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

描述（由申请人提供）：本申请拟利用纳米技术开发两种新的针对肺细胞的预防性和治疗性非病毒基因转移策略。肺特别适合这些治疗策略，因为与环境的直接接触为吸入cDNA和siRNA共轭纳米复合物提供了一个入口。甲型和乙型人类流感病毒耐药毒株的出现，以及H5N1禽流感病毒对一类或两类已批准的抗病毒药物具有大流行潜力，这突出了制定新的抗病毒策略的重要性。R21相的主要目的是在阳离子纳米粒子（即金纳米棒，GNR）和阴离子遗传物质（即cDNA或siRNA）之间构建静电复合物。这些纳米复合物将被设计成可以在大气道（如支气管）上皮细胞中被吸收并表达生物活性，而很少或没有不良的细胞或肺反应。最近刚刚合成的siRNA/cDNA构建体具有双重作用，即抑制流感毒力因子NS1的翻译，以及通过激活RIG-I途径独立刺激I型干扰素的产生。这种抗病毒先天免疫途径的刺激是由于附着在siRNA 5'端的三磷酸（PPP）片段。我们将在体内优先将GNR-5'PPP-NS1siRNA或其对应的cDNA纳米复合物施用于气管和支气管上皮，从而提高治疗的安全性。这些纳米技术方法的扩展也可以应用于治疗其他感染性和非感染性急性肺损伤。R33期的重点将是证明在流感之前和期间使用5'PPP-NS1siRNA和cDNA-nanoplex靶向体内大气道上皮细胞的治疗效果。除了评估呼吸道对流感病毒的清除外，R33期还将专门研究5'PPP-NS1siRNA或cdna -纳米复合物刺激先天抗病毒免疫的能力，从而改变炎症细胞因子环境、适应性免疫反应和抗菌宿主防御，以及预防或降低病毒诱导的呼吸道损伤程度和细菌清除障碍。我们预测，这些大型气道上皮靶向纳米复合物将导致预防和治疗选择，可以预防或显著降低流感症状的发病率和严重程度，包括高致病性H5N1“禽流感”和继发性细菌性肺炎的风险，后者是流感继发死亡的主要原因。我们的目标是在R33期完成后，开发一种纳米颗粒介导的新型抗病毒预防和治疗策略，并向FDA提交新药研究申请，以进行本申请中提出的实验的1期临床试验。

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.