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 生物学推荐它作为基因治疗的安全有效的载体。在 不存在与辅助病毒（腺病毒）wtAAV 的共同感染 以潜伏状态整合并持续存在于宿主细胞基因组中， 如果通过重组 AAV (rAAV) 复制，该特性将具有吸引力 向量。我们最近证明了肌肉作为 rAAV 基因治疗体内平台，无论是在小鼠还是在大 动物模型（血友病B犬的教堂山品系）。整体 拟议研究的目标是改善 AAV 肌肉基因治疗 通过研究 rAAV 涉及的分子步骤来治疗血友病 原代肌细胞中的转导以及通过优化 rAAV/F.IX 载体。 具体目标 I. 体内 ssDNA 转化为 HMW DNA 的分析： A： 确定体内骨骼肌 rAAV 载体的分子命运。 这一目标需要检查 rAAV 感染的总基因组 DNA 肌肉来确定输入单链DNA转化为单链DNA的时间过程 能够持久存在的形式（HMW DNA）。转基因表达水平和 DNA 复制活性的研究将同时进行，以确定 转基因表达随时间明显放大的机制 rAAV 基因传递后的非分裂细胞（成熟肌肉）中。乙： 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导致 rAAV 载体持续表达的未经证实的假设 通过转录形成整合的rAAV序列。使用鼠标模型 在我们的实验室开发，该实验室拥有人类整合站点 wtAAV，我们将寻求证明集成的稳定载体（rAAV/F.IX） 进入骨骼肌细胞基因组。 具体目标二。检测低水平的 F.IX 表达是否遵循 更高的比活性 F.IX 可以改善 rAAV 基因治疗 变体以及通过使用替代衣壳重复施用 rAAV/F.IX 结构。答：已构建了因子 IX 变体来研究 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 被因子 VII 取代，F.IX 包括一个点 催化结构域的突变将在体内进行研究 使用 rAAV 载体进行递送。 B：使用转基因重新施用 替代血清型 rAAV。虽然细胞免疫反应并不限制 rAAV 转基因表达、中和抗体可预见地产生。由 使用衣壳病毒的替代血清型来包装转基因 （AAV2、AAV3、AAV4），顺序给药可能会逃避开发 AAV 中和抗体，并允许增强转基因 重新施用治疗载体后的表达。

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.