Delivery of biologically active nucleic acids to epidermal cells

描述(申请人提供)：利用核酸治疗人类疾病的广泛潜力尚未实现。二十年来，以调节培养细胞基因表达为目的的核酸输送一直是许多实验室的常规做法。超过350种罕见的单基因疾病会导致皮肤疾病，而常见的炎症性疾病，如牛皮癣，具有众所周知的分子通路，可能成为核酸疗法的靶点。不难想象，有超过350种不同的核酸药物设计用于治疗特定的皮肤病。然而，除了用于疫苗，没有核酸被批准用于人类皮肤。给药似乎是皮肤核酸疗法取得进展的主要障碍。皮内注射似乎将核酸输送到表皮细胞，但巨大的皮肤表面使这种方法不切实际。目前正在研究各种创新的物理和化学方法，以加强对皮肤的释放，但缺乏一致和相关的皮肤模型和分析工具，阻碍了对方案的比较研究和优化。没有一个研究人员可能拥有所需的技能、知识和设备来比较他/她自己最喜欢的核酸/递送方法的递送选择，S已经证明这种递送方法在细胞培养中具有生物学活性，在某些情况下还用于组织模型。如果没有这种经过验证的系统，在评估新设计的特定核酸序列和旨在提高其有效性的化学修饰方面的进展将极其困难。这项建议的目标是弥合试管知识和临床应用之间的差距。我们将开发试剂、模型分析系统和分析工具，用于测试核酸递送到表皮细胞的有效和可转移的算法。因为我们的模型是由成功的交付激活的，所以在单个细胞水平上的变化将是可检测的。将有三个平行的轨道：第一个轨道将测试报告质粒DNA的传递；第二个轨道将测试靶向mRNA的生物活性核酸的传递；第三个轨道将测试靶向染色体DNA的生物活性核酸的传递。我们将开发一种分子报告器，只要稍加修改，就可以用来测试任何小核酸的生物相关性和序列特异性活性。我们将开发移植到小鼠身上的小鼠皮肤和人类皮肤等价物的模型，作为标准测试。我们将使用新的灵敏和定量成像设备来比较递送方法，这些方法包括但不限于：电穿孔、声波渗透、离子导入、液体喷射、微针和外用制剂。这项申请的主要美国参与者Transderm、耶鲁、斯坦福、Lucid、Stratech以及来自合作机构(UCSB、Alnylam PharmPharmticals(马萨诸塞州剑桥)、Univ.美国佛罗里达州、老道明大学、特拉弗萨治疗公司(圣地亚哥)、NT Omics公司(西雅图)等，有可能使成千上万的皮肤病患者受益，并肯定会影响当地经济。近年来，斯坦福大学和耶鲁大学向当地经济注入了超过30亿美元的资金。斯坦福大学和耶鲁大学的调查人员的发现导致了数十家公司的成立，这些公司继续提供就业机会，刺激经济。目前的提案将通过在Transderm创造或保留4个就业机会，在耶鲁创造或保留2.35个就业机会，在斯坦福创造或保留3个就业机会，从而对经济产生积极影响。根据加州生物医学行业的数据，生物医学组织每有一名员工，就会有另外三到五名员工受雇于为该行业服务的公司。这项提议可能会对我们的两个小公司合作者Stratech和Lucid产生明确的就业影响。如果这项工作的目标成功，预计皮肤病核治疗的新研发步伐将大大加快，催生许多新的就业机会和小公司。

DESCRIPTION (provided by applicant): The broad potential for using nucleic acids to treat human disease has not been realized. Delivery of nucleic acids for the purpose of regulating gene expression in cultured cells has been routine in many laboratories for twenty years. Over 350 rare monogenic disorders cause disease in the skin, and common inflammatory diseases affecting several percent of the population, such as psoriasis, have well-understood molecular pathways that could be the targets of nucleic acid therapeutics. It is not hard to imagine well over 350 distinct nucleic acid drugs designed to treat specific skin diseases. Yet, apart from use in vaccines, no nucleic acids are approved for use in human skin. Delivery appears to be the major obstacle to progress in developing nucleic acid therapeutics for skin. Intradermal injections appear to deliver nucleic acids to epidermal cells, but the large cutaneous surface makes such an approach impractical. A wide range of innovative physical and chemical methods are being investigated with the aim of enhancing delivery to skin, but the lack of consistent and relevant skin models and analysis tools has impeded comparative studies and optimization of protocols. No one investigator is likely to have the skill, knowledge and equipment needed to readily compare delivery options for his/her own favorite nucleic acid/delivery method, which s/he has shown to be biologically active in cell culture, and in some cases, tissue models. Without such validated systems, progress in evaluating newly designed specific nucleic acid sequences and chemical modifications designed to improve their effectiveness will be extremely difficult. The goal of this proposal is to bridge the gap between test tube knowledge and clinical application. We will develop reagents, model assay systems and analysis tools that can be used in a validated and transferable algorithm for testing delivery of nucleic acids to cells in the epidermis. Because our models are activated by successful delivery, changes at the single cell level will be detectable. There will be three parallel tracks: the first track will test delivery of reporter plasmid DNA; the second track will test delivery of biologically active nucleic acids that target mRNA; the third track will test delivery of biologically active nucleic acids that target chromosomal DNA. We will develop a molecular reporter, which with small modifications, can be used to test biologically relevant, sequence-specific activity of any small nucleic acid. We will develop models of mouse skin and human skin equivalents grafted onto mice as standard assays. We will use new sensitive and quantitative imaging devices to compare delivery methods that include, but need not be limited to: electroporation, sonoporation, iontophoresis, liquid jets, microneedles and topical formulations. The work and discoveries made by the major U.S. participants in this application, TransDerm, Yale, Stanford, Lucid, Stratatech, as well as the investigators from collaborating institutions (UCSB, Alnylam Pharmaceuticals (Cambridge, MA), Univ. S. Florida, Old Dominion Univ., Traversa Therapeutics (San Diego), NT Omics (Seattle), etc) have the potential to benefit many tens of thousands of patients with skin disease, and will certainly impact the local economies. In recent years, Stanford and Yale Universities pumped more than $3 billion into their local economies. Discoveries by investigators at Stanford and Yale have led to the creation of dozens of companies, which continue to provide jobs and stimulate the economy. The current proposal would positively impact the economy by creating or retaining 4 jobs at TransDerm, 2.35 jobs at Yale, and 3 jobs at Stanford. According to the California Biomedical Industry, for every one employee of a biomedical organization, another three to five will be employed in firms that service that industry. This proposal could have definite employment implications for two of our small company collaborators, Stratatech and Lucid. If the goal of this work succeeds, it is expected that the pace of new research and development of nucleic treatments for skin disease will accelerate greatly, spawning many new jobs and small companies.