Gene regulation using novel drugs modulating premature translational termination

使用调节提前翻译终止的新药物进行基因调控

• 批准号: 8703838
• 负责人: JOSEPH DOUGHERTY
• 金额: $ 18.95万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8703838
• 关键词: Alleles; Animals; Apomorphine; Apoptosis; Asses; Attention; Blood - brain barrier anatomy; Cell Culture Techniques; Cells; Clinical Trials; Clone Cells; Codon Nucleotides; Corpus striatum structure; Disease; Disease model; Dopamine; Drug usage; Gene Expression; Gene Expression Regulation; Gene Transfer; Genes; HIV-2; Hereditary Disease; Image; Immunohistochemistry; In Vitro; Lentivirus Vector; Luciferases; Mammalian Cell; Midbrain structure; Modeling; Monitor; Mus; Neurons; Nonsense Codon; Oxidopamine; Parkinson Disease; Pharmaceutical Preparations; Phase II Clinical Trials; Population; Production; Proteins; Regulation; Rotation; Series; Symptoms; System; Technology; Terminator Codon; Testing; Therapeutic Agents; Time; Tissues; Trans-Activators; Transcription Initiation; Transducers; Transduction Gene; Translations; Tyrosine 3-Monooxygenase; base; clinical application; clinical efficacy; design; dopaminergic neuron; flexibility; in vivo; interest; neurotrophic factor; novel; premature; promoter; public health relevance; research study; response; small molecule; therapeutic gene; vector

DESCRIPTION (provided by applicant): Systems providing inducible gene regulation in mammalian cells have been of great experimental value. To date most systems regulate gene expression at the level of transcription initiation. This requires a specialized promoter that is activated by an exogenous transactivator in response to addition of a small molecule. Although these systems have been quite useful, there are some drawbacks. These systems necessitate not only the introduction of the therapeutic gene but also the transactivator gene. They are also restricted to utilizing the specialized promoter obviating the use of a potentially more relevant promoter such as a tissue-specific promoter. Furthermore, high background expression is often a problem. Experimentally this can be overcome by identifying and expanding cell clones with low backgrounds and high levels of induction. However, for clinical applications, this in not typically a viable option indicating that other approaches for achieving regulated expression should be explored. There is a large body of evidence showing that cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation. It was theorized that a cell's ability to make this distinction could be exploited to develop drugs to promote readthrough at a PTC but not at a normal codon. Such drugs could then be used to treat genetic disorders caused by PTCs. Already, small molecules have been developed with this ability, and one has been shown to display clinical efficacy in phase 2 clinical trials while being well tolerated. Here it is hypothesized that the use of drugs specific for PTC readthrough can be combined with gene transfer technology to develop an inducible expression system for use in vivo. The idea is to incorporate a PTC into a gene of interest so that active protein will only be made after administration of a readthrough drug. This system should: 1) afford low levels of background expression with effective levels of induction, 2) require that only the therapeutic gene be transducer, and 3) provide flexibility in selection of the promoter used to drive expression. More specifically, inducible regulation of PTC containing genes will be analyzed using a series of PTC readthrough drugs both in vitro and in vivo after transduction with HIV-2 derived vectors. Regulated expression in vivo will rely heavily upon whole-animal imaging to allow real-time monitoring of expression. Attention will focus upon controlling expression of GDNF since there is mounting evidence that GDNF expression can be very beneficial for treating Parkinson's disease. Although experiments delineated in this proposal use HIV-2 vectors in the context of a Parkinson's disease model, this approach should have broad utility for a variety of gene transfer systems and should be applicable to most disorders that can benefit from regulated gene expression. Public Health Relevance: There are diseases where being able to switch the production of a therapeutic agent on and off is very beneficial. This project aims at developing such a switch.

描述（由申请人提供）：提供哺乳动物细胞中诱导基因调节的系统具有很高的实验价值。迄今为止，大多数系统在转录启动水平下调节基因表达。这需要一个专门的启动子，该启动子因添加小分子而被外源性反式激活者激活。尽管这些系统非常有用，但仍有一些缺点。这些系统不仅需要引入治疗基因，还需要反式激活基因。他们还局限于利用专门的启动子来避免使用潜在的更相关的启动子，例如组织特异性启动子。此外，高背景表达通常是一个问题。在实验上，这可以通过识别和扩展具有低背景和高诱导水平的细胞克隆来克服。但是，对于临床应用，这通常不是一个可行的选择，表明应探讨其他实现受管制表达的方法。有大量证据表明，在翻译过程中，细胞已经开发出一种区分过早终止密码子（PTC）和正常终止密码子的机制。从理论上讲，可以利用细胞做出这种区别的能力来开发药物以在PTC上促进读取，但在正常密码子上却不能。然后，这些药物可用于治疗由PTC引起的遗传疾病。已经以这种能力开发了小分子，并且已经证明一种在2阶段临床试验中显示出临床功效，同时耐受性良好。在这里假设，可以将特定于PTC读取的药物与基因转移技术结合使用，以开发可诱导的表达系统以供体内使用。这个想法是将PTC纳入感兴趣的基因，以便只有在服用读出药物后才能制作活性蛋白。该系统应：1）具有有效诱导水平的背景表达较低，2）仅需要治疗基因为传感器，而3）在选择用于驱动表达的启动子的选择方面具有灵活性。更具体地说，将使用一系列在体外和体内使用HIV-2衍生的载体在体外和体内分析含有PTC基因的诱导型调节。体内调节的表达将在很大程度上依赖整个动物成像以实时监测表达。注意将集中于控制GDNF的表达，因为有越来越多的证据表明GDNF表达对治疗帕金森氏病非常有益。尽管在帕金森氏病模型的背景下，该提案中描述的实验使用了HIV-2载体，但该方法应该对各种基因转移系统具有广泛的实用性，并且应适用于大多数可以从受调节基因表达中受益的疾病。 公共卫生相关性：有些疾病能够开关和关闭治疗剂的生产非常有益。该项目旨在开发这种转换。