Topically-delivered Targeted Gene Suppression of Immune Activation in Psoriasis

描述（由申请人提供）：AuraSense Therapeutics （AST）与西北大学一起，正在应用最近开发的一项技术，使用寡核苷酸功能化纳米颗粒（称为球形核酸或SNAs）来控制蛋白质表达。这些易于合成的纳米颗粒可以有DNA或RNA外壳，流体动力学直径小于50纳米。SNA构建体是调控基因表达的一种强有力的新途径。传统药物开发是一个越来越昂贵的命题。近年来，制药商一直在积极探索基因调控的潜力，以产生治疗癌症的线索。靶基因的鉴定进展迅速，但整个制药行业面临着安全有效地使用基于寡核苷酸的基因调控技术的重大挑战。主要障碍包括核酸稳定性、免疫相关毒性和递送挑战。寡核苷酸在血液循环和进入细胞时容易降解。保持它们的稳定性对于任何基因调控策略的治疗应用至关重要。不幸的是，传统基因调控中使用的“裸”基因序列不仅不稳定，而且会引起免疫反应，导致半衰期极短，疗效差。对于治疗皮肤疾病，局部给药是一个好主意，不仅可以集中在疾病部位进行干预，而且可以最大限度地减少全身毒性的风险；表皮屏障通常阻止核酸进入皮肤。产品SNA结构包括密集的球形阵列的高度定向寡核苷酸，模板上的纳米颗粒。这些材料已经被证明是显著的，因为它们很容易进入细胞，抵抗核酸酶降解，毒性低，导致最小的免疫刺激。关键的是，sna还能够穿透角质层并击倒表皮中的目标，使它们成为治疗皮肤病的潜在强大工具。利用最近关于IL-17和IL-22细胞因子在银屑病发展中的关键作用的前沿发现，AST和西北大学将设计同时靶向这些细胞因子（IL17RA和IL22RA1）的皮肤受体的SNAs，作为银屑病的局部治疗。这些SNAs将在体外和该疾病的小鼠模型中评估基因敲除效力、毒性和治疗效果。经过一段时间的初步开发，AST的目标是通过与艾伯维（Abbvie）、诺华（Novartis）或默克（Merck）等大型制药公司合作，将SNA技术商业化。本提案的具体目的如下：目的1：生成有效抑制小鼠和人角质形成细胞中的基因靶点的IL17RA/IL22RA1 SNAs。AST将设计和测试含有靶向小鼠和人IL17RA和IL22RA1的SiRNA，这是银屑病炎症通路中的关键受体。SNAs的有效性将通过与非靶向siRNA双链功能化的SNAs相比，靶向SNAs处理的角化细胞中mRNA和蛋白质水平的降低来衡量。目的2：评价IL17RA、IL22RA1和IL17RA/IL22RA1杂交SNAs对重组人表皮（RHE）银屑病模型的影响。使用细胞因子诱导的人体3-D模型，该模型在组织学、生化和遗传学上与牛皮癣相似，将进一步评估Aim 1中发现的最有效的个体和杂交人类SNAs。SNA的疗效将根据靶向受体mRNA和蛋白表达的减少以及细胞因子激活的下游产物，以及对银屑病异常分化的纠正来评估。目的3：在小鼠模型中评估IL17RA/IL22RA1 SNAs抑制银屑病表型的能力。我们将测试在Aims 1和2中发现的最有效的IL17RA和IL22RA1 SNAs，使用吡喹莫德诱导的模型来预防和治疗牛皮癣的能力。与二期工作的关系：如果这些研究显示出希望，我们将在另外两种小鼠模型（IL-23诱导小鼠耳中牛皮癣和人源化小鼠系统与人表皮移植）中进一步测试sna的功效，启动临床前毒性研究，理想情况下，为这些sna申请ID。

DESCRIPTION (provided by applicant): AuraSense Therapeutics (AST), along with Northwestern University, is applying a recently developed technology using oligonucleotide-functionalized nanoparticles (called spherical nucleic acids or SNAs) to control protein expression. These easily synthesized nanoparticles can have either DNA or RNA shells, and are less than 50 nm in hydrodynamic diameter. SNA constructs are a powerful new way of regulating gene expression. Significance Conventional drug development is an increasingly expensive proposition. In recent years, pharmaceutical manufacturers have pursued gene regulation's potential with aggressive efforts to generate therapeutic leads for cancer. Identification of target genes has proceeded quickly, but the pharmaceutical industry as a whole faces major challenges involving the safe and effective use of oligonucleotide-based gene regulation technology. The major barriers include nucleic acid stability, immune-related toxicity, and delivery challenges. Oligonucleotides are subject to degradation while circulating in the bloodstream and upon entry into cells. Preserving their stability is critical for therapeutic application of any gene regulation strategy. Unfortunately, the "naked" genetic sequences used in traditional gene regulation are not only unstable, but elicit an immune response, leading to extremely short half-lives and poor efficacy. For treating skin disorders, topical delivery is idea, not only by concentrating the intervention at the disease site, but also by minimizing the risk of systemic toxicity; the epidermal barrier has generally precluded the entry of nucleic acids into skin. The Product SNA constructs consist of a dense spherical array of highly oriented oligonucleotides, templated on a nanoparticle. These materials have already proven to be remarkable in that they readily enter cells, resist nuclease degradation, have low toxicity and lead to minimal immunostimulation. Critically, SNAs are also capable of penetrating the stratum corneum and knocking down targets in the epidermis, making them potentially very powerful tools to treat skin diseases. Leveraging recent cutting-edge discoveries about the key roles of IL-17 and IL-22 cytokines in the development of psoriasis, AST and Northwestern will design SNAs that concurrently target the skin-based receptors for these cytokines (IL17RA and IL22RA1) as topical treatment of psoriasis. These SNAs will be evaluated for gene knockdown potency, toxicity and therapeutic effect in vitro and in a mouse model of the disease. Long Term Goal After a period of initial development, AST aims to commercialize the SNA technology by partnering with a large pharmaceutical company such as Abbvie, Novartis or Merck. The Specific Aims of this proposal are the following: Aim 1: Generate IL17RA/IL22RA1 SNAs that efficiently suppress gene targets in mouse and human keratinocytes. AST will design and test SiRNA containing SNAs that target mouse and human IL17RA and IL22RA1, key receptors in the psoriasis inflammation pathway. The efficacy of the SNAs will be measured by the reduction in mRNA and protein levels in keratinocytes treated with targeted SNAs vs. SNAs functionalized with non-targeting siRNA duplexes. Aim 2: Evaluate the effect of IL17RA, IL22RA1 and IL17RA/IL22RA1 hybrid SNAs on a reconstituted human epidermis (RHE) model of psoriasis. Using a cytokine-induced human 3-D model that histologically, biochemically, and genetically resembles psoriasis, the most potent individual and hybrid human SNAs identified in Aim 1 will be further assessed. SNA efficacy will be evaluated based on the reduction in mRNA and protein expression of targeted receptors and the downstream products of cytokine activation, as well as by correction of the abnormal differentiation seen in psoriasis. Aim 3: Evaluate the abilit of IL17RA/IL22RA1 SNAs to suppress the psoriatic phenotype in a mouse model. We will test the ability of the most efficacious IL17RA and IL22RA1 SNAs, as identified in Aims 1 and 2, to prevent and treat psoriasis using an imiquimod-induced model. Relationship to Phase II Work: Should these studies show promise, we will further test the efficacy of SNAs in two additional mouse models (induction of psoriasis in mouse ears by IL-23 and a humanized mouse system with human epidermal grafting), initiate pre-clinical toxicity studies and, ideally, apply for an ID for these SNAs.