Targeted-delivery of small interference RNA against anthrax (1 R21 AI118228-01A1)

 描述：针对炭疽的靶向递送小干扰RNA。炭疽病是一种由非洲芽孢杆菌引起的严重疾病，这种细菌会形成孢子。炭疽病最常发生在野生和家养哺乳动物物种中；但也可能发生在人类接触受感染的动物或动物组织时，或当炭疽孢子作为生物恐怖武器传播时。随着获得许可的疫苗BioThrax的复杂免疫计划，需要一种新的、易于管理的炭疽疫苗。由于炭疽病是一种很少在人类身上自然发生的疾病，因此制定暴露后预防措施更为现实。 或者是治疗，而不是像目前的疫苗那样进行大规模免疫。暴露后，巨噬细胞摄取炭疽孢子并传播到这些孢子萌发的淋巴结处。然后，炭疽杆菌被释放到血液中，产生毒素，这些毒素是疾病毒力的关键因素：保护性抗原(PA)、浮肿因子(EF)和致死因子(LF)。PA是结合两种宿主细胞受体的受体毒素成分：炭疽毒素受体1(ANTXR1或肿瘤内皮标记物8/TEM8)和炭疽毒素受体2(ANTXR2或毛细血管形态发生蛋白2/CMG2)。结合后，PA被切割，与受体结合的部分形成一个七聚体孔，与EF或LF结合。毒素复合体被内吞并输送到细胞质中。LeTx和EdTx的活性导致免疫系统功能障碍、水肿、休克和死亡。我们的初步研究表明，用RNA干扰(RNAi)技术用特异性的抗ANTXR小干扰RNA(SiRNA)抑制ANTXR的表达可以防止炭疽毒素的细胞毒性。我们推测，解毒的炭疽毒素可以作为抗ANTXR siRNA的载体。为了评估这一假设，我们提出了以下三个特定目标：特定目标1：为ANTXR靶向siRNA传递产生解毒的炭疽毒素。具体目的2：评价其对炭疽毒素诱导的ANTXR沉默细胞毒性的抑制作用。具体目的3：评价抗ANTXR siRNA治疗小鼠炭疽病的疗效。我们预计，拟议的宿主靶向治疗策略将防止因自然感染或生物恐怖袭击而暴露于野生型甚至抗药性炭疽芽胞的患者发生严重疾病和死亡。此外，这项技术可以开发为治疗其他抗菌素耐药病原体的平台，这些病原体使用造孔毒素作为毒力因子。

 DESCRIPTION: Targeted-delivery of small interference RNA against anthrax. Anthrax is a serious disease caused by Bacillus afgfnthracis, a bacterium that forms spores. Anthrax most commonly occurs in wild and domestic mammalian species; but can also occur in humans when they are exposed to infected animals or animal tissues, or when anthrax spores are dispersed as a bioterrorist weapon. The complicated immunization schedule with the licensed vaccine BioThrax calls for a new and easily administered anthrax vaccine. Since anthrax is a disease that rarely occurs naturally in humans, it is more realistic to develop a post exposure prophylaxis or therapy instead of mass immunization, as with the current vaccine. Following exposure, macrophages ingest anthrax spores and travel to the lymph node where these spores germinate. The B. anthracis bacteria are then released into the bloodstream and produce toxins that are key factors in the virulence of disease: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA is the receptor binding toxin component that attaches to either of two host cell receptors: anthrax toxin receptor 1 (ANTXR1 or tumor endothelial marker 8/TEM8) and anthrax toxin receptor 2 (ANTXR2 or capillary morphogenesis protein 2/CMG2). After binding, PA is cleaved and the receptor-bound portions form a heptameric pore that binds EF or LF. The toxin complexes are endocytosed and delivered into the cytosol. The activities of LeTx and EdTx result in malfunction of the immune system, edema, shock, and death. Our preliminary study has shown that inhibition of ANTXR expression by RNA interference (RNAi) technology using specific anti-ANTXR small interference RNA (siRNA) could prevent cytotoxicity of anthrax toxins. We hypothesize that a detoxified anthrax toxin could be used as a delivery vehicle for anti-ANTXR siRNA. In order to evaluate this hypothesis, we propose the following three specific aims: Specific Aim 1: To generate detoxified anthrax toxins for ANTXR-targeted siRNA delivery. Specific Aim 2: To assess the inhibitory effect on anthrax toxin induced cytotoxicity in ANTXR-silenced cells. Specific Aim 3: To evaluate efficacy of the anti-ANTXR siRNA treatment in a mouse model of anthrax. We anticipate that the proposed host-targeted treatment strategy will prevent severe illness and death in patients exposed to both wild type and even antibiotic-resistant B. anthracis spores by natural infection or a bioterrorist attack. Furthermore, this technology can be developed as a platform to treat other antimicrobial-resistant pathogens that employ pore-forming toxins as virulence factors.