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失明中占很大比例。治疗这些疾病的一个关键障碍是无法有效、经济和安全地将药物和基因治疗DNA构建物输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障碍，局部滴眼液是无效的。因此，目前治疗这些疾病的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有危险的副作用。该项目的长期目标是开发一种安全、经济、有效的基因传递方法，使用新颖的非病毒50纳米（s50）胶囊技术，这种技术具有细胞特异性，可以在没有内体包裹的情况下运输到细胞核。我们的初步数据表明，这些纳米胶囊作为局部滴眼液施用于活体大鼠的泪膜上，导致基因载体在视网膜和RPE细胞中的传递和表达。局部应用导致DNA传递到后眼目标是令人惊讶的和潜在的变革。如果应用于临床，它将为许多致盲疾病提供安全、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，从安全性和有效性方面优化s50胶囊外用给药方案。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，以及我们之前优化的眼部注射方案。我们将使用LCA2小鼠视网膜变性模型。LCA2小鼠模型是有用的，因为它们由于缺乏单一基因产物（RPE65）而具有严重的视力缺陷，但它们具有延迟的形态变性。因此，替代基因载体的成功传递可以创造视觉功能，防止变性。如果这项I期研究的结果令人鼓舞，我们将提出使用新型s50胶囊技术进行II期研究，将治疗性RPE65表达载体传递给RPE65缺乏的小鼠，并评估靶基因的表达和功能、视觉功能和形态。

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.