的报告基因文库。一个文库将包含LRP类型的人造基因，另一个文库将包含类似鱼精蛋白(一种具有高精氨酸浓度的蛋白质)的基因。将对文库进行筛选，以寻找优化的报告基因。由于大脑是一个异质组织，为了进一步验证我们的假设，有必要对广泛的不同细胞类型中的基因表达进行成像。为此，表达水泡系统口炎病毒G糖蛋白(VSV-G)的慢病毒将作为穿梭载体，在细胞特异性启动子(神经元的NSE和星形胶质细胞的GFAP)下表达基因。CEST MRI将以频率选择性的方式在大鼠体内评估细胞特异性基因的表达，并将通过组织学进行验证。由于慢病毒除了报告基因外还可以携带治疗基因，我们预计我们的方法适用于实时监测基因替代治疗的效率、安全性和基因表达水平。许多神经病理过程是复杂的，经常需要替换一个以上的基因，有时甚至在多种细胞类型中也是如此。因此，以非侵入性、连续的方式同时成像多个基因可能会极大地帮助监测基因替代治疗的结果。公共卫生相关性：基因替代疗法在缓解许多无法治愈的神经退行性疾病的预后方面具有巨大潜力。目前这项提议的主要目标是开发一种方法，利用专门为磁共振成像(MRI)设计的人造报告基因，对基因输送到中枢神经系统(CNS)进行非侵入性成像。这项新技术应该适用于实时监测基因替代治疗中的基因表达的效率、安全性和水平。这种新的成像方法的好处可以进一步扩展到不同的器官和各种应用，如监测细胞移植或药物治疗后的细胞存活。

DESCRIPTION (provided by applicant): Gene replacement therapy has great potential to alleviate the prognosis of many incurable neurodegenerative diseases. In this approach, a defective gene is replaced in situ by a gene of which its products can restore normal function. Up to date there is no direct method to monitor non-invasively the accuracy of the gene delivery and it's expression level for prolonged time periods and co-register it with anatomical structures in the brain. The main goal of the current proposal is to develop a methodology for the non-invasive imaging of gene delivery to the central nervous system (CNS) based on artificial Magnetic Resonance Imaging (MRI) reporter genes such as the Lysine Rich Protein (LRP) reporter gene, which we have previously developed for tracking cells. This approach is based on the chemical exchange saturation transfer (CEST) contrast mechanism. A radiofrequency pulse is applied to saturate specific amide protons, reducing the water signal. Since different exchangeable protons have different resonance frequencies, this allows creation of a family of reporter genes that are distinguishable from each other in a frequency-selective manner. To test our hypothesis that transgenic expression can be monitored directly in the rat brain with a CEST reporter gene, viral delivery and expression of LRP will be monitored using CEST MRI and will be validated by bioluminescence imaging using luciferase. Next, two libraries of reporter genes having different radiofrequency selectivity will be generated. One library will contain artificial genes of the LRP-type, and the other will contain genes that are similar to protamine (a protein with high arginine concentration). The libraries will be screened for optimized reporter genes. Since the brain is a heterogeneous tissue, to further test our hypothesis it is imperative to image gene expression in a broad range of different cell types. To this end, lentiviruses expressing the Vesicular System Stomatitis Virus G glycoprotein (VSV-G) will be used as a shuttle vector, with gene expression under cell-specific promoters (NSE for neurons and GFAP for astrocytes). Cell-specific gene expression will be assessed by CEST MRI in vivo in rats in a frequency- selective manner and will be validated with histology. Due to the capacity of the lentivirus to carry therapeutic genes in addition to the reporter gene, we anticipate that our approach is applicable for real-time monitoring of the efficiency, safety, and levels of gene expression in gene replacement therapy. Many neuropathological processes are complex and frequently require the replacement of more than one gene, occasionally even in multiple cell types. Thus, imaging multiple genes simultaneously in a non-invasive, serial manner may greatly aid monitoring the outcome of gene replacement therapy. PUBLIC HEALTH RELEVANCE: Gene replacement therapy has great potential to alleviate the prognosis of many incurable neurodegenerative diseases. The main goal of the current proposal is to develop a methodology for the non-invasive imaging of gene delivery to the central nervous system (CNS) using artificial reporter genes designed specifically for Magnetic Resonance Imaging (MRI). This new technology should be applicable for real-time monitoring of the efficiency, safety and levels of gene expression in gene replacement therapy. The benefits of this novel imaging approach could be further expanded to different organs and variety of applications, such as monitoring cell survival in response to cell transplantation or drug treatment.