FIV Vectors for the Treatment of Hemophilia A

描述(申请人提供)：尽管包括我们在内的几个实验室已经在动物模型中证明了血友病A的基因治疗的可行性，但在临床上没有观察到转基因在治疗水平上的长期表达。在血友病患者中缺乏足够的表达水平的因子(FVIII)表明，需要进一步改进现有的基因递送系统才能产生治疗产品。在这个项目的第一阶段，我们启动了一项研究来解决FVIII的表达水平和持续时间的问题，研究结果表明，FVIII的表达受到不稳定的mRNA、与内质网(ER)伴侣的相互作用以及通过与甘露糖结合的凝集素LMAN1的相互作用促进ER到高尔基体转运的要求的限制。在基因治疗应用中，用于FVIII表达的载体通常使用B结构域缺失(BDD)cDNA，其中该结构域的编码序列被移除，以产生用于蛋白质生产和基因转移的更短的结构。值得注意的是，BDD FVIII在细胞内的表达与整个FVIII分子的表达具有相同的局限性。然而，在一个短的B结构域间隔区中加入几个天冬酰胺连接的寡糖，增加了ER到高尔基体的转运，导致功能性FVIII的分泌水平在体外和体内都比全长或B结构域缺失的FVIII高15-25倍。在血友病犬中，通过添加部分B结构域可以显著提高治疗性FVIII的产量这一假说在第一阶段启动了测试，这是与人类疾病最相关的动物模型，也是临床研究的先决条件。这些构建体是用犬FVIII基因序列准备的，以减少引入血友病狗时复杂的跨物种因素。在第一阶段，对制备高滴度病毒的参数进行了优化，并分析了VSV-G和GP64伪型载体的天然免疫应答。第一阶段的完成旨在使该项目能够在血友病小鼠和狗身上进行FVIII表达水平和持续时间的研究，并选择最有利的FVIII基因进行安全和长期表达。在这项第二阶段的研究中，目的是评估不含B结构域或包含两种不同长度的B结构域的犬FVIII的FIV表达水平、持续时间和临床益处。此外，可能进一步显著提高转导细胞的FVIII产量的其他元素将被整合到转移载体中并进行测试。这一第二阶段还包括开发方法，为这些研究和后续研究提供更多的病毒载体，这将需要大量的载体。 公共卫生意义：凝血因子VIII基因疗法在血友病A患者中的临床应用一直存在凝血因子VIII产生不足的问题。使用能够在宿主细胞中长期表达的FIV慢病毒载体，旨在克服表达不良问题的改良的凝血因子VIII基因序列将在血友病犬身上进行测试，这种动物模型与人类疾病非常相似。我们的目标是推动这项研究确定的最好的产品在临床上应用于人类患者。

DESCRIPTION (provided by applicant): Although several labs including ours have demonstrated the feasibility of gene therapy for hemophilia A in animal models, long-term expression of the transgene at therapeutic levels was not observed in the clinic. The lack of sufficient expression levels Factor VIII (FVIII) in hemophilic patients indicates that further improvements of the current gene delivery systems are needed to generate a therapeutic product. In the Phase I of this project we initiated a study to address the issues of FVIII expression level and duration based on findings showing that FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose- binding lectin LMAN1. Vectors for FVIII expression in gene therapy applications typically have used a B domain deleted (BDD) cDNA in which the coding sequence for this domain is removed to yield a shorter construct for protein production and gene transfer. Of note, the expression of BDD FVIII within the cell has the same limitations as the whole FVIII molecule. However, the inclusion of several asparagine-linked oligosaccharides within a short B-domain spacer increased ER to Golgi transport resulting in secretion of functional FVIII at levels 15- to 25-fold higher than full-length or B domain deleted FVIII both in vitro and in vivo. Phase I initiated the testing of the hypothesis that therapeutic FVIII production can be significantly enhanced by addition of parts of the B domain in hemophilic dogs, the most relevant animal model to the human disease and a prerequisite test for clinical studies, Two FIV vectors were constructed and tested which contain canine FVIII with different lengths of the B domain added. The constructs were prepared with the canine FVIII genetic sequence to reduce complicating cross-species factors when introduced into the hemophilic dog. Also, in Phase I the parameters for preparing high titer virus were optimized and the innate immune response to VSV- G and GP64 pseudotyped vectors were analyzed. The completion of the Phase I aims puts the project in position to conduct the FVIII expression level and duration studies in hemophilic mice and dogs and to select the most favorable FVIII cDNA for safe and long term expression. In this Phase II study the aims are to evaluate expression level, duration and clinical benefit from FIV delivery of canine FVIII with no B domain or with two different lengths of the B domain included. Also, additional elements that may further enhance FVIII production significantly from transduced cells will be incorporated into the transfer vector and tested. This Phase II also includes the development of methods to provide increased production of the virus vector for these and subsequent studies, which will require large amounts of the vector. PUBLIC HEALTH RELEVANCE: Clinical application of Factor VIII gene therapies to hemophilia A patients have suffered from insufficient production of Factor VIII. Using an FIV lentiviral vector capable of long-term expression in host cells, Factor VIII gene sequences with improvements designed to overcome the problem of poor expression will be tested in hemophilic dogs, the animal model which closely resembles the human disease. The goal is to advance the best product as determined from this study toward application in the clinic to human patients.