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

描述（由申请人提供）：尽管 RNA 干扰作为治疗皮肤疾病的新型治疗方法具有很高的潜力，但递送问题阻碍了其向临床的进展。传递功能活性 siRNA 的最有效和最有效的方法是通过皮内注射。我们已经证明，皮内注射大量注射产生的高压足以将核酸递送至皮肤（类似于高压尾静脉注射将siRNA递送至肝脏中观察到的情况）。事实上，在最近完成的 1b 期临床试验中，将我们领先的临床 siRNA 抑制剂 TD101 皮内注射到足部病变部位，可显着改善先天性厚甲症状。与这种给药方式相关的剧烈疼痛（需要口服止痛药和区域神经阻滞来减轻疼痛）似乎阻碍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，并且还提供了似乎有利于角质形成细胞摄取 siRNA 的流体动力压力。我们和其他人开发了患者友好型技术，使 siRNA 能够穿过角质层屏障，包括局部制剂 (GeneCream) 以及可渗透到表皮并释放其 siRNA 货物的中空可溶解微针阵列。不幸的是，如果没有与大量注射相关的压力，则很少有功能性 siRNA 进入细胞。我们的长期目标是找到对患者友好的技术，将 siRNA 有效且高效地递送到适当的皮肤区室，并促进角质形成细胞的摄取并掺入 RNA 诱导沉默复合物 (RISC)。在这一第一阶段提案中，我们开发了一种人类表皮等效系统（即再生人类皮肤），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递送技术（促进细胞对siRNA的摄取）以及其他商业化的递送摄取技术，包括Invivofectamine、Accell等。其中最有前途的将与我们的角质层递送技术（GeneCream、微针阵列和/或离子电渗疗法）结合使用。皮肤等同物将从转导（使用 EGFP 报告基因）或未经处理的正常原代人角质形成细胞以及源自先天性厚甲症患者活检的角质形成细胞制备。在第二阶段，第一阶段确定的最佳技术将在小鼠模型中进行测试，包括带有人类皮肤移植物的免疫功能低下的小鼠。这些技术的最有效组合将接受毒理学和药代动力学研究，为临床评估做好准备。 公共卫生相关性：尽管对导致大量皮肤病的潜在基因和突变有了令人兴奋的发现，但几乎没有新的临床治疗方法出现。该提案的目的是利用 RNA 干扰选择性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等效物（器官型皮肤模型），其中可以轻松监测基因表达，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。最佳技术组合将在动物毒理学和药代动力学测定的 GLP 条件下进一步评估，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.