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.

项目摘要 角膜疤痕和青光眼是数百万退伍军人，活跃军事和 平民。眼睛受伤仍然是战场和临床挑战，有可能严重 降低视力和生活质量。眼部创伤和脑损伤，导致的视觉缺陷是主要的 我们的退伍军人和部队之间有视力丧失的原因，从事持久自由和的行动 伊拉克自由。除了急性创伤之外，老年退伍军人与年龄相关的老年人显着增加 与普通人群相比，青光眼。包括治疗在内的总成本估计为2.4 武装部队每年十亿美元。角膜和青光眼之间的联系是a）几乎所有 由于伤口愈合期间产生的感染和药物，严重的眼部受伤 用于治疗炎症的施用将导致青光眼的发作，b）两个疤痕 适应症具有相似的生物学基础，因此在这两个迹象方面都可以节省成本 同时。我们正在提议使用自我交付的siRNA来预防和反向疤痕 角膜并防止在巩膜中疤痕，以降低眼睛内的眼内压力。 我们的自我传递（用胆固醇修饰以进入细胞）siRNA修饰用于体内使用的siRNA是 通过实用专利保护，以防止眼睛疤痕和第二次临时专利 修改的自传递siRNA序列和结构。 与慢性纤维化相似的急性疤痕的特征是免疫细胞浸润和持久性 称为肌纤维细胞的细胞。病理肌纤维细胞表现出增加的细胞粘合剂和组织 通过与细胞外基质和细胞内肌动蛋白结合产生的力收缩 通过整合素（跨膜蛋白）的细胞骨架。我们发现了康复的关键点 可以热靶向控制细胞凋亡，免疫浸润和整联蛋白 - 介导的病理肌纤维细胞发育。总的来说，我们在主要人角膜的研究 细胞，猪角膜器官培养以及兔子表明，图形会影响 去泛素酶（DUB），USP10。 USP10从p53和αV-积聚蛋白中去除泛素。 角膜赢得后，用一剂在体内击倒USP10的体内siRNA 凋亡减少，免疫浸润，纤维化标记和角膜疤痕以及试点研究 在青光眼过滤量中显示出再生愈合。实现实现的目标 最有效，最特定的USP10靶向疗法，在AIM 1中，我们建议阐明USP10 导致结果的域以及USP10绑定伙伴G3BP2对USP10的影响 配音活动。在AIM 2中，我们将扩大对USP10敲低的研究，以确定角膜是否是角膜 通过第二剂的自我交付siRNA，可以完全防止炎症和疤痕 通过新颖的技术逆转以使sirna进入疤痕。在AIM 3中，我们将测试USP10 敲除可以防止炎症和在进行的青光眼手术后的炎症和疤痕 减轻与青光眼相关的眼内压升高。这些成功完成 目标将大大提高我们对促进疤痕的中心机制的理解，并 将导致开发新型的自我保存siRNA方法，以防止角膜和 退伍军人和平民人口中与青光眼相关的失明。