AAV DIRECTED MUSCLE GENE THERAPY FOR HEMOPHILIA B

腺相关病毒（AAV）是一种依赖性细小病毒，其独特的 生物学推荐它作为一种安全有效的基因治疗载体。在 不存在与辅助病毒（腺病毒）wtAAV的共感染 整合并以潜伏状态存在于宿主细胞基因组中， 如果通过重组AAV（rAAV）复制，则具有吸引力的特性 向量。我们最近证明了肌肉作为一种 用于rAAV基因治疗的体内平台，在小鼠和大的 动物模型（血友病B狗的查佩尔山品系）。整体 该研究的目的是改善AAV肌肉基因治疗， 通过研究rAAV中涉及的分子步骤， 在原代肌细胞中的转导和通过优化rAAV/F.IX载体。 具体目标一。体内ssDNA转化为HMW DNA的分析： 确定骨骼肌rAAV载体在体内的分子命运。 这一目标将需要检查来自rAAV感染的小鼠的总基因组DNA。 肌肉，以确定输入ssDNA转化为 高分子量DNA（HMW DNA）。转基因表达水平和 的DNA复制活动将进行平行研究，以确定一个 转基因表达随时间表观放大的机制 在rAAV基因递送后的非分裂细胞（成熟肌肉）中。B： 确定骨骼肌在体内整合rAAV的能力。的 野生型AAV整合到宿主细胞基因组中的能力已经导致 未经证实的假设，即从rAAV载体持续表达， 通过转录形成整合的rAAV序列。使用小鼠模型 在我们的实验室里开发的，它有人类整合位点， wtAAV，我们将寻求证明稳定的载体（rAAV/F.IX）整合 骨骼肌细胞基因组中。 具体目标二。为了测试低水平的F.IX表达是否在 rAAV基因治疗可以通过更高的比活性来改善F.IX 通过重复施用具有替代衣壳的rAAV/F.IX， 结构.答：已构建因子IX变体以研究 蛋白质与邻近的凝血级联蛋白的相互作用。 构建体，包括具有F.IX的EGF-1结构域的嵌合蛋白 由因子VII和F.IX（包括一个单点）取代 将在体内后研究催化结构域中的突变 使用rAAV载体递送。B：使用 替代血清型rAAV。虽然细胞免疫反应并不限制 rAAV转基因表达，中和抗体可预测地产生。通过 使用衣壳病毒的替代血清型包装转基因 (AAV2，AAV 3，AAV 4），顺序施用可避免发展成 中和抗体的AAV，并允许增强的转基因 在再施用治疗性载体后的表达。

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.