Oral immune modulatory therapy using antigens bioencapsulated in plant cells

描述（由申请人提供）：目前对许多遗传性蛋白质缺乏症的治疗是基于重组蛋白的静脉输注。输注的蛋白质是昂贵的，并且经常遇到中和治疗的抗体应答，从而使治疗复杂化并进一步增加成本。它们也会引起严重的过敏甚至危及生命的过敏反应。口服免疫调节疗法代表了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，并且不需要对患者细胞进行免疫抑制或遗传操作。该提案利用了基于使用植物叶绿体作为高效生物反应器的独特概念，用于生产和口服递送治疗性蛋白抗原，而不需要发酵、纯化、冷链或无菌注射。生物包囊抗原在胃中受到保护，不受酸或酶的影响，但当植物细胞壁被肠道细菌消化时，会在肠道中释放。我们建议进一步推进这种方法的有效交付的抗原配体融合肠道相关淋巴组织。我们还在开发适用于临床转化的口服给药系统（基于可食用作物莴苣）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代表严重并发症，其显著增加发病率和死亡率。例如，由于因子IX（F.IX）基因中的缺失或早期终止密码子而患有血友病B的患者具有增加的抑制物形成风险。大多数F.IX抑制剂不仅效价高且难以消除，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F.IX，产生烟草叶绿体和生物封装在植物细胞中，防止抑制剂的形成和过敏性蛋白质替代治疗血友病B（在PNAS 107：7101的特点）。这些结果说明了我们的方法对遗传性蛋白质缺陷的免疫调节治疗的有效性。 本提案的目标是开发一种预防或减轻致病性抗体反应的血友病B口服免疫调节方案（包括过敏反应）;剖析抗原呈递和免疫调节的潜在机制;寻找有效抗原递送的替代经粘膜载体;测试具有类似抗体/过敏反应的不同疾病的方法（溶酶体贮积症庞贝氏病）;开发转质体莴苣以表达F.IX用于将来在人类中的口服递送;研究冻干转基因材料的稳定性和微生物污染;以及在血友病B的大型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几种疾病的预防，如血友病和溶酶体贮积症，从而消除了对免疫抑制的需要。

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.