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静脉注射重组蛋白。输注的蛋白质价格昂贵，半衰期短(从而限制了治疗效果的持续时间)，经常成为抗体反应的靶点(从而使治疗复杂化/中和，产生免疫毒性，并进一步增加成本)；并且需要反复静脉注射，这是痛苦和不便的。治疗后，20-30%的血友病A患者(凝血因子VIII缺乏症)和1.5-3%的血友病B患者(凝血因子IX缺乏症)形成针对输入的因子的抑制性抗体(抑制物)。抑制剂的形成是治疗的严重并发症，并增加了疾病的发病率和死亡率。旁路试剂是可用的，但价格昂贵，而且不能常规给药，因为它们有血栓形成的风险。临床免疫耐受诱导(ITI)方案包括长时间频繁的高剂量因子给药，而且非常昂贵。生物工程已经创造了强大的试剂，可以非侵入性和经济高效地将蛋白质(或编码蛋白质的核酸)口服到肠道。在生物工程研究伙伴关系的应用中，我们建议推进这一治疗和口服耐受诱导的方法。最近，Daniell和Herzog实验室发现，口服生物囊化因子IX(以叶绿体转基因植物叶片材料的形式)有效地防止了血友病B小鼠在随后的蛋白质替代治疗中形成抑制性抗体和过敏反应。Leong实验室开发了一种用于血友病的口服基因疗法，该疗法基于包装在壳聚糖纳米颗粒中的质粒载体。因此，肠上皮口服给药既可以部分恢复血友病动物的止血(从而预防自发性出血)，也可以提供免疫调节(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)。为了建立对血友病A的有效口服疗法(血友病A是一种更流行的疾病，抑制物形成的风险要高得多)，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。具体地说，我们建议：i)确定纳米颗粒口服基因治疗在血友病小鼠模型中纠正凝血和抑制抗F.VIII抗体形成的能力；ii)培育用于口服输送和预防/逆转蛋白质替代中抑制物形成的F.VIII叶绿体转基因生菜；以及iii)扩大犬血友病A的口服治疗范围。最终，我们打算整合这些方法来提供一种完全基于口服的治疗方案，以完成对出血的预防和抑制物的形成。这种方法很容易适应其他遗传性蛋白质缺陷或某些过敏症。

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.