摘要
将细菌作为载体用于基因治疗的可行性越来越被认可。这主要归因于细菌的一些本质特性,例如肿瘤靶向功能、携带大的基因或蛋白负荷的能力、用于一系列常见实验室菌株的完善的基因工程的可行性。然而,为了该领域的进步,一些细菌作为载体用于基因治疗相关的问题仍需要解决。其中,对活宿主中细菌的非侵入性检测/成像系统的发展需要解决。体内光学成像已拥有先进的临床前研究,且通常涉及到为发光(如lux操纵子)或荧光蛋白(GFP等)进行基因表达构建的细菌的工程化。基因改造的要求可能被限制在工程化的实验不适合和技术不可行(例如由于缺乏合适的工程工具)。我们在此描述一个利用内生菌酶的活性来特定激活外源荧光成像探针的新型策略。红移的、猝熄的荧光团CytoCy5S通过细菌特异性硝基还原酶 (NTR) 而降低荧光形式。NTR酶存在于大多细菌属中,不存在于哺乳动物中,这使得体内革兰氏阴性菌和革兰氏阳性菌的高度特异性检测。本研究中,在体外所有被检查的细菌属——大肠杆菌、沙门氏菌、李斯特菌、双歧杆菌和梭状芽孢杆菌中,均检测到剂量反应性细菌特异性信号。NTR-敲除菌株的检查由酶特异性探针验证。体内全身成像成功在各种感染模型中随时间推移的细菌的特异性、剂量反应性监测,且观测到对细菌和宿主均无毒性。本研究表明了将先天NTR活性作为一种策略用于光学成像的野生型细菌的概念,而这个概念也可延伸到将NTR特异性探针用于其他成像方式。
关键词: 癌症,CytoCy5S,荧光,NTR,光学成像。
Current Gene Therapy
Title:In Vivo Bacterial Imaging without Engineering; A Novel Probe-Based Strategy Facilitated by Endogenous Nitroreductase Enzymes
Volume: 15 Issue: 3
Author(s): Michael Stanton, Michelle Cronin, Panos Lehouritis and Mark Tangney
Affiliation:
关键词: 癌症,CytoCy5S,荧光,NTR,光学成像。
摘要: The feasibility of utilising bacteria as vectors for gene therapy is becoming increasingly recognised. This is primarily due to a number of intrinsic properties of bacteria such as their tumour targeting capabilities, their ability to carry large genetic or protein loads and the availability of wellestablished genetic engineering tools for a range of common lab strains. However, a number of issues relating to the use of bacteria as vectors for gene therapy need to be addressed in order for the field to progress. Amongst these is the need for the development of non-invasive detection/imaging systems for bacteria within a living host. In vivo optical imaging has advanced preclinical research greatly, and typically involves engineering of bacteria with genetic expression constructs for luminescence (e.g. the lux operon) or fluorescent proteins (GFP etc.). This requirement for genetic modification can be restrictive, where engineering is not experimentally appropriate or technologically feasible (e.g. due to lack of suitable engineering tools). We describe a novel strategy exploiting endogenous bacterial enzymatic activity to specifically activate an exogenously administered fluorescent imaging probe. The red shifted, quenched fluorophore CytoCy5S is reduced to a fluorescent form by bacterial-specific nitroreductase (NTR) enzymes. NTR enzymes are present in a wide range of bacterial genera and absent in mammalian systems, permitting highly specific detection of Gram-negative and Gram–positive bacteria in vivo. In this study, dose-responsive bacterial-specific signals were observed in vitro from all genera examined – E. coli, Salmonella, Listeria, Bifidobacterium and Clostridium difficile. Examination of an NTR-knockout strain validated the enzyme specificity of the probe. In vivo whole-body imaging permitted specific, dose-responsive monitoring of bacteria over time in various infection models, and no toxicity to bacteria or host was observed. This study demonstrates the concept of exploiting innate NTR activity as a reporting strategy for wild-type bacteria using optical imaging, while the concept may also be extended to NTR-specific probes for use with other imaging modalities.
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Michael Stanton, Michelle Cronin, Panos Lehouritis and Mark Tangney , In Vivo Bacterial Imaging without Engineering; A Novel Probe-Based Strategy Facilitated by Endogenous Nitroreductase Enzymes, Current Gene Therapy 2015; 15 (3) . https://dx.doi.org/10.2174/1566523215666150126122712
DOI https://dx.doi.org/10.2174/1566523215666150126122712 |
Print ISSN 1566-5232 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5631 |
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Programmed Cell Death (PCD) is recognized as a pivotal biological mechanism with far-reaching effects in the realm of cancer therapy. This complex process encompasses a variety of cell death modalities, including apoptosis, autophagic cell death, pyroptosis, and ferroptosis, each of which contributes to the intricate landscape of cancer development and ...read more
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