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: 7858056
• 负责人: Kyson Xiaohuai Chou
• 金额: $ 81.04万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858056
• 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; abstracting; 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; 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）： 适当的转基因表达对于实现治疗作用并确保许多基因治疗策略的安全至关重要。当前控制转基因表达的调节系统通常需要使用表达反式激活蛋白的盒式盒式盒子的特殊启动子和共递送。尽管这些系统已被证明有效，但要求禁止对多个转基因的差异调节。此外，跨激活器共同传递的要求大大增加了所选基因转移载体所需的有效载荷。为了开发一种没有这种缺点的新型调节系统，我们正在基于替代剪接的新调节系统探索。迄今为止，我们开发了能够调节转基因表达的最小替代剪接内含子。此外，我们已经证明了使用两个不同的ASO独立控制两个不同转基因的表达的可行性。使用AAV矢量传递我们的新调节系统，我们以受控的方式成功地表达了小鼠眼中的标记基因。因此，我们提议进一步开发我们的新调节系统，并将所得技术应用于眼部疾病的基因治疗。我们的假设是，我们的新调节系统可以开发以独立调节多个转基因的表达，并且通过不同途径抑制血管生成的多种有效因素的差异表达将对治疗眼部新血管形成具有协同作用。我们的长期目标是开发新型的输送系统，以利用AAV介导的持续基因转移的优势，并能够控制所传递的治疗基因的表达。该提案中描述的研究将使我们能够进一步优化适合于临床相关疾病基因治疗的新型调节系统。该提案的具体目的如下：1。在眼睛模型中研究我们诱导系统的转基因表达动力学。我们已经在体外和体内成功证明了我们的诱导系统。我们将重点介绍在眼模中使用标记基因研究动力学，包括在各种诱导条件下的发作，持续时间和转基因表达水平。 2。开发我们的诱导系统以差异调节多个转基因的表达。在我们的调节系统中，通过使用靶向替代剪接位点的反义寡核苷酸来调节转基因消息的替代剪接来控制转基因表达。由于可以改变替代剪接位点及其侧翼序列，因此我们将开发具有不同序列的内含子，以允许对多个转基因的差异调节。 3。调节眼模中多个转基因的表达。我们将首先使用正常眼中的标记基因验证我们提出的多变压调节系统，然后在眼病模型中差异地调节多个有效阻滞剂的表达，以提供支持新型调节系统的生存因子。 公共卫生相关性： 调节转基因表达对于达到治疗作用并确保许多基因治疗策略的安全至关重要。我们正在开发一种新型调节系统，以独立调节多个转基因的表达，例如那些编码有效因素通过不同的途径抑制血管生成的因素，并可能在治疗眼部新生血管中具有协同作用。