Pachyonychia congenita clinical trial using therapeutic self-delivery siRNAs

描述(由申请人提供)：RNA干扰(RNAi)有可能彻底改变显性遗传疾病的治疗。小分子抑制性RNA(SiRNAs)是一种高效、选择性强的单核苷酸特异性分子。目前正在对包括皮肤在内的一些适应症进行使用siRNAs的临床试验。在第一阶段资金的推动下，开展了一项针对先天性肥甲症(PC)的小型1b期临床试验。这种极端的皮肤病是由编码角蛋白6、16和17的基因突变引起的，包括单核苷酸变化。PC患者的主要主诉是发生在脚部压力点或其附近的虚弱疼痛的老茧和水泡。通过瘤内注射siRNA(TD101)，将脚底这些确定的区域作为局部siRNA治疗的靶点，取得了令人鼓舞的结果。不幸的是，与皮损注射相关的疼痛(需要口腔止痛药和区域神经阻滞才能进行治疗)阻碍了这种给药模式的广泛使用。这一观察结果促使人们加紧努力，寻找对患者友好(即，几乎没有痛苦)的分娩方式。这项临床试验是人类首次在皮肤中使用siRNA，也是第一次针对突变基因的siRNA。在该提案的第二阶段，我们延长了第一阶段和1b阶段临床试验的进展。我们发现，未经修饰或稳定的siRNA不容易被皮肤角质形成细胞摄取，但经修饰的，即所谓的“自我传递”(Sd)siRNA则容易被皮肤角质形成细胞摄取。SD-siRNA的进一步优化将在小鼠和人类皮肤模型中进行，随后将合成GMP材料，用于小鼠和兔的毒理学研究和临床试验，在这些试验中，siRNA将由Transderm公司开发和生产的可溶解微针阵列或局部GeneCream“配方”输送。GeneCream和微针阵列的设计都是为了有效地传递SD-siRNA，几乎没有疼痛(微针突起设计为只穿透到非神经支配的表皮)。虽然PC是一种罕见的疾病，但这种疾病的性质使其成为第一个人siRNA皮肤临床试验的理想原型皮肤病(定义的突变，在有限的、定义的区域表达)，我们完全期待所学到的教训将很容易推广到其他皮肤病。 与公共卫生相关：尽管发现了导致许多遗传性皮肤病的潜在突变，但几乎没有有效的治疗方法出现。可以开发siRNAs来针对特定的疾病相关突变的发现预示着一种新的治疗范例的出现。我们已经确定了一种有效的、选择性的siRNA，它针对角蛋白6a基因中的单个核苷酸突变，该基因是导致罕见的皮肤疾病先天性厚甲并阻止其表达的原因。这种siRNA(FDA称为TD101)已在1b期临床试验中成功测试。尽管有疗效的迹象，但给药途径的痛苦性质(皮内大量注射)要求我们开发一种对患者更友好的给药系统。该提案的第二阶段允许进行临床前研究，使用装载有自我递送版本的TD101 siRNA的微针进行第二次临床试验。应用siRNA技术治疗前列腺癌的经验教训应该很容易推广到许多其他显性皮肤病，并可能推广到其他由突变或过度表达的致病基因引起的疾病。

DESCRIPTION (provided by applicant): RNA interference (RNAi) has the potential to revolutionize treatment of dominant genetic disorders. Small inhibitory RNAs (siRNAs) are highly potent and selective, demonstrating remarkable single-nucleotide specificity. Clinical trials using siRNAs are currently underway for a number of indications including skin. Facilitated by Phase 1 funding, a small one patient phase 1b clinical trial was undertaken for pachyonychia congenita (PC). This ultrarare skin disorder is caused by mutations, including single nucleotide changes, in the genes encoding keratins 6, 16, and 17. The major complaint of PC patients is the debilitating painful callusing and blistering which occurs on or near the pressure points of the feet. These defined regions on the soles of the feet were targeted for local siRNA treatment by intralesional injection of siRNA (TD101) with encouraging results. Unfortunately, the pain associated with injections into lesions (oral pain medication and regional nerve blocks were required to allow treatment), prevents widespread use of this mode of administration. This observation has led to intense efforts to identify patient-friendly (i.e., little or no pain) delivery options. This clinical trial was the first human use of siRNA in skin and also the first siRNA to target a mutated gene. In Phase 2 of this proposal, we extend the progress of Phase 1 and the Phase 1b clinical trial. We have found that unmodified or stabilized siRNAs are not taken up readily by skin keratinocytes but that modified, so-called "self-delivery" (sd) siRNAs are. Additional optimization of the sd-siRNA will be undertaken in mouse and human skin models followed by synthesis of GMP material for mouse and rabbit toxicology studies and clinical trials in which the siRNA will be delivered by dissolvable microneedle arrays or a topical GeneCream" formulation, both developed and manufactured at TransDerm. GeneCream and microneedle arrays are both designed to effectively deliver sd-siRNA with little or no pain (microneedle protrusions are designed to only penetrate to the non-innervated epidermis). Although PC is a rare disease, the nature of the disorder makes it an ideal prototype skin disorder (defined mutations with expression in limited, defined areas) for first-in-man siRNA skin clinical trials, and we fully expect the lessons learned will be readily generalized to other skin disorders. PUBLIC HEALTH RELEVANCE: Despite the discovery of the underlying mutations responsible for many inherited skin diseases, few effective treatments have emerged. The discovery that siRNAs can be developed to target specific disease-related mutations portends the advent of a new treatment paradigm. We have identified a potent and selective siRNA that targets a single nucleotide mutation in the keratin 6a gene that is responsible for the rare skin disorder pachyonychia congenita and blocks its expression. This siRNA (known to the FDA as TD101) was successfully tested in a Phase 1b clinical trial. Despite signs of efficacy, the painful nature of the administration route (intradermal injection of large volumes) required that we develop a more patient-friendly delivery system. Phase 2 of this proposal allows preclinical studies enabling a second clinical trial using microneedles loaded with a self-delivery version of TD101 siRNA. The lessons learned in applying siRNA technology for treatment of PC should be readily generalizable to a host of other dominant skin disorders and likely to other disorders resulting from mutated or overexpressed disease-causing genes.