Influenza therapy by Au-nanorod 5'PPP-NS1-siRNA/cDNA targeting of bronchial cells

描述(申请人提供)：这项申请建议开发两种新的预防性和治疗性非病毒基因转移策略，利用纳米技术在体内靶向肺细胞。肺特别适合于这些治疗策略，因为与环境的直接接触为吸入cDNAs和siRNA结合的纳米复合体提供了一个入口。人类甲型和乙型流感病毒耐药株的出现，以及对一种或两种经批准的抗病毒药物具有大流行潜力的H5N1禽流感病毒的出现，突显了开发新的抗病毒战略的重要性。R21阶段的主要目标是在阳离子纳米颗粒(即金纳米棒，GNR)和阴离子遗传物质(即cDNAs或siRNA)之间构建静电复合体。这些纳米网络将被设计成可以在大气道(即支气管)上皮细胞中被摄取并表达生物活性，而几乎没有或没有不良的细胞或肺反应。最近合成的siRNA/cDNA体具有抑制流感毒力因子NS1翻译的双重作用，并通过激活RIG-I途径独立地刺激I型干扰素的产生。这一抗病毒天然免疫途径的刺激是由于siRNA 5‘端连接的三磷酸(PPP)部分所致。我们将在体内优先将GNR-5‘PPP-NS1siRNA或其对应的cDNA纳米丛应用于气管和支气管上皮，从而增加治疗的安全性。这些纳米技术方法的推广也可用于治疗其他感染性和非传染性急性肺损伤。R33阶段的重点将是展示在流感前和流感期间使用5‘PPP-NS1siRNA和cDNA-纳米复合体靶向体内大呼吸道上皮细胞的治疗效果。除了评估流感病毒从呼吸道的清除情况外，R33阶段还将具体检查5‘PPP-NS1siRNA或cDNA-纳米网络刺激天然抗病毒免疫的能力，导致炎性细胞因子环境的改变、适应性免疫反应和抗菌宿主防御，以及预防或减轻病毒引起的呼吸损伤和细菌清除障碍的程度。我们预测，这些以大呼吸道上皮为靶标的纳米网络将导致预防和治疗方案，可以预防或显著降低流感症状的发病率和严重性，包括高致病性H5N1“禽流感”和继发性细菌性肺炎的风险，继发性细菌性肺炎是流感继发死亡的主要原因。我们的目标是在R33阶段完成后，有一种纳米颗粒介导的新型抗病毒预防和治疗策略，可供FDA提交研究新药申请，作为本申请中建议的实验的结果，进行第一阶段临床试验。 与公共卫生相关：流感是美国和世界人民的头号杀手之一，流感病毒耐药株(包括“禽流感”)的出现要求我们开发新的预防和治疗方法来应对这种疾病。这项应用提出了一种新的方法，利用气雾剂吸入器将基因(CDNAs)或其即时信息(SiRNA)转移到排列在肺大通道上的细胞。将cDNA或siRNA附着在小的(纳米尺寸)金棒上将有助于将抗流感治疗传递到呼吸道细胞内，在那里它将发挥作用。这种治疗增强了对流感病毒的免疫力，并干扰了病毒对呼吸道造成损害的能力。

DESCRIPTION (provided by applicant): This application proposes to develop two novel prophylactic and therapeutic non-viral gene transfer strategies that target pulmonary cells in vivo employing nanotechnology. The lung is especially well suited for these treatment strategies as direct contact with the environment provides a portal for inhalation administration of cDNA and siRNA conjugated nanoplexes. The emergence of drug-resistant strains of human influenza A and B viruses, as well as avian H5N1 influenza viruses with pandemic potential to one or both classes of approved antiviral agents underscores the importance of developing novel antiviral strategies. The primary objective of the R21 phase is to construct an electrostatic complex between a cationic nanoparticle (i.e., Gold Nanorods, GNR) and anionic genetic material (i.e., cDNA or siRNA). These nanoplexes will be engineered such that they can be taken-up and express bioactivity in large airway (i.e., bronchial) epithelial cells with little or no untoward cellular or pulmonary responses. The siRNA/cDNA constructs, which have just recently been synthesized, have dual actions of suppressing the translation of the influenza virulence factor, NS1, as well as independently stimulating type I interferon production through activation of the RIG-I pathway. Stimulation of this antiviral innate immune pathway occurs as a result of a triphosphate (PPP) moiety attached to the 5' end of the siRNA. We will preferentially administer the GNR-5'PPP-NS1siRNA or its counterpart cDNA nanoplexes to the tracheal and bronchial epithelium in vivo, thereby increasing the safety of the treatment. Extension of these nanotechnological approaches can also be applied to treat other infectious, as well as non-infectious acute lung injuries. The focus in the R33 phase will be to demonstrate the therapeutic efficacy of using 5'PPP-NS1siRNA and cDNA-nanoplex targeting of large airway epithelial cells in vivo before and during influenza. In addition to assessing the clearance of influenza virus from the respiratory tract, the R33 phase will specifically examine the ability of 5'PPP-NS1siRNA or cDNA-nanoplexes to stimulate innate antiviral immunity, resulting in alteration of the inflammatory cytokine milieu, adaptive immune response, and antibacterial host defense, as well as prevent or reduce the degree of viral induced respiratory injury and impairment of bacterial clearance. We predict that these large airway epithelial-targeted nanoplexes will lead to prophylactic and therapeutic options that can prevent or significantly reduce the morbidity and severity of symptoms of influenza including the highly pathogenic H5N1 "bird flu" and the risk of secondary bacterial pneumonia, which is the major cause of death secondary to influenza. It is our goal to have a nanoparticle mediated novel antiviral prophylactic and therapeutic strategy at the completion of the R33 phase available for Investigational New Drug filing with the FDA to go for Phase 1 clinical trials as a result of the experiments proposed in this application. PUBLIC HEALTH RELEVANCE: Influenza is one of the top killers of people in the USA and the world, and the emergence of drug-resistant strains of influenza virus (including the "Bird Flu") requires that we develop new preventative and treatment approaches to this disease. This application proposes to develop a novel method to transfer a gene (cDNA) or its immediate message (siRNA) to cells that line the large airways of the lung employing an aerosol inhaler. Attaching cDNA or siRNA to small (nanometer size) gold rods will help deliver the anti-influenza treatment to inside the airway cells where it will work. This treatment stimulates immunity against influenza virus, as well as interferes with the virus' ability to do damage to the airways.