Novel factor VIII variants for improved efficacy in gene therapy for hemophilia A

 描述（由申请方提供）：血友病是一种X-连锁出血性疾病，由凝血因子蛋白因子VIII（FVIII）（血友病A，HA）或因子IX（FIX）（血友病B，HB）缺乏引起，全球每5,000例男性中有1例。循环中少量凝血因子蛋白（>正常值的1%）可显著改善疾病表型。在犬模型中进行的HB基因治疗的临床前研究为使用腺相关病毒（AAV）载体进行的临床试验提供了基础。这些正在进行的临床试验证明了治疗水平FIX的持续表达。然而，80%的血友病是由于FVIII缺乏，血友病A（HA）。HA的基因治疗提出了三个不同的挑战：（1）人FVIII（hFVIII）的固有特性使得其与类似大小的其他蛋白质相比难以表达，（2）大尺寸的FVIII cDNA易于重排，这阻碍了AAV产生，和（3）蛋白质治疗后患者中抗FVIII抗体（抑制剂）形成的高速率。在早期的研究中，在HA犬中AAV递送犬FVIII导致cFVIII的长期剂量依赖性表达，其显著改善疾病表型，然而，实现这些目标的AAV剂量仍然相对较高。这种高载体剂量可能是翻译至人类的障碍，因为人类可能对AAV具有预先存在的免疫力，因此在施用AAV载体时可能对AAV衣壳具有免疫应答。我们最近鉴定了两种新的人FVIII修饰，其克服了将FVIII cDNA包装到AAV载体中并表达hFVIII蛋白的挑战。首先，独特的密码子优化的hFVIII DNA序列表达高于野生型hFVIII，但也更有效地包装到AAV载体中。该密码子优化的cDNA序列的AAV包装产生具有比野生型hFVIII高两倍的产率的同源AAV载体。第二，hFVIII蛋白中的细胞内蛋白酶切割识别位点的变体赋予增加的分泌和生物活性。我们假设这些hFVIII变体的AAV递送将在比天然hFVIII更低的AAV剂量下具有治疗性，与目前使用的B结构域缺失的FVIII构建体相比，免疫原性没有增加。本提案的目的是确定hFVIII的这些新型变体的AAV递送在均耐受人FVIII的独特血友病A小鼠模型（特定目的1）和血友病A犬模型（特定目的2）中的有效性和安全性。这些研究有可能成功降低AAV载体剂量，这将对避免抗AAV免疫应答和AAV介导的FVIII基因递送后的长期疗效产生深远影响，这将为将血友病A的这种基因治疗方法转化为临床提供必要的进展。

 DESCRIPTION (provided by applicant): Hemophilia is an X-linked bleeding disorder caused by a deficiency in the clotting factor protein factor VIII (FVIII) (hemophilia A, HA) or factor IX (FIX) (hemophilia B, HB) present in 1 in 5,000 males worldwide. Small amounts of clotting factor protein (>1% of normal) in the circulation provide substantial improvement of the disease phenotype. Preclinical studies of gene-based therapies for HB in dog models provided the basis for ongoing clinical trials using adeno-associated viral (AAV) vectors. These ongoing clinical trials demonstrated sustained expression of therapeutic levels of FIX. However, 80% of all hemophilia is due to FVIII deficiency, hemophilia A (HA). Gene therapy for HA presents three distinct challenges: (1) intrinsic properties of human FVIII (hFVIII) make it difficult to express compared to other proteins of similar size (2) the large size of the FVIII cDNA is prone to rearrangements which hampers AAV production and (3) high rates of anti-FVIII antibody (inhibitor) formation in patients following protein therapy. In earlier studies, AAV delivery of canine FVIII in HA dogs resulted in long-term dose-dependent expression of cFVIII that significantly improves the disease phenotype, however, the dose of AAV to achieve these goals remains relatively high. This high vector dose may be an obstacle to translating to humans since humans may have pre-existing immunity to AAV and thus may have an immune response to the AAV capsid upon AAV vector administration. We recently identified two novel human FVIII modifications that overcome the challenges of packaging the FVIII cDNA into AAV vectors and expressing hFVIII protein. First, a unique codon-optimized hFVIII DNA sequence expresses higher than wild type hFVIII but also packages more efficiently into AAV vectors. AAV packaging of this codon-optimized cDNA sequence results in a homogenous AAV vector that has a two-fold higher yield than wild type hFVIII. Second, variants of an intracellular protease cleavage recognition site in the hFVIII protein confer increased secretion and biological activity. We hypothesize that AAV delivery of these hFVIII variants will be therapeutic at lower AAV doses than native hFVIII, with no increases in immunogenicity over the currently used B-domain deleted FVIII constructs. The goal of this proposal is to determine the efficacy and safety of AAV delivery of these novel variants of hFVIII in unique hemophilia A mouse models (Specific Aim 1) and hemophilia A dog models (Specific Aim 2) that are both tolerant to human FVIII. These studies have the potential for success in reducing the AAV vector dose which will have a profound impact on avoiding the anti-AAV immune responses and on the long-term efficacy after AAV-mediated gene delivery of FVIII which will provide the necessary advances to translate this gene therapy approach for hemophilia A into the clinic.