Evaluation of siRNA uptake and functional activity in a human organotypic skin mo

描述(由申请人提供)：尽管RNA干扰作为一种治疗皮肤病的新方法具有很高的潜力，但交付方面的担忧阻碍了它们进入临床。传递具有功能活性的siRNA的最有效和最有效的方法是皮内注射。我们已经证明，大量皮内注射产生的高压足以将核酸输送到皮肤(类似于将siRNA输送到肝脏的高压尾静脉注射)。事实上，在最近完成的1b期临床试验中，皮内注射大量我们的主要临床siRNA抑制剂TD101到足部皮损，导致先天性厚甲症状显著改善。与这种给药模式相关的剧烈疼痛(需要口腔止痛药和区域神经阻滞来缓解疼痛)似乎阻止了这种方法的未来使用。皮内注射的使用绕过了角质层屏障，并提供了一种流体动力压力，似乎有助于角质形成细胞摄取siRNA。我们和其他人已经开发出患者友好的技术，允许siRNA穿过角质层屏障输送，包括局部制剂(GeneCream)以及穿透表皮并释放其siRNA货物的中空可溶微针阵列。不幸的是，如果没有与大容量注射相关的压力，几乎没有功能的siRNA进入细胞。我们的长期目标是找到对患者友好的技术，使siRNA能够有效和高效地输送到适当的皮肤间隔，并促进角质形成细胞的摄取和整合到RNA诱导沉默复合体(RISC)中。在这个第一阶段的方案中，我们开发了一个相当于人类表皮的系统(即再生的人类皮肤)，其中可以监测先前存在的基因表达(CD44、突变角蛋白6a和/或报告)。该模型系统将用于评估和优化Traversa的PTD-DRBD siRNA传递技术(促进细胞对siRNA的摄取)以及其他商业化的传递摄取技术，包括英维夫他明、Accell等。其中最有前途的将与我们的角质层传递技术(GeneCream、微针阵列和/或离子导入)结合使用。皮肤等效物将从转导的(带有EGFP报告的)或未经处理的正常原代人类角质形成细胞以及来自先天性厚甲患者活检组织的角质形成细胞制备。在第二阶段，第一阶段确定的最好的技术将在小鼠模型中进行测试，包括携带人类皮肤移植的免疫低下的小鼠。这些技术的最有效组合(S)随后将接受毒理学和药代动力学研究，为临床评估做准备。 与公共卫生相关：尽管对导致大量皮肤病的潜在基因和突变有令人兴奋的发现，但几乎没有出现任何新的临床治疗方法。这项建议的目的是利用RNA干扰来选择性地抑制疾病相关基因的表达。在第一阶段，我们开发了人类皮肤的等效物(器官型皮肤模型)，在这种模型中，可以很容易地监测基因表达以成功传递功能性siRNA，并测试促进皮肤角质形成细胞摄取siRNA的siRNA技术，与允许跨越角质层皮肤屏障的现有技术相结合。在第二阶段，我们将在动物模型中测试第一阶段开发的技术。最佳技术组合(S)将在GLP条件下进一步评估动物毒理学和药代动力学分析，为人体试验做准备。

DESCRIPTION (provided by applicant): Although RNA interference offers high potential as a novel therapeutic approach for treating skin disorders, delivery concerns have hampered their progression to the clinic. The most efficient and effective way to deliver functionally-active siRNA is through intradermal injection. We have shown that the high pressure generated by intradermal injection of large volumes is sufficient to deliver nucleic acid to skin (similar to what is observed in high pressure tail vein injection that delivers siRNA to the liver). Indeed, the intradermal injection of large volumes of our lead clinical siRNA inhibitor TD101 to foot lesions led to marked improvement in pachyonychia congenita symptoms in a recently completed Phase 1b clinical trial. The intense pain associated with this mode of administration (oral pain medication and regional nerve blocks were required to mitigate the pain) appears to prevent future use of this method. The use of intradermal injection bypasses the stratum corneum barrier and also provides a hydrodynamic pressure that appears to facilitate siRNA uptake by keratinocytes. We, and others, have developed patient-friendly technologies that allow siRNA to be delivered across the stratum corneum barrier, including a topical formulation (GeneCream) as well as hollow dissolvable microneedle arrays that penetrate into the epidermis and release their siRNA cargo. Unfortunately, without the pressure associated with injection of high volumes, little functional siRNA enters the cell. Our long-term goal is to identify patient-friendly technologies that will allow effective and efficient siRNA delivery into the appropriate skin compartment and also facilitate uptake by keratinocytes and incorporation into the RNA induced silencing complex (RISC). In this Phase I proposal, we develop a human epidermal equivalent system (i.e. regenerated human skin), in which pre-existing gene expression (CD44, mutant keratin 6a and/or reporter) can be monitored. This model system will be used to evaluate and optimize Traversa's PTD-DRBD siRNA delivery technology (facilitates siRNA uptake by cells) as well as other commercially-available delivery uptake technologies including Invivofectamine, Accell, etc. The most promising of these will be used in combination with our stratum corneum delivery technologies (GeneCream, microneedle arrays and/or iontophoresis). Skin equivalents will be prepared from transduced (with EGFP reporter) or untreated normal primary human keratinocytes as well as keratinocytes derived from pachyonychia congenita patient biopsies. In Phase 2, the best technologies identified in Phase 1 will be tested in mouse models including immunocompromised mice harboring human skin grafts. The most effective combination(s) of these technologies will then be subjected to toxicology and pharmacokinetics studies in preparation for evaluation in the clinic. PUBLIC HEALTH RELEVANCE: Despite the exciting discoveries of the underlying genes and mutations responsible for a large number of skin disorders, few if any novel clinical treatments have emerged. The purpose of this proposal is to exploit RNA interference to selectively inhibit expression of disease-relevant genes. In Phase 1, we develop human skin equivalents (organotypic skin model) in which gene expression can be readily monitored for successful delivery of functional siRNA and test siRNA technologies that facilitate siRNA uptake by skin keratinocytes in combination with existing technologies that allow delivery across the stratum corneum skin barrier. In Phase 2, we will test the technology developed in Phase 1 in animal models. The best combination(s) of technologies will be further evaluated under GLP conditions in animal toxicology and pharmacokinetic assays in preparation for human trials.