Topically-delivered Mutation-specific Gene Targeting for Epidermolytic Ichthyosis

描述(由申请人提供)：AuraSense Treateutics(AST)和西北大学正在应用一项最近开发的技术，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具有DNA或RNA外壳，其流体力学直径小于50 nm。SNA结构是调控基因表达的一种强有力的新方法。这些构建物由高度定向的寡核苷酸组成的致密球形阵列，以纳米颗粒为模板，具有易于进入细胞、抗核酸酶降解、低毒和最小免疫刺激作用等显著特点。关键的是，SNAs还能够穿透生物屏障，如角质层和血脑屏障，增加可通过基因调控治疗的器官数量。AST和西北大学将设计针对KRT10 R156H(热点)突变的SNA，用于治疗表皮松解性鱼鳞病(EI)，这是一种改变生活的孤儿皮肤病，是显性负性角蛋白基因疾病的原型。这些SNAs将在体外和重建的人类表皮疾病模型中进行评估。在第一阶段，我们将选择通过识别R156H位点选择性地击倒突变KRT10的siRNA SNAs，并将测试局部应用的突变特异性敲除对患者KCs产生的3-D器官型RAFT培养的功能影响。目的1：利用KRT10SNAs对EI患者永生化KCs中热点KRT10R156H/c.467G&gt；A突变进行特异性敲除。首先，将候选SNAs和对照(打乱的SNAs或载体)添加到原型c.467G&gt；EI永生化细胞或对照永生化KCs的24孔板中，并在72小时通过等位基因特异性RT-qPCR筛选突变和野生型基因的敲除。目标是通过RT-PCR检测突变型和野生型K10基因分别超过70%和低于20%的敲除。目的2：抑制重组人表皮替代物中的K10热点突变。AIM 1中表现最好的两个SNA将在培养的重组人表皮等价物(3-D RAFT培养)中进行测试，这些细胞来自正常和EI患者的角质形成细胞。我们的目标是通过RT-PCR观察到突变基因和野生型K10基因的敲除超过70%和不到20%，并在临床和组织学上逆转重建的三维人类表皮模型中的缺陷。在第二阶段的研发工作中，我们将在人源化的移植小鼠中验证这种方法对EI的控制能力。我们还将在第二阶段展示SNA的安全性，使我们能够在计划第二阶段完成后进入商业合作伙伴关系，并拥有足够的数据来提交IND。

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. These constructs consist of a dense spherical array of highly oriented oligonucleotides, templated on a nanoparticle, and are remarkable in their ability to readily enter cells, resistance to nuclease degradation, low toxicity and minimal immunostimulation. Critically, SNAs are also capable of penetrating biological barriers such as the stratum corneum and blood brain barrier, increasing the number of organs that can be treated using gene regulation. AST and Northwestern will design SNAs against the KRT10 R156H (hotspot) mutation for the treatment of epidermolytic ichthyosis (EI), a life-altering orphan skin disease that is a prototype for dominant negative keratin gene disorders. These SNAs will be evaluated in vitro and in a reconstituted human epidermal model of the disease. In Phase I we will choose the siRNA SNAs that knock down selectively the mutant KRT10 by recognizing specifically the R156H site and will test the functional impact of topically-applied mutation-specific knockdown in 3-D organotypic raft cultures generated from patient KCs. Aim 1: Optimize the specific knockdown of the hotspot KRT10 R156H/c.467G>A mutation in immortalized KCs isolated from patients with EI using KRT10 SNAs. Initially, candidate SNAs, and controls (scrambled SNAs or vehicle) will be added in 24-well plates to a prototype c.467G>A EI immortalized cell line or control normal immortalized KCs, and knockdown of the mutant and wild type genes will be screened by allele-specific RT- qPCR at 72 h. The goal is to observe greater 70% knockdown and less than 20% knockdown of the mutant and wild-type K10 gene, respectively, as measured with RT-PCR. Aim 2: Suppress the K10 hot-spot mutation in reconstituted human epidermal equivalents. The two SNAs that perform best from Aim 1 will be tested in cultured reconstituted human epidermal equivalents (3-D raft cultures) from normal and EI patient keratinocytes. Our goal is to observe greater 70% knockdown of the mutant gene and less than 20% knockdown of the wild-type K10 gene as measured with RT-PCR, and to reverse the defect clinically and histologically in the reconstituted 3-D human epidermal model. Relationship to Phase II. In a Phase II research and development effort, we will validate this approach in humanized grafted mice for their ability to control EI. We will also demonstrate SNA safety in Phase II, allowing us to enter into a commercial partnership upon completion of Phase II of the program and have sufficient data for filing an IND.