Oral immune modulatory therapy using antigens bioencapsulated in plant cells

描述（由申请人提供）：目前许多遗传性蛋白质缺乏症的治疗是基于重组蛋白的静脉输注。注入的蛋白质很昂贵，并且经常遇到抗体反应，使治疗中和，从而使治疗复杂化并进一步增加成本。它们还会引起严重的过敏甚至危及生命的过敏反应。口服免疫调节疗法是预防此类反应的一种理想的抗原特异性方法，因为它是非侵入性的，不需要免疫抑制或对患者细胞进行遗传操作。该方案利用了一个独特的概念，即利用植物叶绿体作为高效生物反应器，生产和口服治疗性蛋白抗原，而不需要发酵、纯化、冷链或无菌注射。生物包封抗原在胃中受到酸或酶的保护，但当植物细胞壁被共生细菌消化时，就会在肠道中释放出来。我们建议进一步推进这种方法，使抗原-配体融合物有效地递送到肠道相关淋巴组织。我们也在开发一种适合临床翻译的口服给药系统（基于可食用作物生菜）方面取得了实质性进展。在x连锁出血性疾病血友病的治疗中，针对治疗性凝血因子的抑制性抗体的形成是一种严重的并发症，它大大增加了发病率和死亡率。例如，由于因子IX （F.IX）基因缺失或提前终止密码子而患有B型血友病的患者，其抑制剂形成的风险增加。大多数fix抑制剂不仅高滴度且难以消除，而且这些抗体通常与潜在的危及生命的过敏反应有关。最近，我们证明口服由烟草叶绿体产生并被生物包裹在植物细胞中的fix，可以防止血友病B蛋白替代治疗中抑制剂的形成和过敏反应（PNAS 107:7101）。这些结果说明了我们的方法对免疫调节治疗遗传性蛋白质缺乏症的有效性。本提案的目标是为B型血友病制定一种口服免疫调节方案，以预防或减轻致病性抗体反应（包括过敏反应）；剖析抗原呈递和免疫调节的潜在机制；寻找可替代的经黏膜抗原递送载体；对具有类似抗体/过敏反应的不同疾病（溶酶体贮积障碍庞贝病）的方法进行测试；开发表达fix的转基因生菜，用于人类口服；研究冻干转基因材料的稳定性和微生物污染情况；并在大型血友病b动物模型中进行转化研究。我们假设我们的低成本方法可以用于预防几种疾病，如血友病和溶酶体贮积症，从而消除对免疫抑制的需要。

DESCRIPTION (provided by applicant): Current treatment for many inherited protein deficiencies is based on intravenous infusion of recombinant proteins. The infused protein is expensive and is often encountered by antibody responses that neutralize therapy, thereby complicating treatment and further increasing costs. They also cause severe allergic or even life-threatening anaphylactic reactions. Oral immune modulatory therapy represents an ideal antigen-specific approach to prevent such responses because it is non-invasive and does not require immune suppression or genetic manipulation of the patient's cells. This proposal takes advantage of a unique concept based on the use of plant chloroplasts as highly efficient bioreactors for production and oral delivery of therapeutic protein antigens without a need for fermentation, purification, cold chain, or sterile injections. Bioencapsulated antigens are protected in the stomach from acids or enzymes, but are released in the gut when plant cell walls are digested by commensal bacteria. We propose to further advance this method for effective delivery of antigen-ligand fusions to the gut-associated lymphoid tissue. We have also made substantial progress towards development of an oral delivery system (based on the edible crop lettuce) suitable for clinical translation. In treatment of the X-linked bleeding disorder hemophilia, formation of inhibitory antibodies against the therapeutic clotting factor represents a serious complication that substantially increases morbidity and mortality. For example, patients with hemophilia B due to deletion or early stop codon in the factor IX (F.IX) gene are at increased risk for inhibitor formation. Not only are most F.IX inhibitors high-titer and difficult to eliminate, but these antibodies are often associated with potentially life-threatening anaphylactic reactions. Recently, we demonstrated that oral delivery of F.IX, produced by tobacco chloroplasts and bioencapsulated in plant cells, prevents formation of inhibitors and anaphylaxis in protein replacement therapy for hemophilia B (featured in PNAS 107:7101). These results illustrate the effectiveness of our approach towards immune modulatory therapy for inherited protein deficiencies. The goals of this proposal are to develop an oral immune modulatory protocol for hemophilia B that prevents or mitigates pathogenic antibody responses (including anaphylaxis); to dissect the underlying mechanisms of antigen presentation and immune regulation; to find alternative transmucosal carriers for effective antigen delivery; to test the approach for a different disease with similar antibody/anaphylactic response (the lysosomal storage disorder Pompe disease); to develop transplastomic lettuce to express F.IX for future oral delivery in humans; to investigate stability and microbial contamination of the freeze-dried transgenic material; and to perform translational studies in a large animal model of hemophilia B. We hypothesize that our low cost approach can be utilized in a prophylactic fashion in several diseases, such as hemophilia and lysosomal storage disorders, thereby eliminating the need for immune suppression.