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: 7858506
• 负责人: Assaf A Gilad
• 金额: $ 20.77万
• 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/7858506
• 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巨大潜力。在这种方法中，有缺陷的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递送的准确性及其长时间的表达水平，并将其与大脑中的解剖结构共配准。目前建议的主要目标是开发一种方法，用于基于人工磁共振成像（MRI）报告基因（如富赖氨酸蛋白（LRP）报告基因）的非侵入性成像的基因递送到中枢神经系统（CNS），我们以前开发的跟踪细胞。这种方法是基于化学交换饱和转移（CEST）对比机制。施加射频脉冲以使特定酰胺质子饱和，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，这允许产生以频率选择性方式彼此可区分的报告基因家族。为了检验我们的假设，即转基因表达可以用CEST报告基因在大鼠脑中直接监测，将使用CEST MRI监测病毒递送和LRP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，将产生具有不同射频选择性的报告基因的两个文库。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（一种具有高精氨酸浓度的蛋白质）相似的基因。将筛选文库中的优化报告基因。由于大脑是一种异质组织，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，表达水泡系统口炎病毒G糖蛋白（VSV-G）的慢病毒将用作穿梭载体，在细胞特异性启动子（神经元的NSE和星形胶质细胞的GFAP）下进行基因表达。将通过CEST MRI以频率选择性方式在大鼠体内评估细胞特异性基因表达，并将用组织学进行验证。由于慢病毒携带治疗基因的能力，除了报告基因，我们预计，我们的方法是适用于实时监测的效率，安全性和基因替代治疗中的基因表达水平。许多神经病理过程是复杂的，并且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。因此，以非侵入性的连续方式同时对多个基因进行成像可以极大地帮助监测基因替代疗法的结果。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。目前的建议的主要目标是开发一种方法，用于非侵入性成像的基因传递到中枢神经系统（CNS），使用专门为磁共振成像（MRI）设计的人工报告基因。这项新技术应适用于实时监测的效率，安全性和基因表达水平的基因替代治疗。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。

项目成果

CEST phase mapping using a length and offset varied saturation (LOVARS) scheme.

• DOI: 10.1002/mrm.23312
• 发表时间: 2012-10
• 期刊: MAGNETIC RESONANCE IN MEDICINE
• 影响因子: 3.3
• 作者: Song, Xiaolei;Gilad, Assaf A.;Joel, Suresh;Liu, Guanshu;Bar-Shir, Amnon;Liang, Yajie;Gorelik, Michael;Pekar, James J.;van Zijl, Peter C. M.;Bulte, Jeff W. M.;McMahon, Michael T.
• 通讯作者: McMahon, Michael T.

Non-invasive temperature mapping using temperature-responsive water saturation shift referencing (T-WASSR) MRI.

• DOI: 10.1002/nbm.3066
• 发表时间: 2014-03
• 期刊: NMR IN BIOMEDICINE
• 影响因子: 2.9
• 作者: Liu, Guanshu;Qin, Qin;Chan, Kannie W. Y.;Li, Yuguo;Bulte, Jeff W. M.;McMahon, Michael T.;van Zijl, Peter C. M.;Gilad, Assaf A.
• 通讯作者: Gilad, Assaf A.