DNA Delivery for Treatment of Proliferative Vitreoretinopathy and Ocular Scarring

描述(申请人提供)：尽管在过去十年中出现了许多用于治疗眼科疾病的新的治疗分子，但它们在临床上有效应用面临的主要问题之一是药物输送。通常，生物活性分子在体内的半衰期很短，需要重复给药。在眼内给药的情况下，这通常意味着患者依从性差，费用高。因此，在几个月到几年的时间内控制释放治疗分子预计将在患者护理和眼病治疗中产生重大影响。这项提议的重点是研究用于眼内治疗分子输送的微米和纳米级聚合物纤维。与其他纳米和微米级传递载体相比，纤维具有优势，因为如果出现并发症，它们可以移动或移除。 与几种眼病有关的主要靶点之一是转化生长因子-β(TGF-β)。这些疾病包括角膜疤痕，白内障和青光眼手术后的疤痕，以及增殖性玻璃体视网膜病变。通过使用反义寡核苷酸、适体等小分子药物来降低转化生长因子β的表达及其作用，在治疗这些疾病方面已显示出一定的成功。虽然针对转化生长因子-β的siRNA的传递也被证明是有效的，但由于siRNA对普遍存在的核糖核酸酶的敏感性以及在体内需要大量的分子来有效地敲除，所以siRNA作为一种长期治疗手段是有限的。在这项研究中，我们建议检查DNA的传递，它编码siRNA来抑制转化生长因子-β的表达。DNA的传递解决了稳定性问题，因为DNA比RNA更稳定，并且解决了药物数量的问题，因为单个DNA分子可以转录成许多siRNA。 这项研究的目的是开发一种微尺度的药物输送平台，该平台可以很容易地植入治疗各种眼部疾病。具体地说，这项研究的假设是，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表达下调。这一假设将在三个特定的目标中得到验证：1)评估通过静电纺丝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在下调表达方面的作用，以及3)在人角膜成纤维细胞模型中评估释放的DNA在抑制转化生长因子-β表达方面的作用。之所以选择这个眼睛模型，是因为研究人员对它很熟悉。然而，能够成功地传递针对一种疾病的治疗性DNA的药物传递平台可以迅速应用于其他疾病的治疗，如湿性黄斑变性中的血管内皮生长因子基因敲除，或糖尿病视网膜病变。 与公共健康相关：这项提议与公共健康问题密切相关，因为它的长期目标是提供一个药物输送平台，为治疗眼内疾病提供持续的分子释放。短期目标是，在眼内瘢痕疾病的细胞培养模型中，聚合物纳米纤维是否能够传递DNA以抑制转化生长因子-β的表达，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。

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.