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疾病的新方法具有很高的潜力，但对递送的担忧阻碍了其临床进展。传递功能活性siRNA最有效的方法是皮内注射。我们已经证明，大容量皮内注射产生的高压足以将核酸传递到皮肤（类似于在高压尾静脉注射中观察到的将siRNA传递到肝脏）。事实上，在最近完成的一项1b期临床试验中，将我们的主要临床siRNA抑制剂TD101皮内注射到足部病变处，可以显著改善先天性肿甲的症状。与这种给药方式相关的剧烈疼痛（需要口服止痛药和局部神经阻滞来减轻疼痛）似乎阻止了这种方法的未来使用。皮内注射绕过角质层屏障，也提供了一种流体动力压力，似乎有助于角化细胞摄取siRNA。我们和其他公司已经开发了患者友好型技术，允许siRNA通过角质层屏障传递，包括局部配方（GeneCream）以及空心可溶解微针阵列，这些微针阵列可以穿透表皮并释放siRNA。不幸的是，如果没有与高容量注射相关的压力，很少有功能性siRNA进入细胞。我们的长期目标是确定对患者友好的技术，使siRNA能够有效和高效地递送到适当的皮肤隔间中，并促进角质形成细胞的吸收，并结合到RNA诱导沉默复合物（RISC）中。在这个I期计划中，我们开发了一个人类表皮等效系统（即再生的人类皮肤），其中可以监测预先存在的基因表达（CD44，突变角蛋白6a和/或报告基因）。该模型系统将用于评估和优化Traversa的PTD-DRBD siRNA传递技术（促进siRNA被细胞摄取）以及其他商业上可用的传递摄取技术，包括Invivofectamine， Accell等。其中最有希望的将与我们的角质层输送技术（GeneCream、微针阵列和/或离子电泳）结合使用。皮肤等效物将由转导的（带有EGFP报告细胞）或未经处理的正常原代人角质形成细胞以及先天性肿甲患者活检获得的角质形成细胞制备。在第二阶段，在第一阶段确定的最佳技术将在小鼠模型中进行测试，包括携带人类皮肤移植物的免疫功能低下小鼠。然后，这些技术的最有效组合将进行毒理学和药代动力学研究，为临床评估做准备。

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.