FACTOR IX MOUSE MODELS FOR HEMOPHILIA B GENE THERAPY

目前使用病毒载体对血友病进行基因替代疗法的努力显示出在相关动物模型（血友病B小鼠和狗模型）中生物活性分泌蛋白（即>1%的因子IX）的长期基因表达（超过1年）而没有显著毒性的前景。特别是动物研究强调了消除载体表达基因产物的瞬时抗体和优化载体递送和表达的重要性，这是确保人类临床试验成功的紧迫挑战。理想的动物模型和更有效的载体盒的产生可以极大地推进这一发展阶段。最近，由于对因子VIII的亲和力增加或催化活性提高，我们已经成功地开发了具有更高比活性的因子IX（FIX）分子。预期对胶原蛋白W具有三倍高结合亲和力的单点突变体在较低浓度的血浆因子IX水平下维持止血。组合这些变体应产生活性额外增加的FIX分子。为了在体内有效地测试这些构建体，我们使用敲除技术设计了FIX缺陷动物模型，该技术允许基因盒的特异性重新插入（敲入）。使用该模型，我们可以评估上述拟定突变体的生物活性，这应该可以更好地了解体内FIX活性，并有助于确定这些基因盒用于病毒载体递送的潜在终身有效性和安全性。该提议的另一个目的是使用该方法产生正常以及临床相关的突变人FIX小鼠。预计我们将能够生成定制设计的人源化FIX小鼠模型（CRM+/CRM-;抑制剂阴性耐受或抑制剂阳性），从而模拟目前在临床中观察到的自发突变体，用于小鼠中的彻底表征。这些动物对于研究载体所需的治疗水平和突变型人FIX小鼠背景中可能产生的潜在免疫应答非常重要。因此，本提案的主要重点将与检测在“敲入”FIX缺陷小鼠模型中表达的人和变体因子IX基因产物的分子和生物学后果相关。长期目标是更好地了解FIX在体内的分子作用，以期增强人类的有效基因治疗。

Current efforts towards gene replacement therapy for the hemophilias using viral vectors show promise for long-term gene expression (over l 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 successe 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 successfiil 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 W is anticipated to maintain hemostasis at a lower concentration of plasma factor IX levels. Combining these variants 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-in) 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 clinicaly 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.