Delivery of biologically active nucleic acids to epidermal cells

描述（由申请人提供）：利用核酸治疗人类疾病的广阔潜力尚未实现。二十年来，在许多实验室中，为调节培养细胞中的基因表达而递送核酸已成为常规。超过350种罕见的单基因疾病会导致皮肤疾病，而影响到百分之几人口的常见炎症性疾病，如牛皮癣，已经有了很好的分子途径，可以成为核酸治疗的目标。不难想象，有350多种不同的核酸药物被设计用来治疗特定的皮肤疾病。然而，除了用于疫苗之外，没有任何核酸被批准用于人体皮肤。递送似乎是发展皮肤核酸疗法的主要障碍。皮内注射似乎可以将核酸输送到表皮细胞，但由于皮肤表面太大，这种方法不切实际。人们正在研究一系列创新的物理和化学方法，目的是增强皮肤的递送，但缺乏一致和相关的皮肤模型和分析工具阻碍了比较研究和方案的优化。没有一个研究者可能有足够的技能、知识和设备来比较他/她自己喜欢的核酸/递送方法的递送选择，这种方法在细胞培养中，在某些情况下，在组织模型中具有生物活性。如果没有这样的验证系统，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的进展将是极其困难的。本提案的目标是弥合试管知识与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，可用于验证和可转移的算法，以测试核酸在表皮细胞中的传递。因为我们的模型是由成功的分娩激活的，在单细胞水平上的变化将被检测到。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的生物活性核酸的递送；第三条轨道将测试针对染色体DNA的生物活性核酸的递送。我们将开发一种分子报告基因，稍加修饰，就可用于测试任何小核酸的生物学相关、序列特异性活性。我们将开发小鼠皮肤模型和移植到小鼠身上的人类皮肤模型作为标准测定。我们将使用新的敏感和定量成像设备来比较递送方法，包括但不限于：电穿孔、超声穿孔、离子电泳、液体喷射、微针和局部配方。这项应用的主要美国参与者TransDerm、耶鲁、斯坦福、Lucid、Stratatech以及合作机构（UCSB、Alnylam Pharmaceuticals （Cambridge， MA）、university S. Florida、Old Dominion university、Traversa Therapeutics （San Diego）、NT Omics （Seattle）等）的研究人员所做的工作和发现，有可能使成千上万的皮肤病患者受益，并且肯定会对当地经济产生影响。近年来，斯坦福大学和耶鲁大学向当地经济投入了30多亿美元。斯坦福大学（Stanford）和耶鲁大学（Yale）研究人员的发现催生了数十家公司，这些公司继续提供就业机会，刺激经济增长。目前的提案将对经济产生积极影响，在TransDerm创造或保留4个工作岗位，在耶鲁创造2.35个工作岗位，在斯坦福创造3个工作岗位。根据加州生物医学行业的数据，每有一名生物医学组织的员工，就会有另外三到五名员工受雇于为该行业提供服务的公司。这个提议可能会对我们的两个小公司合作者Stratatech和Lucid的就业产生明确的影响。如果这项工作的目标成功，预计新的研究和开发的核酸治疗皮肤病的步伐将大大加快，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.