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

人体器官型皮肤模型中 siRNA 摄取和功能活性的评估

基本信息

批准号：
7915054
负责人：
ROGER L KASPAR
金额：
$ 34.09万
依托单位：
TRANSDERM, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7915054
关键词：
Analgesics Animal Model Animals Biological Assay Biological Models Biopsy Bypass CD44 gene Cell Culture Techniques Cells Clinic Clinical Clinical Treatment Clinical Trials Collaborations Complex Disease Double-Stranded RNA Binding Domain Drug Formulations Drug Kinetics Epidermis Ethanol Evaluation Florida Fluorescence Microscopy Freezing Future Gene Expression Gene Mutation Genes Genetic Goals Human Immunocompromised Host Immunofluorescence Immunologic Immunohistochemistry Injection of therapeutic agent Iontophoresis Keratin Label Lead Lesion Letters Link Liposomes Liver Luciferases Messenger RNA Methodology Methods Modeling Monitor Mus Natural regeneration Nerve Block Nucleic Acids Pain Patients Penetration Peptides Pharmacologic Substance Phase Preparation Proteins RNA Interference RNA-Induced Silencing Complex Reporter Skin Skin Transplantation Skin graft Small Interfering RNA Stratum corneum Symptoms System Tail Technology Testing Time Toxicology Transfection Transgenic Organisms Universities Veins Xenograft procedure design foot in vivo inhibitor/antagonist intense pain intradermal injection keratinocyte mouse model mutant novel novel therapeutic intervention oral pain pressure prevent protein expression public health relevance research clinical testing skin disorder uptake

项目摘要

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.

描述（由申请人提供）：尽管RNA干扰提供了一种用于治疗皮肤疾病的新型治疗方法，但分娩问题阻碍了他们进入诊所的发展。传递功能活性siRNA的最有效方法是通过皮内注射。我们已经表明，通过皮内注射大量产生的高压足以将核酸递送至皮肤（类似于在高压尾静脉注射中观察到的，将siRNA传递给肝脏）。实际上，在最近完成的1B期临床试验中，皮内注射大量我们的铅临床siRNA抑制剂TD101至脚部病变导致Pachyonychia congenatia症状显着改善。与这种给药方式相关的剧烈疼痛（口服止痛药物和区域神经阻滞需要缓解疼痛）似乎可以防止将来使用这种方法。使用皮内注射绕过角质屏障，还提供了流体动力压力，似乎可以促进角质形成细胞的siRNA摄取。我们和其他人已经开发了对患者友好的技术，可以使siRNA在角质屏障中传递，包括局部配方（Genecream）以及渗透到表皮并释放其sirna货物的空心溶解的微针阵列。不幸的是，如果没有与高体积注射有关的压力，那么功能性siRNA几乎没有进入细胞。我们的长期目标是确定患者友好的技术，这些技术将使有效，有效的siRNA递送到适当的皮肤室中，并促进角质形成细胞的摄取，并掺入RNA诱导的沉默络合物（RISC）中。在这个阶段的建议中，我们可以开发人类表皮等效系统（即重生的人皮），其中可以监测预先存在的基因表达（CD44，突变体角蛋白6a和/或报告剂）。该模型系统将用于评估和优化Traversa的PTD-DRBD siRNA递送技术（促进细胞的siRNA摄取）以及其他商业上可用的递送吸收技术，包括Invivofofectamine，Accell等。这些技术最有希望的是与我们的Cornem cornem cornecoper and Cornecoper and Cornecoper and/Microone earlem and/Microoneem and/Microoneem and/cornem and/cornemem and/cornem and/cornem and/cornem and/cornise dor。皮肤当量将由转导的（与EGFP报告基因）或未经治疗的正常原代人角质形成细胞以及源自Pachyonychia congenanita患者活检的角质形成细胞制备。在第2阶段中，将在第1阶段确定的最佳技术进行在小鼠模型中测试，包括具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进行毒理学和药代动力学研究，以准备在诊所进行评估。公共卫生相关性：尽管对造成大量皮肤疾病的基础基因和突变有令人兴奋的发现，但如果出现任何新颖的临床治疗，就很少了。该建议的目的是利用RNA干扰来有选择地抑制疾病相关基因的表达。在第1阶段，我们开发了人类皮肤当量（器官皮肤模型），其中可以轻松地监测基因表达，以成功地递送功能性siRNA和测试siRNA技术，从而促进皮肤角质形成细胞的siRNA摄取，并与现有的技术结合使用，这些技术允许在层层层层层层皮肤屏障中递送。在第2阶段，我们将测试动物模型中第1阶段开发的技术。在动物毒理学和药代动力学测定中，将进一步评估技术的最佳组合，以准备人类试验。