Differential regulation of multiple transgenes for treatment of eye disease

多种转基因的差异调节治疗眼病

• 批准号: 7635025
• 负责人: Kyson Xiaohuai Chou
• 金额: $ 81.09万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7635025
• 关键词: Alternative Splicing; Angiopoietin-2; Animal Model; Antisense Oligonucleotides; Cells; Data; Dependovirus; Development; Disease; Disease model; Ensure; Exons; Eye; Eye diseases; Failure; Gene Transduction Agent; Gene Transfer; Genes; Goals; In Vitro; Introns; Kinetics; Lead; Mediating; Modeling; Mus; Pathway interactions; Process; Proteins; RNA Splicing; Regulation; Research; Safety; Site; System; Technology; Therapeutic Effect; Therapeutic Studies; Tissues; Trans-Activators; Transgenes; adeno-associated viral vector; angiogenesis; base; bench to bedside; clinically relevant; gene therapy; gene transfer vector; human disease; in vivo; mRNA Precursor; neovascularization; novel; ocular neovascularization; pre-clinical; promoter; public health relevance; red fluorescent protein; therapeutic gene; transgene expression

DESCRIPTION (provided by applicant): Proper transgene expression is essential to achieve therapeutic effect and ensure safety in many gene therapy strategies. Current regulation systems for controlling transgene expression typically require use of a special promoter and co-delivery of a cassette expressing a trans-activator protein. While these systems have been proven effective, the requirements prohibit differential regulation of multiple transgenes. Additionally, the requirement of co-delivering a trans-activator adds significantly to the payload required of the chosen gene transfer vector. To develop a new type of regulation system without such drawbacks, we are exploring a novel regulation system based on alternative splicing. To date, we have developed minimal alternative splicing introns capable of regulating transgene expression. Furthermore, we have demonstrated the feasibility of using two different ASO to independently control the expression of two different transgenes. Using AAV vector for delivering our novel regulation system, we have successfully expressed a marker gene in mouse eye in a controlled manner. Therefore, we are proposing to further develop our novel regulation system and apply the resulting technology for gene therapy of ocular diseases. Our hypothesis is that our novel regulation system could be developed to independently regulate the expression of multiple transgenes, and that differential expression of multiple potent factors inhibiting angiogenesis via different pathways would have a synergistic effect in treating ocular neovascularization. Our long-term goal is to develop novel delivery systems that exploit the advantages of AAV mediated persistent gene transfer with the ability to control the expression of the therapeutic genes being delivered. The studies described in this proposal would allow us to further optimize our novel regulation system suitable for gene therapy of clinically relevant diseases. The Specific Aims of the proposal are as follows: 1. Study the kinetics of transgene expression for our inducible system in an eye model. We have already successfully demonstrated our inducible system both in vitro and in vivo. We will focus on using marker genes in an eye model to study the kinetics including the onset, duration and level of transgene expression under various conditions of induction. 2. Develop our inducible system to differentially regulate the expression of multiple transgenes. In our regulation system, transgene expression is controlled by using antisense oligonucleotides targeting alternative splice site to modulate the alternative splicing of transgene message. Since the alternative splice site and its flanking sequences can be varied, we will develop introns with different sequences to allow differential regulation of multiple transgenes. 3. Regulate the expression of multiple transgenes in eye models. We will first validate our proposed multiple-transgene regulation system using marker genes in normal eyes and then differentially regulate the expression of multiple potent blockers for survival factors supporting neovascularization in an eye disease model in an effort to better validate this novel regulation system. PUBLIC HEALTH RELEVANCE: Regulating transgene expression is essential to achieve therapeutic effect and ensure safety in many gene therapy strategies. We are developing a novel regulation system to independently regulate the expression of multiple transgenes, such as those encoding potent factors inhibiting angiogenesis via different pathways and potentially having a synergistic effect in treating ocular neovascularization.

描述（由申请人提供）： 在许多基因治疗策略中，适当的转基因表达对于实现治疗效果和确保安全性是必不可少的。目前用于控制转基因表达的调节系统通常需要使用特殊启动子和共递送表达反式激活蛋白的盒。虽然这些系统已被证明是有效的，但这些要求禁止对多种转基因进行差异调节。此外，共递送反式激活因子的要求显著增加了所选基因转移载体所需的有效载荷。为了开发一种新型的调控系统，而没有这些缺点，我们正在探索一种新的调控系统的基础上选择性剪接。到目前为止，我们已经开发了最小的选择性剪接内含子能够调节转基因表达。此外，我们已经证明了使用两种不同的阿索独立控制两种不同转基因表达的可行性。使用AAV载体传递我们的新调控系统，我们已经成功地在小鼠眼中以可控的方式表达标记基因。因此，我们建议进一步开发我们的新调控系统，并将所得技术应用于眼部疾病的基因治疗。我们的假设是，我们的新的调控系统可以独立地调节多种转基因的表达，并通过不同的途径抑制血管生成的多种有效的因子的差异表达将有协同作用，在治疗眼部新生血管。我们的长期目标是开发新的递送系统，其利用AAV介导的持续基因转移的优点，具有控制被递送的治疗基因的表达的能力。本提案中描述的研究将使我们能够进一步优化适用于临床相关疾病基因治疗的新型调控系统。该提案的具体目标如下：1。在眼模型中研究我们的诱导系统的转基因表达动力学。我们已经成功地证明了我们的诱导系统在体外和体内。我们将集中于使用标记基因在眼睛模型中研究动力学，包括在各种诱导条件下转基因表达的开始、持续时间和水平。2.开发我们的诱导系统，以差异调节多种转基因的表达。在我们的调控系统中，转基因表达是通过使用针对选择性剪接位点的反义寡核苷酸来调节转基因信息的选择性剪接来控制的。由于可变剪接位点及其侧翼序列可以变化，我们将开发具有不同序列的内含子，以允许多种转基因的差异调节。3.调节多种转基因在眼模型中的表达。我们将首先验证我们提出的多转基因调控系统，在正常眼睛中使用标记基因，然后差异调节多种有效的阻滞剂的表达，支持新血管形成的生存因子在眼部疾病模型，以更好地验证这种新的调控系统。 公共卫生关系： 在许多基因治疗策略中，调节转基因表达对于实现治疗效果和确保安全性是必不可少的。我们正在开发一种新的调节系统，以独立地调节多种转基因的表达，例如那些编码通过不同途径抑制血管生成的有效因子的转基因，并可能在治疗眼部新生血管形成中具有协同作用。