Nuclease-Resistant Aptamers for Anthrax Opsonization

描述（研究者提供）：炭疽芽孢杆菌抗吞噬聚d -谷氨酸（PDGA）胶囊是吸入性炭疽的主要毒力因子。虽然产生针对PDGA的抗体可能对植物性炭疽有价值，但这种抗体需要“人源化”。一种更简单、更便宜的方法是开发抗核酸酶（“屏蔽”）的DNA适体，在“杂交”适体的另一端带有Fc-TR或C3bR结合域，以对抗PDGA。在第一阶段，Operational Technologies （OpTech）与德克萨斯大学圣安东尼奥分校生物化学系（UTSA）合作，提议通过SELEX工艺开发针对PDGA、小鼠Fc-yR和C3bR的2'-氨基嘧啶修饰的屏蔽DNA适体。OpTech将连接适配体，并比较在存在和不存在屏蔽适配体以及血清调理的pga偶联微珠的情况下，RAW 264.7巨噬细胞样细胞系对pga偶联微珠的吞噬作用。由于聚阴离子PDGA似乎抑制了吞噬溶酶体的形成和杀死摄入的细菌，用2'-氨基修饰的适体靶向PDGA可能有助于部分中和聚阴离子电荷，并通过呼吸爆发杀死细菌。在II期，OpTech将克隆所有适配体并对其进行测序，然后在西南研究基金会（Southwest Research Foundation）对携带剧毒炭疽的RAW 264.7细胞中的适配体调控和细菌杀伤进行评估。由于炭疽和其他一些传染性细菌利用胶囊来逃避吞噬，任何廉价的试剂都可以作为有效的调理素，在医学、兽医或农业社区都具有巨大的价值。如果成功，OpTech将拥有一种在炭疽接触后治疗方面具有巨大商业价值的药物物质。例如，针对透明质酸或其他胶囊材料的屏蔽适体概念的进一步发展，也将导致对许多链球菌相关感染的更好治疗。这种屏蔽适体甚至可以用于吸入器，以增强肺泡巨噬细胞吞噬。

DESCRIPTION (provided by investigator): The anti-phagocytic poly-D-glutamic acid (PDGA) capsule of Bacillus anthracis is a major virulence factor of inhalation anthrax. Although generation of antibodies to PDGA might be of value in opsonizing vegetative anthrax, such antibodies would require "humanization". A simpler and less expensive approach would be the development of nuclease-resistant ('shielded') DNA aptamers against PDGA with an Fc-TR or C3bR binding domain at the other end of a 'hybrid' aptamer. In Phase I, Operational Technologies (OpTech) in conjunction with the Biochemistry Department of the University of Texas at San Antonio (UTSA), proposes to develop 2'- amino pyrimidine modified shielded DNA aptamers against PDGA, murine Fc-yR, and C3bR by the SELEX process. OpTech will link the aptamers and compare phagocytosis of PDGA-conjugated microbeads by the RAW 264.7 macrophage-like cell line in the presence and absence of shielded aptamers and versus serum opsonized PDGA-conjugated microbeads. Since polyanionic PDGA appears to inhibit phagolysosome formation and killing of ingested bacteria, targeting PDGA with 2'-amino-modified aptamers may serve to partially neutralize the polyanionic charge and enable bacterial killing by the respiratory burst. In Phase II, OpTech will clone and sequence all aptamers, then assess aptamer-opsonization and bacterial killing in RAW 264.7 cells with virulent anthrax at the Southwest Research Foundation. Since anthrax and some other infectious bacteria utilize capsules to evade phagocytosis, any inexpensive reagent that acts as an effective opsonin would be of tremendous value in the medical, veterinary or agricultural communities. If successful, OpTech will possess a pharmaceutical substance of great commercial value in the post-exposure treatment of anthrax. Further development of the concept for shielded aptamers against hyaluronic acid or other capsule materials, for example, would also lead to better treatments for many Streptococcus-related infections. Such shielded aptamers could even be used in inhalers to enhance alveolar macrophage phagocytosis.