DNA Delivery for Treatment of Proliferative Vitreoretinopathy and Ocular Scarring

描述（由申请人提供）：虽然在过去十年中已经可获得许多用于治疗眼部疾病的新治疗分子，但是它们在临床中的有效应用所面临的主要问题之一是药物递送。通常，生物活性分子在体内具有短的半衰期，并且需要重复施用。在眼内给药的情况下，这通常意味着患者依从性差和成本高。因此，预期治疗性分子在数月至数年的时间内的受控释放在患者护理和眼部疾病的治疗中具有显著影响。该提案的重点是研究用于眼内递送治疗分子的微米和纳米级聚合物纤维。与其他纳米和微米级输送载体相比，纤维的优势在于，如果出现并发症，它们可以被移动或移除。 转化生长因子-β（TGF-β）是参与几种眼部疾病的主要靶点之一。这些疾病包括角膜瘢痕形成、白内障和青光眼手术后的瘢痕形成以及增殖性玻璃体视网膜病变。降低TGF-β通过使用反义寡核苷酸、适体和其它小分子药物的表达及其作用在治疗这些疾病方面已经显示出一些成功。虽然针对TGF-β的siRNA的递送也已被证明是有效的，但siRNA作为长期治疗是有限的，这是由于其对普遍存在的RNA酶的易感性以及需要具有大量的分子用于体内有效敲低。在这项研究中，我们建议检查传递的DNA编码的siRNA敲低TGF-β的表达。DNA的递送解决了稳定性的问题，因为DNA比RNA稳定得多，并且解决了药物量的问题，因为单个DNA分子可以转录成许多siRNA。 该研究的目的是开发一种微尺度药物递送平台，该平台可以容易地植入以治疗各种眼部疾病。具体而言，本研究的假设是，所提出的治疗性DNA可以从聚合物纳米纤维中掺入和释放，并且释放的DNA可以导致眼细胞模型中的表达敲低。该假设将在三个具体目标中进行测试：1）评估从电纺形成的纳米纤维中释放的DNA的生物活性，2）在不稳定的GFP模型中评估释放的DNA在敲低表达中的作用，以及3）在人角膜成纤维细胞模型中评估释放的DNA在敲低TGF-²表达中的作用。然而，能够成功递送针对一种疾病的治疗性DNA的药物递送平台可以迅速应用于治疗其他疾病，例如在湿性黄斑变性或糖尿病视网膜病变中VEGF敲除。 公共卫生相关性：该提案与公共卫生问题密切相关，因为其长期目标是提供药物递送平台，用于提供分子的持续释放以治疗眼内疾病。短期目标是聚合物纳米纤维是否可以递送DNA以在眼内瘢痕形成疾病（包括角膜瘢痕形成、手术后瘢痕形成和增殖性玻璃体视网膜病变）的细胞培养模型中敲低TGF-β的表达。

DESCRIPTION (provided by applicant): While many new therapeutic molecules for treatment of ocular diseases have become available over the past decade, one of the major problems facing their effective application in clinic has been drug delivery. Often bioactive molecules have a short half-life in vivo and require repeated administration. In the case of intraocular administration this often means poor patient compliance and high cost. As such, controlled release of therapeutic molecules over a period of months to years is expected to have a significant impact in patient care and in the treatment of ocular disease. The focus of this proposal is to investigate micro and nanoscale polymer fibers for the intraocular delivery of therapeutic molecules. Fibers have an advantage over other nano and microscale delivery vectors in that they can be moved or removed if complications arise. One of the primary targets implicated in several ocular diseases is Transforming Growth Factor - beta (TGF-¿). These diseases include corneal scarring, scarring following cataract and glaucoma surgery, as well as proliferative vitreoretinopathy. Decreasing TGF-¿ expression and its effect through use of antisense oligonucleotides, aptamers and other small molecule drugs has shown some success in treatment of these diseases. While delivery of siRNA against TGF-¿ has also been demonstrated to be effective, siRNA is limited as a long term therapy due to its susceptibility to ubiquitous RNAses as well as the need to have a large number of molecules for effective knockdown in vivo. In this study we propose to examine delivery of DNA which codes for the siRNA to knockdown TGF-¿ expression. Delivery of DNA addresses issues of stability since DNA is much more stable than RNA, and addresses issues of drug amount since a single DNA molecule can be transcribed into many siRNAs. The objective of the proposed research is to develop a microscale drug delivery platform which can easily be implanted to treat a wide range of ocular diseases. Specifically the hypothesis of this study is that the proposed therapeutic DNA can be incorporated and released from polymer nanofibers, and that released DNA can result in expression knockdown in ocular cell models. The hypothesis will be tested in three specific aims: 1) Evaluate the bioactivity of DNA released from nanofibers formed by electrospinning, 2) In a destabilized GFP model evaluate effect of released DNA in knocking down expression, and 3) In a human corneal fibroblast model evaluate the effect of released DNA in knockdown of TGF-¿ expression. This ocular model was chosen due to the investigators' familiarity with it. However, a drug delivery platform which can successfully deliver therapeutic DNA against one disease could rapidly be applied toward treatment of other diseases, such as VEGF knockdown in wet-type macular degeneration, or in diabetic retinopathy. PUBLIC HEALTH RELEVANCE: This proposal is critically concerned with the issue of public health in that its long term goal is to provide a drug delivery platform for providing sustained release of molecules to treat intraocular diseases. The short term goal is whether polymer nanofibers can deliver DNA to knock down expression of TGF-¿ in cell culture models of intraocular scarring disorders including corneal scarring, post-surgical scarring, and proliferative vitreoretinopathy.