Use of alternative splicing and rAAV for longterm control of transgene expression

使用选择性剪接和 rAAV 来长期控制转基因表达

• 批准号: 7305361
• 负责人: Kyson Xiaohuai Chou
• 金额: $ 21.9万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7305361
• 关键词: Adenoviruses; Alternative Splicing; Animal Model; Animals; Antisense Oligonucleotides; Artificial Chromosomes; Dependovirus; Development; Elements; Exons; Gene Transfer; Genes; Goals; Herpesviridae; Heterogeneous Nuclear RNA; Immune response; In Vitro; Introns; Length; Mediating; Modification; Mus; Mutate; Numbers; Plasmids; Positioning Attribute; Process; Proteins; RNA Splicing; Regulation; Retroviridae; Series; Site; Subfamily lentivirinae; System; Testing; Therapeutic Effect; Time; Trans-Activators; Virus; adeno-associated viral vector; base; gene therapy; gene transfer vector; improved; in vivo; mRNA Precursor; novel; research study; therapeutic gene; transgene expression

DESCRIPTION (provided by applicant): Proper transgene expression is essential to achieve therapeutic effect in many gene therapy strategies. Current systems for regulating transgene expression typically require co-delivery of a cassette expressing a trans-activator protein adding significantly to the payload required of the chosen gene transfer vector. The concern of payload is exacerbated for adeno-associated virus (AAV) which has a packaging limit of less than 5 kb. To avoid the drawback, we are exploring a novel regulation system based on alternative splicing. Alternative splicing is the inclusion or skipping of an alternative exon in between two exons that are constitutively included during the process of pre-mRNA splicing. The inclusion of an alternative exon will result in synthesis of a truncated protein in our system. Other studies have shown that inclusion of the alternative exon could be blocked by anti-sense oligonucleotides (AON) complementary to an essential element of alternative splicing. Thus, skipping of the alternative exon is forced to take place resulting in synthesis of a functional full length protein. Controlling transgene expression mediated by adeno-associated virus (AAV) is confronted by a challenge of using minimal elements. 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 hypothesis of our proposed studies is that by incorporating an optimized alternative splicing mechanism into the AAV delivery system, both long- term and controllable transgene expression could be achieved in vivo. The Specific Aims are: 1) Optimize a small intron capable of alternative splicing, and 2) Incorporate optimized introns capable of alternative splicing into AAV vector to control transgene expression over time in an animal model. The experiments described in this proposal would allow us to develop and optimize the alternative splicing as a general means of controlling transgene expression mediated by AAV. Such a mechanism could also be used by other gene transfer vectors including lentivirus and other retroviruses, adenovirus, herpes virus as well as artificial chromosome. Proper transgene expression is essential to achieve therapeutic effect in many gene therapy strategies. Current systems typically have a demanding payload required of the chosen gene transfer vector. This concern is exacerbated for AAV which has a packaging limit of less than 5 kb. To overcome the current drawback, we are proposing a novel regulation system for controlling the expression of the therapeutic genes being delivered.

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至关重要。目前用于调节转基因表达的系统通常需要共递送表达反式激活蛋白的盒，从而显著增加所选基因转移载体所需的有效载荷。对于包装极限小于5kb的腺相关病毒（AAV），有效载荷的问题加剧。为了避免这一缺陷，我们正在探索一种基于选择性剪接的新型调控系统。选择性剪接是在前体mRNA剪接过程中组成性包含的两个外显子之间包含或跳过选择性外显子。包含选择性外显子将导致在我们的系统中合成截短的蛋白质。其他研究表明，包含的选择性外显子可以被阻断的反义寡核苷酸（AON）互补的选择性剪接的基本要素。因此，被迫发生选择性外显子的跳跃，导致功能性全长蛋白质的合成。腺相关病毒（AAV）介导的转基因表达控制面临着使用最少元件的挑战。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。我们提出的研究的假设是，通过将优化的可变剪接机制结合到AAV递送系统中，可以在体内实现长期和可控的转基因表达。具体目标是：1）优化能够选择性剪接的小内含子，和2）将优化的能够选择性剪接的内含子并入AAV载体中以控制动物模型中随时间的转基因表达。本提案中描述的实验将使我们能够开发和优化选择性剪接作为控制由AAV介导的转基因表达的一般手段。这种机制也可用于其他基因转移载体，包括慢病毒和其他逆转录病毒、腺病毒、疱疹病毒以及人工染色体。在许多基因治疗策略中，适当的转基因表达是实现治疗效果所必需的。目前的系统通常具有所选基因转移载体所需的苛刻的有效载荷。对于包装极限小于5kb的AAV，这种担忧加剧。为了克服目前的缺点，我们提出了一种新的调节系统，用于控制正在交付的治疗基因的表达。