Oral Therapy for Hemophilia A

描述（由申请人提供）：目前x连锁出血性疾病血友病的标准治疗是基于重组蛋白的静脉给药。注射的蛋白质价格昂贵，半衰期短（从而限制了治疗效果的持续时间），经常是抗体反应的目标（从而使治疗复杂化/中和，产生免疫毒性，并进一步增加成本）；而且需要反复静脉注射，既痛苦又不方便。经治疗后，20-30%的A型血友病患者（缺乏因子VIII）和1.5-3%的B型血友病患者（缺乏因子IX）形成针对输注因子的抑制性抗体（“抑制剂”）。抑制剂的形成是治疗的严重并发症，并增加疾病的发病率和死亡率。旁路试剂是可用的，但价格昂贵，不能常规给予，因为它们有血栓形成的风险。临床免疫耐受诱导（ITI）方案包括长期频繁的高剂量因子施用，并且非常昂贵。生物工程已经创造出了强大的试剂，可以将蛋白质（或编码蛋白质的核酸）口服到肠道，这种试剂无创且成本低廉。在生物工程研究伙伴关系申请中，我们建议将这种方法用于治疗和口服耐受诱导。最近，Daniell和Herzog实验室发现，口服生物包封因子IX（以叶绿体转基因植物叶片材料的形式）可以有效地阻止血友病B小鼠在随后的蛋白质替代治疗中形成抑制抗体和过敏反应。梁实验室开发了一种基于包装成壳聚糖纳米颗粒的质粒载体的血友病口服基因疗法。因此，口服肠上皮可以提供治疗，部分恢复血友病动物的止血（从而预防自发性出血）和免疫调节（从而在不使用免疫抑制药物的情况下防止有害的免疫反应）。为了建立有效的口服治疗血友病A（这是一种更为普遍的血友病，并且具有更高的抑制剂形成风险），我们组建了一个跨学科的团队，包括植物生物技术、生物工程、免疫学和动物模型。具体来说，我们建议i)确定基于纳米颗粒的口服基因治疗在血友病小鼠模型中纠正凝血和抑制F.VIII抗体形成的能力；ii)产生fviii叶绿体转基因生菜，用于口服和预防/逆转蛋白质替代中抑制剂的形成；iii)将口服治疗扩大到犬血友病a。最终，我们打算整合这些方法，提供一种完全基于口服的治疗方案，以预防出血并防止抑制剂的形成。这种方法可以很容易地适用于其他遗传性蛋白质缺乏或某些过敏。

DESCRIPTION (provided by applicant): Current standard treatment of the X-linked bleeding disorder hemophilia is based on intravenous administration of recombinant protein. The infused protein is expensive, has a short half-life (thereby limiting duration of the therapeutic effect), is often targeted by antibody responses (thereby complicating/neutralizing therapy, creating immunotoxicities, and further increasing costs); and repeated intravenous access is required, which is painful and inconvenient. Upon treatment, 20-30% of patients with hemophilia A (factor VIII deficiency) and 1.5-3% of hemophilia B patients (factor IX deficiency) form inhibitory antibodies ("inhibitors") against the infused factor. Inhibitor formation represents a serious complication of treatment and increases morbidity and mortality of the disease. Bypass reagents are available, but are expensive and cannot be routinely given because they pose a thrombotic risk. Clinical immune tolerance induction (ITI) protocols consist of frequent high-dose factor administrations for a long period of time and are very expensive. Bioengineering has created powerful reagents for non-invasive and cost-effective oral delivery of the protein (or a nucleic acid encoding the protein) to the gut. Within this Bioengineering Research Partnership application, we propose to advance this approach for treatment and for oral tolerance induction. Recently, the Daniell and Herzog labs found that oral delivery of bioencapsulated factor IX (in form of chloroplast transgenic plant leaf material) effectively prevented formation of inhibitory antibodies and anaphylactic reactions in subsequent protein replacement therapy in hemophilia B mice. The Leong lab developed an oral gene therapy for hemophilia based on plasmid vectors packaged into chitosan nanoparticles. Thus, oral delivery to the intestinal epithelium can provide both therapy that partially restores hemostasis in animals with hemophilia (thereby providing prophylaxis against spontaneous bleeding) and immune modulation (thereby preventing deleterious immune responses without use of immune suppressive drugs). In order to establish effective oral therapy for hemophilia A (which is the more prevalent form of the disease and has a much higher risk of inhibitor formation), we assembled an interdisciplinary team, encompassing plant biotechnology, bioengineering, immunology, and animal models. Specifically, we propose to i) determine the ability of nanoparticle-based oral gene therapy to correct coagulation and suppress antibody formation to F.VIII in murine models of hemophilia; ii) generate F.VIII chloroplast transgenic lettuce for oral delivery and prevention/reversal of inhibitor formation in protein replacement; and iii) scale up oral therapies to canine hemophilia A. Ultimately, we intend to integrate the approaches to provide an entirely oral-based treatment regimen that accomplished prophylaxis against bleeds and prevents inhibitor formation. This approach could readily be adapted to other inherited protein deficiencies or certain allergies.