A Self-Delivery siRNA for Promoting Regenerative Healing in the Eye

用于促进眼部再生愈合的自我递送 siRNA

• 批准号: 10406147
• 负责人: AUDREY M BERNSTEIN
• 金额: --
• 依托单位: SYRACUSE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10406147
• 关键词: 3-Dimensional; Actins; Acute; Aging; Animals; Apoptosis; Award; Binding; Biological; Blindness; Brain Injuries; Bulla; Burr hole procedure; C-terminal; Cell Adhesion; Cell Proliferation; Cells; Cholesterol; Chronic; Cicatrix; Clinical; Clinical Trials; Collagen; Cornea; Corneal Injury; Cost Savings; Cytoskeleton; Data; Development; Disease; Dose; Endothelium; Exhibits; Extracellular Matrix; Eye; Eye Injuries; Family suidae; Fibroblasts; Fibronectins; Fibrosis; Filtering Surgery; Filtration; Flow Cytometry; Freedom; Gel; General Population; Genes; Glaucoma; Goals; Health; Hour; Human; Immune; Immunohistochemistry; Infiltration; Inflammation; Injury; Integral Membrane Protein; Integrin alphaV; Integrins; Lead; Legal patent; Length; Link; Mediating; Methodology; Microscopy; Military Personnel; Mitomycin C; Modification; Myofibroblast; N-terminal; Natural regeneration; Nerve Regeneration; Operative Surgical Procedures; Organ Culture Techniques; Oryctolagus cuniculus; Outcome; Pathologic; Pathway interactions; Penetration; Pharmaceutical Preparations; Physiologic Intraocular Pressure; Pilot Projects; Play; Population; Publishing; Quality of life; Recycling; Role; Sclera; Services; Small Interfering RNA; Speed; Structure; Surgical Flaps; TP53 gene; Techniques; Testing; Therapeutic; Therapeutic Intervention; Thick; Time; Tissues; Toxin; Translations; Trauma; Tumor-infiltrating immune cells; Ubiquitin; Ubiquitination; Veterans; Viral; Vision; Visual; Visual impairment; Work; age related; alkalinity; antifibrotic treatment; base; corneal burn; corneal scar; cost; disability; experimental study; glaucoma surgery; healing; improved; in vivo; inhibitor; irritation; knock-down; neovascularization; novel; operation; overexpression; pressure; prevent; regenerative; siRNA delivery; slit lamp imaging; standard of care; surgery outcome; targeted treatment; therapeutic target; tonometry; wound; wound closure; wound healing

Project Summary Corneal scarring and glaucoma accounts for ocular disability in millions of veterans, active military, and civilians. Injuries to the eye remain a battlefield and clinical challenge with the potential to severely reduce vision and quality of life. Ocular trauma and brain injury with resulting visual deficits are major causes of vision loss among our veterans and troops engaged in Operations Enduring Freedom and Iraqi Freedom. In addition to acute trauma, aging veterans have a significant increase in age-related glaucoma compared to the general population. The total cost, including treatment is estimated at 2.4 billion annually for the Armed Services. The link between cornea and glaucoma is that a) almost any severe eye injury because of the inflammation generated during wound healing and the drugs administered to treat the inflammation will lead to the onset of glaucoma, and b) the two scarring indications have similar biological underpinnings and thus it is a cost savings to work on both indications at the same time. We are proposing the use of a self-delivery siRNA to prevent and reverse scarring in the cornea and to prevent scarring in the sclera bleb made to reduce intraocular pressure in the eye. Our self-delivery (modified with cholesterol to gain entry into cells) siRNA modified for in vivo use is protected with a utility patent to prevent scarring in the eye and a second provisional patent for the modified self-delivery siRNA sequence and structure. Acute scarring, similar to chronic fibrosis, is characterized by immune cell infiltration and the persistence of cells termed myofibroblasts. Pathological myofibroblasts exhibit increased cell adhesion and tissue contraction through the force generated by binding to the extracellular matrix and the intracellular actin cytoskeleton via integrins (transmembrane proteins). We have discovered a key point in the healing pathway that can be therapeutically targeted to control cell apoptosis, immune infiltration, and integrin- mediated pathological myofibroblast development. Collectively, our studies in primary human corneal cells, pig corneal organ culture, and in rabbits demonstrate that wounding induces the expression of the deubiquitinase (DUB), USP10. USP10 removes ubiquitin from both p53 and αv-integrins. Knockdown of USP10 with one dose of self-delivery siRNA in vivo after corneal wounding significantly reduced apoptosis, immune infiltration, fibrotic markers, and corneal scarring, and a pilot study demonstrated regenerative healing in the glaucoma filtration bleb. Towards the goal of realizing the most effective and specific USP10-targeted therapeutic, in Aim 1, we propose to elucidate the USP10 domains that lead to scarring outcomes and the effect of USP10 binding partner, G3BP2 on USP10 DUB activity. In Aim 2, we will expand our studies on USP10 knockdown to determine if corneal inflammation and scarring can be totally prevented with a second dose of self-delivery siRNA and reversed by novel techniques to permit siRNA entry into a scar. In Aim 3, we will test if USP10 knockdown can prevent inflammation and scarring in the bleb after glaucoma surgery that is performed to relieve intraocular pressure elevation associated with glaucoma. Successful completion of these Aims will significantly improve our understanding of the central mechanisms that promote scarring, and will lead to the development of novel self-delivery siRNA approaches to preventing corneal and glaucoma-related blindness in the veteran and civilian populations.

项目概要 角膜疤痕和青光眼导致数百万退伍军人、现役军人和军人眼部残疾 眼睛受伤仍然是一个战场和临床挑战，有可能造成严重后果。 导致视力缺陷的眼外伤和脑损伤是导致视力和生活质量下降的主要原因。 参与持久自由行动的退伍军人和部队视力丧失的原因 除了急性创伤之外，老年退伍军人与年龄相关的疾病也显着增加。 与一般人群相比，青光眼的总费用（包括治疗）估计为 2.4。 每年为武装部队提供 10 亿美元 角膜和青光眼之间的联系是：a) 几乎任何疾病。 由于伤口愈合过程中产生的炎症和药物造成的严重眼损伤 治疗炎症会导致青光眼的发作，并且b）这两种疤痕 适应症具有相似的生物学基础，因此研究这两种适应症可以节省成本 同时，我们建议使用自传递 siRNA 来预防和逆转疤痕形成。 角膜并防止巩膜形成疤痕，降低眼内压。 我们的自我递送（用胆固醇修饰以进入细胞）经过修饰用于体内使用的 siRNA 是 受一项实用专利保护，以防止眼睛留下疤痕，并获得第二项临时专利保护 修饰的自递送siRNA序列和结构。 急性疤痕与慢性纤维化类似，其特点是免疫细胞浸润和持续存在 称为肌成纤维细胞的细胞表现出细胞粘附和组织增加。 通过与细胞外基质和细胞内肌动蛋白结合产生的力进行收缩 通过整合素（跨膜蛋白）的细胞骨架我们发现了愈合的关键点。 可以治疗靶向控制细胞凋亡、免疫浸润和整合素的途径 总的来说，我们对原代人角膜的研究。 细胞、猪角膜器官培养物和兔子证明，受伤会诱导表达 去泛素酶 (DUB)，USP10 可去除 p53 和 αv 整合素中的泛素。 角膜受伤后体内一剂自递送 siRNA 显着敲低 USP10 减少细胞凋亡、免疫浸润、纤维化标记物和角膜疤痕，以及一项试点研究 青光眼滤过泡的再生愈合正在朝着实现这一目标的方向发展。 最有效和特异性的 USP10 靶向治疗，在目标 1 中，我们建议阐明 USP10 导致疤痕结果的结构域以及 USP10 结合伴侣 G3BP2 对 USP10 的影响 在目标 2 中，我们将扩大对 USP10 敲低的研究，以确定角膜是否有效。 通过第二剂自我递送 siRNA 可以完全预防炎症和疤痕形成 通过新技术逆转，允许 siRNA 进入疤痕。在目标 3 中，我们将测试 USP10 是否有效。 敲低可以预防青光眼手术后滤泡内的炎症和疤痕 缓解与青光眼相关的眼压升高。 目标将显着提高我们对促进疤痕形成的核心机制的理解，以及 将导致新型自我递送 siRNA 方法的开发，以预防角膜和 退伍军人和平民中与青光眼相关的失明。