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

 描述(由申请人提供)：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)或凝血因子IX(FIX)(血友病B，Hb)缺乏引起，目前全世界每5,000名男性中有1人缺乏凝血因子蛋白因子VIII(FVIII)(血友病A，HA)或凝血因子IX(FIX，血友病B，Hb)。循环中的少量凝血因子蛋白(&gt；正常的1%)可显著改善疾病表型。在狗模型上进行的基于基因的乙肝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表明，FIX的治疗水平持续存在。然而，80%的血友病是由于FVIII缺乏，血友病A(HA)。HA的基因治疗面临三个不同的挑战：(1)人类FVIII(HFVIII)的固有特性使其难以表达，与其他大小相似的蛋白质相比；(2)FVIII基因的大小容易发生重排，阻碍AAV的产生；(3)蛋白质治疗后患者中高比率的抗FVIII抗体(抑制物)的形成。在早期的研究中，AAV给HA犬体内的犬FVIII导致了cFVIII的长期剂量依赖性表达，显著改善了疾病的表型，然而，实现这些目标的AAV剂量仍然相对较高。这种高的载体剂量可能是向人类转化的障碍，因为人类可能对AAV具有预先存在的免疫力，因此可能对AAV载体接种后的AAV衣壳产生免疫反应。我们最近发现了两种新的人类FVIII修饰，它们克服了将FVIII基因包装到AAV载体中并表达hFVIII蛋白的挑战。首先，一个独特的密码子优化的hFVIII DNA序列比野生型hFVIII表达更高，但也更有效地包装到AAV载体中。对这个密码子优化的cDNA序列进行AAV包装后，得到了同源AAV载体，其产量是野生型hFVIII的两倍。第二，hFVIII蛋白中一个胞内蛋白酶裂解识别位点的变体可以增加分泌和生物活性。我们假设，这些hFVIII变异体的AAV传递将以比天然hFVIII更低的AAV剂量治疗，而免疫原性不会比目前使用的B结构域缺失的FVIII构建物增加。这项建议的目的是确定在对人类FVIII耐受的独特的血友病A小鼠模型(特定目标1)和血友病A犬模型(特定目标2)中，AAV传递这些hFVIII的新变种的有效性和安全性。这些研究有可能成功地减少AAV载体的剂量，这将对避免抗AAV免疫反应和AAV介导的FVIII基因传递后的长期疗效产生深远的影响，将为将这种血友病A的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.