AAV DIRECTED MUSCLE GENE THERAPY FOR HEMOPHILIA B

AAV 定向肌肉基因治疗 B 型血友病

基本信息

批准号：
6625215
负责人：
Paul E Monahan
金额：
$ 11.41万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-02-01 至 2003-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6625215
关键词：
adeno associated virus group capsid chimeric proteins coagulation factor IX gene expression gene therapy hemophilia B laboratory mouse neutralizing antibody nonhuman therapy evaluation striated muscles technology /technique development transfection /expression vector

项目摘要

The adeno-associated virus (AAV) is a dependent parvovirus whose unique biology recommends it as a safe and efficient vector for gene therapy. In the absence of co-infection with a helper virus (adenovirus), the wtAAV integrates and persists in the host cell genome in a latent state, a property that would be attractive if reproduced by recombinant AAV (rAAV) vectors. We recently demonstrated the ability of muscle to serve as a platform for rAAV gene therapy in vivo, both in mouse and in a large animal model (the Chapel Hill strain of hemophilia B dogs). The overall goals of the proposed research are to improve AAV muscle gene therapy for hemophilia by investigating the molecular steps involved in rAAV transduction in primary muscle cells and by optimizing rAAV/F.IX vectors. Specific Aim I. Analysis of conversion of ssDNA to HMW DNA in vivo: A: Determine the molecular fate of rAAV vectors of skeletal muscle in vivo. This aim will require examining total genomic DNA from rAAV-infected muscle to determine the time course for conversion of input ssDNA to a form capable of persistence (HMW DNA). Levels of transgene expression and of DNA replication activity will be investigated in parallel to define a mechanism for the apparent amplification of transgene expression over time in non-dividing cells (mature muscle) following rAAV gene delivery. B: Determine the capacity of skeletal muscle to integrate rAAV in vivo. The ability of wild-type AAV to integrate into the host cell genome has led to the unproven assumption that sustained expression from rAAV vectors occurs via transcription form integrated rAAV sequences. Using a mouse model developed in our laboratory, which has the human integration site for wtAAV, we will seek to demonstrate stable vector (rAAV/F.IX) integrated into the skeletal myocyte genome. Specific Aim II. To test whether low levels of F.IX expression following rAAV gene therapy can be improved by higher specific activity F.IX variants and by repeat administration of rAAV/F.IX with alternative capsid structures. A: Factor IX variants have been constructed to study interactions of the protein with neighboring clotting cascade proteins. Constructs, including a chimeric protein with the EGF-1 domain of F.IX replaced by that of factor VII, and F.IX including a single point mutations in the catalytic domain will be investigated after in vivo delivery using rAAV vectors. B: Readministration of transgene using alternative serotype rAAV. While cellular immune response does not limit rAAV transgene expression, neutralizing antibodies predictably develop. By using alternative serotypes of capsid virus for packaging of transgene (AAV2, AAV3, AAV4), sequential administrations may elude the development of neutralizing antibodies to AAV, and allow augmented transgene expression after re-administration of the therapeutic vector.

腺相关病毒（AAV）是依赖的细小病毒，其独特生物学建议它是基因治疗的安全有效载体。在与助手病毒（腺病毒）的没有共同感染，WTAAV 在潜在状态下的宿主细胞基因组中的整合并持续存在如果重组AAV复制（RAAV），那将很有吸引力向量。我们最近证明了肌肉作为一个在小鼠和大型中，在体内Raav Gene Therapy的平台动物模型（血友病B狗的教堂山菌株）。总体拟议的研究的目标是改善AAV肌肉基因疗法血友病通过研究RAAV涉及的分子步骤一级肌肉细胞的转导和优化RAAV/F.IX载体。特定目标I. ssDNA转化为体内HMW DNA的分析：A：确定体内骨骼肌的RAAV载体的分子命运。此目标将需要检查来自RAAV感染的总基因组DNA 肌肉确定输入ssDNA转化为A的时间过程能够持久性的形式（HMW DNA）。转基因表达和 DNA复制活性将平行研究以定义A 随着时间的推移，转基因表达明显扩增的机制在RAAV基因递送后，在非分裂细胞（成熟的肌肉）中。 B：确定骨骼肌在体内整合RAAV的能力。这野生型AAV集成到宿主细胞基因组中的能力已导致未经证实的假设是从RAAV载体中持续表达通过转录形式集成的RAAV序列。使用鼠标模型在我们的实验室中开发的，该实验室具有人类整合地点 WTAAV，我们将寻求展示稳定的向量（RAAV/F.IX）集成进入骨骼心肌基因组。具体目标II。测试较低级别的f.ix表达以下 RAAV基因疗法可以通过较高的特异性活性F.IX改善变体和通过替代capsid的Raav/f.ix重复给药结构。答：已经构建了第IX因子变体来研究蛋白质与相邻的凝血级联蛋白的相互作用。构造，包括具有f.ix的EGF-1结构域的嵌合蛋白被因子VII的代替，而F.ix包括一个点体内将研究催化结构域中的突变使用RAAV矢量交付。 B：使用使用替代血清型RAAV。虽然细胞免疫反应不限制 Raav转基因表达，可以预测地中和抗体。经过使用Capsid病毒的替代血清型来包装转基因（AAV2，AAV3，AAV4），顺序管理可能会避免开发中和对AAV的抗体，并允许增强的转基因重新管理治疗载体后的表达。