Self-assembling nanoparticles for intranasal delivery of influenza fusion inhibitors

 描述(申请人提供)：迫切需要新的流感抗病毒疗法，以补充疫苗接种和现有药物，并加强全球努力，控制流行病和潜在的新流行病。面对现有药物出现的抗病毒耐药性，新的抗流感治疗至关重要。这项研究计划的长期目标是开发一种安全高效的鼻内流感血凝素(HA)衍生抑制剂，用于预防未接种疫苗的高危人群。在初步工作中，我们通过将脂质连接到来自HA C-末端区域的特定多肽，并添加细胞穿透性多肽序列来进行细胞内靶向，生成了流感融合抑制剂。我们发现鼻腔给药我们的先导融合抑制物可以提供抗病毒作用。 在体内，预防与批准的药物瑞乐沙(C)一样有效。融合抑制肽自组装成约30-50 nm的纳米颗粒，并被靶细胞内化。我们计划优化候选抗病毒多肽，并在体内对它们进行评估，为人类使用奠定基础。为此，我们建议提高：1)抗病毒效力；2)肽自组装；3)靶细胞膜插入；4)体内生物分布。我们的目标是产生候选多肽，当给药时，保护人类呼吸道上皮，防止病毒感染。这些目标将通过两个具体的目标来实现：1.使用结构导向突变和蛋白质工程来优化流感多肽融合抑制剂的抗病毒效力和生物利用度。系统的结构方法将被用来在抑制剂结合界面加入特定的残基取代，从而增加抑制剂与其靶标的结合能。利用生物物理分析和生物工程的结合，我们将 优化抑制剂的特性--包括颗粒稳定性和内体定位--以便将IC50降低到纳摩尔值。我们将进行体外和体外研究，以评估工程抑制肽的抗病毒活性。2.评价优化的自组装肽对棉鼠感染流感的保护作用。我们将评估优化后的纳米粒的生物分布和毒性特性，并评估它们在体内的抗流感效力。在迭代过程中，实验结果将指导进一步的优化，产生一套有前景的研究抗流感药物。

 DESCRIPTION (provided by applicant): New antiviral therapy for influenza is urgently needed to complement vaccination and existing drugs, and to strengthen global efforts to control epidemics and potential new pandemics. New anti-influenza treatments are critical in the face of emerging antiviral resistance to the existing drugs. The long-term objective of this research plan is to develop a safe and highly effective intranasal influenza hemagglutinin (HA)-derived inhibitor as prophylaxis for use in high-risk unvaccinated populations. In preliminary work, we have generated influenza fusion inhibitors by conjugating lipid to specific peptides derived from the C-terminal region of HA and adding cell penetrating peptide sequences for intracellular targeting. We showed that intranasal administration of our lead fusion inhibitor provides antiviral prophylaxis as efficient as the approved drug Relenza(c) in vivo. The fusion inhibitory peptides self-assemble into ~30- 50 nm nanoparticles and are internalized by the target cells. We plan to optimize the candidate antiviral peptides and assess them in vivo to lay the groundwork for human use. To do so, we propose to enhance: 1) antiviral potency; 2) peptide self-assembly; 3) target cell membrane insertion; 4) in vivo biodistribution. We aim to generate candidate peptides that, when administered intranasally, protect the human airway epithelium and prevent viral infection. These goals will be accomplished with two specific aims: 1. Use structure-guided mutagenesis and protein engineering to optimize the antiviral potency and bioavailability of influenza peptide fusion inhibitors. A systematic structural approach will be used to incorporate specific residue substitutions at the inhibitor binding interface that increase the binding energy f the inhibitor to its target. Using a combination of biophysical analysis and bioengineering we will optimize the inhibitors' features -- including particle stability and endosomal localization -- in order to lower the IC50 to nanomolar values. We will conduct in vitro and ex vivo studies to assess the antiviral activity of the engineered inhibitory peptides. 2. Evaluate the protection against influenza infection afforded by optimized self- assembling peptides in cotton rats. We will evaluate the bio-distribution and toxicity properties of the optimized nanoparticles, and assess their in vivo anti-influenza potency. In an iterative process, the outcome of the experiments will guide further optimization, yielding a set of promising investigational anti-influenza agents.