Imaging of gene delivery in the central nervous system

中枢神经系统基因传递的成像

• 批准号: 7659274
• 负责人: Assaf A Gilad
• 金额: $ 25.01万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7659274
• 关键词: Achievement; Amides; Arginine; Astrocytes; Behavioral; Biological Assay; Bioluminescence; Blood - brain barrier anatomy; Brain; Cell Survival; Cell Transplantation; Cells; Chemicals; Clinical Trials; Complex; Cytomegalovirus; Disease Progression; Energy Transfer; Enzymes; Family; Frequencies; Gene Delivery; Gene Expression; Genes; Glial Fibrillary Acidic Protein; Glycine; Goals; Histology; Image; In Situ; Label; Libraries; Location; Luciferases; Lysine; Magnetic Resonance Imaging; Metabolic; Methodology; Methods; Modality; Monitor; Mutagenesis; Neuraxis; Neurodegenerative Disorders; Neurons; Neurosciences; Neurotransmitters; Organ; Outcome; Output; Parkinson Disease; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Physiologic pulse; Process; Property; Protamines; Proteins; Protons; Rattus; Regulation; Reporter; Reporter Genes; Reporting; Safety; Shuttle Vectors; Signal Transduction; Stomatitis; Structure; Subfamily lentivirinae; Synthetic Genes; System; Testing; Time; Tissues; Toxin; Transgenic Organisms; Validation; Viral; Virus; Water; alternative treatment; base; brain tissue; cell type; design; directed evolution; enolase; gene function; gene replacement therapy; gene therapy; glycoprotein G; imaging modality; in vivo; new technology; non-invasive monitor; novel; optical imaging; outcome forecast; polypeptide; promoter; protein expression; public health relevance; radiofrequency; response; therapeutic gene; transgene expression; viral gene delivery

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.

描述（由申请人提供）：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很大的潜力。在这种方法中，有缺陷的基因在原位被其产物可以恢复正常功能的基因所取代。到目前为止，还没有直接的方法来监测基因传递的准确性和长时间的表达水平，并将其与大脑的解剖结构共同登记。当前提案的主要目标是开发一种基于人工磁共振成像（MRI）报告基因（如赖氨酸富蛋白（LRP）报告基因）的非侵入性中枢神经系统（CNS）基因传递成像方法，我们之前开发了用于跟踪细胞。这种方法是基于化学交换饱和转移（CEST）对比机制。一个射频脉冲被应用于饱和特定的酰胺质子，减少水信号。由于不同的可交换质子具有不同的共振频率，这就允许创建一个以频率选择方式相互区分的报告基因家族。为了验证我们的假设，即利用CEST报告基因可以直接监测转基因基因在大鼠大脑中的表达，我们将使用CEST MRI监测LRP的病毒传递和表达，并使用荧光素酶进行生物发光成像验证。接下来，将生成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型的人工基因，另一个文库将包含与鱼精蛋白（一种高精氨酸浓度的蛋白质）相似的基因。文库将筛选优化的报告基因。由于大脑是一个异质组织，为了进一步验证我们的假设，必须在广泛的不同细胞类型中对基因表达进行成像。为此，将利用表达水泡系统口炎病毒G糖蛋白（VSV-G）的慢病毒作为穿梭载体，在细胞特异性启动子（神经元为NSE，星形胶质细胞为GFAP）下进行基因表达。细胞特异性基因表达将通过CEST MRI在大鼠体内以频率选择性的方式进行评估，并将通过组织学进行验证。由于慢病毒除了携带报告基因外还携带治疗基因的能力，我们预计我们的方法适用于基因替代治疗中基因表达的效率、安全性和水平的实时监测。许多神经病理过程是复杂的，经常需要替换多个基因，有时甚至在多个细胞类型中。因此，以非侵入性的串行方式同时对多个基因进行成像可能极大地有助于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的潜力。当前提案的主要目标是开发一种方法，利用专门为磁共振成像（MRI）设计的人工报告基因，对中枢神经系统（CNS）的基因传递进行非侵入性成像。这项新技术可用于基因替代治疗中基因表达的效率、安全性和水平的实时监测。这种新型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，例如监测细胞移植或药物治疗后的细胞存活。