Oral immune modulatory therapy using antigens bioencapsulated in plant cells

描述(申请人提供)：目前对许多遗传性蛋白质缺乏症的治疗是基于静脉输注重组蛋白质。注入的蛋白质价格昂贵，而且经常会遇到抗体反应，从而中和治疗，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反应，甚至危及生命的过敏反应。口服免疫调节疗法是预防这种反应的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的遗传操作。这一建议利用了一种独特的概念，即利用植物叶绿体作为高效的生物反应器来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。生物包裹的抗原在胃中受到酸或酶的保护，但在植物细胞壁被共生细菌消化时在肠道中释放。我们建议进一步发展这一方法，以有效地将抗原-配体融合传递到肠道相关淋巴组织。我们还在开发适用于临床翻译的口服递送系统(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，大大增加了发病率和死亡率。例如，由于因子IX(F.IX)基因缺失或过早停止密码子而导致的血友病B患者形成抑制物的风险增加。大多数FIX抑制剂不仅效价高，难以消除，而且这些抗体经常与潜在威胁生命的过敏反应有关。最近，我们证明了口服F.IX，由烟草叶绿体产生，并被生物包裹在植物细胞中，在血友病B的蛋白质替代疗法中防止抑制剂的形成和过敏反应(见PNAS 107：7101)。这些结果说明了我们对遗传性蛋白质缺陷的免疫调节治疗方法的有效性。这项建议的目标是为血友病B开发一种口服免疫调节方案，以预防或减轻致病抗体反应(包括过敏反应)；剖析抗原呈递和免疫调节的潜在机制；寻找有效的抗原递送的替代跨粘膜载体；测试针对具有类似抗体/过敏反应的不同疾病(溶酶体储存障碍庞培病)的方法；开发转体生菜以表达F.IX以用于未来的人类口服；调查冻干转基因材料的稳定性和微生物污染；并在血友病B的大型动物模型中进行翻译研究。我们假设，我们的低成本方法可以以预防的方式用于几种疾病，如血友病和溶酶体储存障碍，从而消除免疫抑制的需要。

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.