PET报告基因显像研究进展

近20年的基因研究已经取得了很多研究成果,使基因治疗从实验室走向临床治疗成为可能.然而体内基因表达的监测仍有许多至关重要的问题没有解决,随着技术的发展,利用特定的报告基因和报告探针行PET显像,去推测基因在体内的表达成为可能.通过PET,不但能够正确理解基因治疗的过程,也为基因治疗的发展和用于临床提供了依据.     

PET报告基因显像

运用正电子发射体层显像(PET)及相关的PET报告基因及报告探针的活体显像技术可提供关于基因治疗定量及定性的信息,并可提供其他技术不能获得的参数,这对更好地理解基因治疗的过程及人类基因治疗的未来发展及临床应用很有意义。     

癌症基因治疗的临床应用

癌症的基因治疗已成为肿瘤研究中最活跃的领域之一，虽然在基础理论和实验研究方面做了大量的工作，但在临床应用方面目前还刚刚起步。这里对癌症基因治疗的临床应用情况作一介绍。工临床应用概况癌症基因治疗的首次人体应用是1989年5月22日由美国国家癌症研究所（NCI）的Rosenberg研究小组开始的。其实，这第一例病人只是将细菌抗新霉素（NeoR）基因在体外培养条件下通过基因转移（GeneTransfer，GT）的方法，成功地插入到病人播散性恶性黑色素瘤中浸润淋巴细胞基因中，再将已经带有Ne－oR基因的淋巴细胞输入病人体内，以观察外源性基…     

光化学基因转染技术

肿瘤基因转染技术虽然发展很快,仍有许多限制因素制约着它的临床应用,特别是缺乏有效而特异的体内基因转递技术,难以保证基因转染效率。大量研究表明,尽管目前基因治疗载体种类很多,但无论病毒载体或是合成载体都存在着生物安全性、体内转染效率等方面的限制。特别是多数基因载     

骨缺损基因治疗的研究进展

基因治疗是修复骨缺损的主要研究方向之一。当前对骨缺损基因治疗的研究热点主要在于对骨诱导因子目的基因、各种载体及靶细胞的研究,将目的基因通过体内或体外途径转入靶细胞,维持骨诱导因子在局部持续有效地表达,从而修复骨缺损。本文就骨缺损基因治疗的实验研究和临床应用进展作一综述。     

肿瘤基因治疗原理及应用

近年来,随着分子生物学、免疫学等现代医学技术的飞速发展,基因治疗已成为肿瘤治疗中的一个热点。自从1989年美国的Rosenberg首次将基因治疗技术应用到黑色素瘤患者身上以来,目前已有一百多项人类疾病基因治疗项目经美国RAC获准进行临床实验,其中大部份与肿瘤有关。1 基因治疗原理1.1 概念 所谓基因治疗,是指应用基因工程和细胞生物学技术,将一些具有治疗价值的外源基因导入体内,通过修复或补充失去正常功能的某些基因,抑制体内某些基因的过盛表达来达到治疗目的的方法。1.2 肿瘤发生机制 肿瘤的发生机制至今尚未完全明确,但目前比较一致的看法认为肿瘤的发生是     

正电子放射性显像剂代谢及其研究方法进展

正电子放射性显像剂主要用于PET的研究,能在分子水平上反映细胞代谢、细胞受体活性、细胞核内的核酸合成以及细胞基因的改变,在临床疾病诊断和治疗中有重要的地位和作用.PET显像剂进入生物体内后会发生代谢转化,了解PET显像剂的代谢途径和转化过程,对于准确分析和解释显像结果及设计开发新型PET显像剂非常重要.该文总结了目前PET显像剂代谢研究的现状,并对PET显像剂代谢研究方法以及分析技术等进行了综述.     

靶向表皮生长因子受体小分子类PET显像剂研究进展

表皮生长因子受体（EGFR）在多种癌症的发生发展中起着重要作用,目前已有多种EGFR靶向药物被美国食品与药品管理局批准用于临床,但因个体敏感程度不同,总体疗效偏低。研究表明EGFR高表达或突变患者对靶向药物敏感,因此明确EGFR表达水平和突变状态对临床用药有重要指导意义。PET成像技术能实现分子水平无创显像,并能通过SUV进行半定量研究,使得在体内无创明确EGFR表达、突变情况,指导靶向药物的精准治疗成为可能。笔者综述了靶向EGFR的小分子类PET显像剂,以期为新的探针研发及其临床应用提供一定帮助。     

PET显像仪的类型及临床应用

近几年来,核医学PET显像发展迅速,在临床应用中发挥着越来越重要的作用。目前,PET显像仪共有三种类型:专用型PET(dedicated PET)、混合型PET(hybrid PET)和组合型PET(combined PET)。PET与CT组合即PET-CT,不仅能反映人体解剖结构改变,更重要的是可以提供体内功能代谢信息,从分子水平揭示疾病发病机理和治疗效应,是临床诊断心脑疾病和肿瘤的重要手段。PET-CT临床应用十分广泛,尤其是在肿瘤、神经系统、心血管系统等病变的临床应用方面具有其独特的功能和作用。     

成肌细胞在肌肉骨骼系统疾病基因治疗中的应用研究进展

基因治疗 ,就是利用分子生物学的方法将目的基因转入靶细胞内 ,通过靶细胞表达目的基因产物治疗疾病的方法。基因治疗主要有两种途径 :即invivo及exvivo方式。invivo途径即体内基因治疗 ,是将外源基因装配于特定的真核细胞表达载体 ,直接导入体内。exvivo途径也称“回体基因治疗” ,是将含有外源基因的载体在体外导入自体或异体 (异种 )细胞 ,经体外细胞扩增后 ,输回自体。此方法易于操作 ,并且使用自体细胞有利于解决安全性问题。自从 80年代初以来 ,靶细胞的选择问题一直伴随着基因治疗研究。目前研究较多的有骨髓干细胞、成纤维细胞、肌…     

Reporter gene imaging: potential impact on therapy

Positron emission tomography (PET)-based molecular-genetic imaging in living organisms has enjoyed exceptional growth over the past 5 years; this is particularly striking since it has been identified as a new discipline only within the past decade. Positron emission tomography is one of three imaging technologies (nuclear, magnetic resonance and optical) that has begun to incorporate methods that are established in molecular and cell biology research. The convergence of these disciplines and the wider application of multi-modality imaging are at the heart of this success story. Most current molecular-genetic imaging strategies are "indirect," coupling a "reporter gene" with a complimentary "reporter probe." Reporter gene constructs can be driven by constitutive promoter elements and used to monitor gene therapy vectors and the efficacy of trans gene targeting and transduction, as well as to monitor adoptive cell-based therapies. Inducible promoters can be used as "sensors" to regulate the magnitude of reporter gene expression and can be used to provide information about endogenous cell processes. Reporter systems can also be constructed to monitor mRNA stabilization and specific protein-protein interactions. Promoters can be cell specific and restrict transgene expression to certain tissue and organs. The translation of reporter gene imaging to specific clinical applications is discussed. Several examples that have potential for patient imaging studies in the near future include monitoring adenoviral-based gene therapy, oncolytic herpes virus therapy, adoptive cell-based therapies and Salmonella-based tumor-targeted cancer therapy and imaging. The primary translational applications of noninvasive in vivo reporter gene imaging are likely to be (a) quantitative monitoring of the gene therapy vector and the efficacy of transduction in clinical protocols, by imaging the location, extent and duration of transgene expression; (b) monitoring cell trafficking, targeting, replication and activation in adoptive therapies, involving ex vivo transduction of harvested immune-competent cells and stem/progenitor cells; (c) assessments of endogenous molecular events using different reporter gene imaging technologies following the development of safe, efficient and target-specific vectors for "diagnostic transductions."     

Monitoring the efficacy of adoptively transferred prostate cancer-targeted human T lymphocytes with PET and bioluminescence imaging.

Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model. METHODS: RM1 murine prostate carcinoma tumors transduced with human prostate-specific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice. Human T lymphocytes were transduced with chimeric antigen receptors (CAR) specific for either hPSMA or human carcinoembryonic antigen (hCEA) and with a fusion reporter gene for herpes simplex virus type 1 thymidine kinase (HSV1tk) and green fluorescent protein, with or without click beetle red luciferase. The localization of adoptively transferred T cells in tumor-bearing mice was monitored with 2'-(18)F-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-ethyluracil ((18)F-FEAU) small-animal PET and bioluminescence imaging (BLI). RESULTS: Cotransduction of CAR-expressing T cells with the reporter gene did not affect CAR-mediated cytotoxicity. BLI of Renilla and click beetle red luciferase expression enabled concurrent imaging of adoptively transferred T cells and systemic tumors in the same animal. hPSMA-specific T lymphocytes persisted longer than control hCEA-targeted T cells in lung hPSMA-positive tumors, as indicated by both PET and BLI. Precise quantification of T-cell distributions at tumor sites by PET revealed that delayed tumor progression was positively correlated with the levels of (18)F-FEAU accumulation in tumor foci in treated animals. CONCLUSION: Quantitative noninvasive monitoring of genetically engineered human T lymphocytes by PET provides spatial and temporal information on T-cell trafficking and persistence. PET may be useful for predicting tumor response and for guiding adoptive T-cell therapy.     

Defining the success of cardiac gene therapy: how can nuclear imaging contribute?

Gene therapy is a promising modality for the treatment of various cardiovascular diseases such as ischaemia, heart failure, restenosis after revascularisation, hypertension and hyperlipidaemia. An increasing number of approaches are moving from experimental and preclinical validation to clinical application, and several multi-centre trials are currently underway. Despite the rapid progress in cardiac gene therapy, many basic tools and principles remain under development. Questions with regard to the optimal method for gene delivery in a given situation remain open, as do questions concerning therapeutic efficacy and the time course and magnitude of gene expression in target and remote areas. Nuclear imaging provides valuable tools to address these open issues non-invasively. Functional effects of molecular therapy at the tissue level can be identified using tracers of blood flow, metabolism, innervation or cell death. The use of reporter genes and radiolabelled reporter probes allows for non-invasive assessment of location, magnitude and persistence of transgene expression in the heart and the whole body. Co-expression of a reporter gene will allow for indirect imaging of the expression of a therapeutic gene of choice, and linkage of measures of transgene expression to downstream functional effects will enhance the understanding of basic mechanisms of cardiac gene therapy. Hence, nuclear imaging offers great potential to facilitate and refine the determination of therapeutic effects in preclinical and clinical cardiovascular gene therapy.     

基因表达正电子发射断层显像

分子生物学与核医学的结合形成了分子核医学,基因表达正电子发射断层(PET)显像是当今分子核医学研究的热点和前沿领域之一。基因表达PET显像包括反义PET显像和报告基因PET显像,反义PET显像由于技术上的问题,远不如报告基因PET显像那样发展迅速,报告基因PET显像已广泛用于动物实验研究,可望不久的将来会用于临床研究。     

The human norepinephrine transporter in combination with 11C-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy.

Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility of the human norepinephrine transporter (hNET) as a reporter gene in combination with the reporter probe 11C-m-hydroxyephedrine (mHED) for PET. METHODS: An adenoviral vector (AdTrack-hNET) containing the hNET gene as reporter gene and the enhanced green fluorescent protein (EGFP) as a substitute for a therapeutic gene was constructed. After COS-7, A2780, and U373 cells were transiently transduced with AdTrack-hNET, hNET protein expression, EGFP fluorescence, and cellular uptake of 11C-mHED were determined. In rats, U373 tumor xenografts were grown and transiently transduced with either AdTrack-hNET or an AdTrack-Luc control adenovirus. Intratumoral accumulation of 11C-mHED was determined by PET and ex vivo biodistribution. The tumors were subsequently examined for EGFP fluorescence. RESULTS: 11C-mHED uptake was positively correlated with AdTrack-hNET viral titer and hNET protein expression. However, large differences in transfection efficiency between cell lines were observed. The highest 11C-mHED uptake was found in hNET transfected U373 cells, in which tracer uptake was >70-fold higher than that in control cells. 11C-mHED accumulation could be inhibited by desipramine, a potent inhibitor of hNET. In all cell lines, 11C-mHED uptake was positively correlated with EGFP fluorescence, implying that imaging of hNET with 11C-mHED would enable monitoring of a coexpressed therapeutic gene. In the animal model, gene transfection efficiencies were very low, as determined by EGFP fluorescence. Still, a significantly higher 11C-mHED uptake in hNET transduced tumors than that in control tumors was demonstrated by ex vivo biodistribution studies. PET with a clinical camera could visualize 1 of 3 hNET transduced tumors, indicating that the transfection efficiency was near the detection limit. CONCLUSION: These results indicate that monitoring of gene therapy using the hNET/11C-mHED reporter gene/probe is feasible, but further investigation with regard to the sensitivity of the technique is required.     

A new acycloguanosine-specific supermutant of herpes simplex virus type 1 thymidine kinase suitable for PET imaging and suicide gene therapy for potential use in patients treated with pyrimidine-based cytotoxic drugs.

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs. METHODS: The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with (3)H-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-ethyluracil ((3)H-FEAU), (3)H-pencyclovir ((3)H-PCV), and (3)H-GCV and by drug sensitivity assays. PET with (18)F-FEAU and (18)F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling. RESULTS: FEAU accumulation was not detected in HSV1-A167Ysr39tk-expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-beta-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate (3)H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with (3)H-PCV in vitro and with (18)F-FHBG in vivo. CONCLUSION: We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with (18)F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.     

Noninvasive molecular imaging to detect transgene expression of lentiviral vector in nonhuman primates.

Noninvasive imaging of a reporter gene is a new and promising technique to quantify transgene expression after gene therapy. This study was performed to demonstrate visualization of lentiviral-marked cells by PET. METHODS: We transduced nonhuman primate CD34+ hematopoietic cells with a lentiviral vector expressing a PET reporter gene, the mutant viral herpes simplex virus type 1-thymidine kinase (HSV1-sr39tk) gene. 1-(2-Fluoro-2-deoxy-beta-D-arabinofuranosyl)-76Br-5-bromouracil (76Br-FBAU) was used as the substrate for the viral tk enzyme. Upon phosphorylation, 76Br-FBAU was retained by cells and imaged by PET. The long half-life of 76Br, 16.2 h, permitted us to perform extended pharmacokinetic and imaging studies. RESULTS: 76Br-FBAU was retained in vascular tissues of the animals with transplanted tk lentiviral vector-transduced CD34+ cells. Elimination of 76Br-FBAU was through renal and hepatic excretion. CONCLUSION: Noninvasive molecular imaging using PET will help us, in the future, to define the contribution and distribution of cells and their progeny to tissue repair and development.     

Assays for noninvasive imaging of reporter gene expression.

Repeated, noninvasive imaging of reporter gene expression is emerging as a valuable tool for monitoring the expression of genes in animals and humans. Monitoring of organ/cell transplantation in living animals and humans, and the assessment of environmental, behavioral, and pharmacologic modulation of gene expression in transgenic animals should soon be possible. The earliest clinical application is likely to be monitoring human gene therapy in tumors transduced with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene. Several candidate assays for imaging reporter gene expression have been studied, utilizing cytosine deaminase (CD), HSV1-tk, and dopamine 2 receptor (D2R) as reporter genes. For the HSV1-tk reporter gene, both uracil nucleoside derivatives (e.g., 5-iodo-2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil [FIAU] labeled with 124I, 131I) and acycloguanosine derivatives [e.g., 8-[18F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (8-[18F]-fluoroganciclovir) ([18F]FGCV), 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG)] have been investigated as reporter probes. For the D2R reporter gene, a derivative of spiperone [3-(2'-[18F]-Fluoroethyl)spiperone ([18F]FESP)] has been used with positron emission tomography (PET) imaging. In this review, the principles and specific assays for imaging reporter gene expression are presented and discussed. Specific examples utilizing adenoviral-mediated delivery of a reporter gene as well as tumors expressing reporter genes are discussed.     

In vivo molecular-genetic imaging: multi-modality nuclear and optical combinations

Multi-modality, noninvasive in vivo imaging is increasingly being used in molecular-genetic studies and will soon become the standard approach for reporter gene imaging studies in small animals. The coupling of nuclear and optical reporter genes, as described here, represents only the beginning of a far wider application of this technology in the future. Optical imaging and optical reporter systems are cost-effective and time-efficient; they require less resources and space than PET or MRI, and are particularly well suited for imaging small animals, such as mice. Optical reporter systems are also very useful for the quantification and selection of transduced cells using FACS, and for performing in vitro assays to validate the function and sensitivity of constitutive and specific-inducible reporter systems. However, optical imaging techniques are limited by depth of light penetration and do not yet provide optimal quantitative or tomographic information. These issues are not limiting for PET- or MRI-based reporter systems, and PET- and MRI-based animal studies are more easily generalized to human applications. Many of the shortcomings of each modality alone can be overcome by the use of dual- or triple-modality reporter constructs that incorporate the opportunity for PET, fluorescence and bioluminescence imaging.