Topical delivery of nanoencapsulated plasmid DNA to posterior ocular targets

描述（由申请人提供）：年龄相关性黄斑变性（AMD）、其他视网膜营养不良和青光眼等疾病在全球范围内造成很大比例的失明。治疗这些疾病的关键障碍是不能有效、经济和安全地将药物和基因治疗DNA构建体递送到视网膜和视网膜色素上皮（RPE）。由于眼睛前段的渗透性和吸收屏障，通过滴眼剂的局部递送一直是无效的。因此，目前用于这些疾病的递送方法需要使用病毒载体进行昂贵的眼部注射，在一些临床试验中已经证明了危险的副作用。这个项目的广泛的，长期的目标是开发一种安全的，经济的，有效的基因传递方法，使用新的，非病毒的亚50纳米（s50）胶囊技术，是细胞特异性和交通到细胞核没有内体截留。我们的初步数据表明，这些纳米胶囊作为局部滴眼液施用到活体大鼠的泪膜上，导致基因载体在视网膜和RPE细胞中的递送和表达。导致DNA递送至眼后部靶标的局部应用是令人惊讶的并且具有潜在的变革性。如果将其用于临床，它将为许多致盲性疾病提供安全和经济的治疗。在这项I期研究中，我们建议使用易于评估的荧光质粒DNA表达载体，在安全性和有效性方面优化局部s50胶囊给药方案。然后，我们将比较优化的局部s50胶囊给药方案的表达水平和炎症标志物，以及我们先前优化的眼部注射方案。我们将使用视网膜变性的LCA 2小鼠模型。LCA2小鼠模型是有用的，因为它们由于简单缺乏单一基因产物（RPE 65）而具有严重的视力缺陷，但它们具有延迟的形态学变性。因此，替换基因载体的成功递送导致视觉功能的产生和退化的预防。如果这项I期研究的结果令人鼓舞，我们将提出II期研究，使用新型s50胶囊技术将治疗性RPE 65表达载体递送给RPE 65缺陷的小鼠，并评估靶基因表达和功能、视觉功能和形态。 公共卫生相关性：这项研究与公共卫生的相关性可能非常高。大部分失明是由视网膜和视网膜色素上皮（RPE）病理引起的。几种治疗性DNA构建体和药物在动物和细胞培养模型中是这些疾病的有效治疗方法，但在临床中的用途有限，因为它们不能有效和安全地递送到靶组织和细胞。该项目的目的是开发一种安全有效的方法来提供这些治疗。

DESCRIPTION (provided by applicant): Diseases such as age related macular degeneration (AMD), other retinal dystrophies, and glaucoma cause a large proportion of blindness worldwide. A critical barrier in treating these diseases is the inability to efficaciously, economically, and safely deliver drugs and gene therapy DNA constructs to the retina and the retinal pigment epithelium (RPE). Topical delivery by eye drops has been ineffective because of permeability and absorption barriers of the anterior segment of the eye. Thus, current delivery approaches for these diseases entail costly injections of the eye using viral vectors that in some clinical trials have demonstrated dangerous side effects. The broad, long-term objective of this project is to develop a safe, economical, and effective gene delivery approach using novel, nonviral sub-50 nanometer (s50) capsule technology that is cell-specific and traffics to the nucleus without endosomal entrapment. Our preliminary data indicate that these nanocapsules administered as topical eye drops onto the tear film of living rats result in delivery and expression of gene vectors in cells of the retina and RPE. Topical application resulting in DNA delivery to posterior ocular targets is surprising and potentially transformative. If brought to the clinic, it should provide safe and economical treatment for many blinding diseases. In this Phase 1 study, we propose to optimize the topical s50-capsule dosing regimen in terms of safety and efficacy using easily-assessed fluorescent plasmid DNA expression vectors. We then will compare expression levels and inflammatory markers of the optimized topical s50-capsule dosing regimen, with an ocular injection protocol that we have previously optimized. We will use the LCA2 mouse model of retinal degeneration. LCA2 mouse models are useful because they have severe vision deficits due to simple lack of a single gene product (RPE65), yet they have delayed morphological degeneration. Thus, successful delivery of replacement gene vector results in creation of visual function and prevention of degeneration. If the results of this Phase I study are encouraging, we will propose Phase II studies using the novel s50 capsule technology to deliver therapeutic RPE65 expression vectors to mice deficient in RPE65, and assess target gene expression and function, visual function, and morphology. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is potentially very high. A large proportion of blindness is caused by retina and retinal pigment epithelium (RPE) pathologies. Several therapeutic DNA constructs and drugs are effective treatments for these diseases in animal and cell culture models, but are of limited use in the clinic because they cannot be delivered to target tissue and cells efficiently and safely. The aim of this project is to develop a safe and effective way to deliver these treatments.