FIV Vectors for the Treatment of Hemophilia A

描述（由申请人提供）：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基因治疗的可行性，但在临床中未观察到治疗水平的转基因长期表达。血友病患者中缺乏足够的因子VIII（FVIII）表达水平表明需要进一步改进目前的基因递送系统以产生治疗产品。在该项目的I期中，我们启动了一项研究，以解决FVIII表达水平和持续时间的问题，该研究基于以下发现：FVIII表达受到不稳定mRNA的限制、与内质网（ER）伴侣的相互作用以及通过与甘露糖结合凝集素LMAN 1相互作用促进ER向高尔基体转运的要求。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去除以产生用于蛋白质生产和基因转移的较短构建体。值得注意的是，BDD FVIII在细胞内的表达具有与整个FVIII分子相同的限制。然而，在短的B结构域间隔区中包含几种天冬酰胺连接的寡糖增加了ER至高尔基体的转运，导致功能性FVIII的分泌水平在体外和体内均比全长或B结构域缺失的FVIII高15- 25倍。第I阶段开始了对以下假设的测试，即通过在血友病狗中添加部分B结构域可以显著增强治疗性FVIII的产生，血友病狗是与人类疾病最相关的动物模型，并且是临床研究的先决条件测试。构建并测试了两种FIV载体，其含有添加了不同长度的B结构域的犬FVIII。用犬FVIII基因序列制备构建体，以减少引入血友病犬时的复杂跨物种因素。此外，在第I阶段，优化了制备高滴度病毒的参数，并分析了对VSV-G和GP 64假型载体的先天免疫应答。I期研究的完成旨在使该项目能够在血友病小鼠和犬中进行FVIII表达水平和持续时间研究，并选择最有利的FVIII cDNA进行安全和长期表达。在该II期研究中，目的是评价不含B结构域或包含两种不同长度的B结构域的犬FVIII的FIV递送的表达水平、持续时间和临床益处。此外，可进一步显著增强转导细胞的FVIII产生的其他元件将掺入转移载体中并进行测试。第二阶段还包括开发方法，为这些和后续研究提供增加的病毒载体产量，这将需要大量的载体。 公共卫生相关性：因子VIII基因疗法对血友病A患者的临床应用已经遭受因子VIII产生不足。使用能够在宿主细胞中长期表达的FIV慢病毒载体，将在血友病犬（与人类疾病非常相似的动物模型）中测试具有旨在克服表达差问题的改进的因子VIII基因序列。我们的目标是将本研究确定的最佳产品推向临床应用于人类患者。

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.