Factor IX mouse models for hemophilia B gene therapy

描述(由申请人提供) 血友病基因替代治疗的研究进展 病毒载体显示出长期基因表达的希望(超过一年) 相关的生物活性分泌蛋白(即因子IX的1%) 动物模型(血友病B小鼠和狗模型) 毒性。特别是动物研究，强调了 消除针对载体表达基因产物的瞬时抗体和 优化载体传递和表达是 确保了人体临床试验的成功。理想动物模型的生成 而更高效的介质盒可以推进这一发展阶段 非常喜欢。最近，我们成功地开发了因子IX(FIX) 由于对因子亲和力增加而具有更高比活性的分子 的催化活性还是提高了。一个带有三重基因的单点突变 较高的IV型胶原结合亲和力有望维持止血 在较低浓度的血浆凝血因子IX水平。结合这些病毒体 应该会产生额外的活性增加的固定分子。至 在体内有效地测试这些结构，我们已经设计出了一种缺陷的修复方法 动物模型使用(“基因敲除”)技术，允许特定的 重新插入(“敲除”)基因盒。有了这个模型，我们可以评估 上述建议的突变体的生物学活性应提供 更好地了解FIX在体内的活性并帮助确定 这些基因盒对病毒的潜在终身有效性和安全性 媒介递送。这项提议的另一个目标是产生 正常和临床相关的突变人类FIX小鼠使用这种方法 接近。预计我们将能够生成定制设计的 人性化修复鼠标模型(CRM+/CRM-；抑制物阴性耐受或 抑制物阳性)，从而模仿现在在 临床，用于在小鼠身上进行彻底的定性。这些动物将成为 对研究从载体和潜力中所需的治疗水平很重要 在突变的人FIX鼠背景中可能产生的免疫反应。 因此，这项提案的主要重点将与测试 人类及其变异型因子IX基因的分子生物学效应 表达“敲入”修复缺陷小鼠模型的产品。长期的 目的是更好地了解FIX在体内的分子作用 希望加强对人类的有效基因治疗。

DESCRIPTION (provided by applicant) Current efforts toward gene replacement therapy for the hemophilias using viral vectors show promise for long-term gene expression (over 1 year) of biologically active secreted proteins (i.e. > 1% of factor IX) in relevant animal models (hemophilia B mouse and dog models) without significant toxicity. Animal studies, in particular, underscore the importance of eliminating transient antibodies to the vector-expressed gene product and optimizing vector delivery and expression as the pressing challenges for assured success of human clinical trials. Generation of ideal animal models and more efficient vector cassettes could advance this phase of development immensely. Recently, we have been successful in developing Factor IX (FIX) molecules with higher specific activity due to increased affinity for Factor VIII or elevated catalytic activity. A single point mutant with threefold higher binding affinity for collagen IV is anticipated to maintain hemostasis at a lower concentration of plasma factor IX levels. Combining these viriants should generate FIX molecules with additional increases in activity. To effectively test these constructs in vivo, we have engineered a FIX deficient animal model using ("knock-out") technology that allows for specific reinsertion ("knock-out") of gene cassettes. With this model, we can assess the biological activity of the above proposed mutants which should provide a better understanding of FIX activity in vivo as well as assist in determining the potential lifelong efficacy and safety of these gene cassettes for viral vector delivery. An additional objective of this proposal is to generate normal as well as clinically relevant mutant human FIX mice using this approach. It is anticipated that we will be able to generate custom designed humanized FIX mouse models (CRM+/CRM-; inhibitor negative tolerant or inhibitor positive) thereby mimicking spontaneous mutants now seen in the clinic, for thorough characterization in the mouse. These animals will be important for studying Therapeutic levels required from vectors and potential immune response that may be generated in mutant human FIX mouse background. Therefore, the major focus of this proposal will be related to testing the molecular and biological consequences of human and variant factor IX gene products expressed in a "knock-in" FIX deficient mouse model. The long-term objective is to better understand the molecular role of FIX in vivo with the hope of enhancing effective gene therapy in humans.