Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

眼外伤引起的角膜瘢痕是退伍军人、现役军人和 每年影响150万美国人目前，超过167，000名退伍军人在法律上是盲人， 数百万人视力严重受损，每年有超过7000名退伍军人失明。的 美国每年治疗失明的费用是1390亿美元。目前的治疗提供短期缓解， 并且常常不能治疗先前存在的角膜瘢痕， 一个标准的护理。在目前的资金支持下，我们已经成功地发现了第一个可能的非手术治疗。 在体内治愈既存角膜瘢痕的疗法，揭示了调节角膜瘢痕的表观遗传机制， 纤维化，建立了新的单基因和双基因疗法来阻断肌成纤维细胞的形成，其特征在于新的 TGFβ DNA结合蛋白，建立了犬体内角膜纤维化模型，并鉴定了许多新的 分子靶点、基因和药物来控制角膜纤维化或变薄，如22篇出版物所证实的。 我们的中心假设是，纳米粒子介导的基因治疗提供了长期的缓解角膜 无明显副作用的失明和长期目标仍然是创新的板凳发展， 床旁转化纳米医学方法来治疗体内角膜前和后瘢痕。这 项目测试5个新的假设，制定为3个独立的目标：1）测试假设，PEI 2- GNP介导的Id 3基因（分化抑制剂）递送到兔基质中将限制转分化 间质成纤维细胞转化为肌成纤维细胞（一种导致不良伤口愈合和纤维化的关键机制）， 通过破坏E蛋白与α-平滑肌肌动蛋白（TGFβ）启动子的结合， 下游靶基因）。2)检验2个假设：（a）PEI 2-GNP-递送局部HGF+ BMP 7基因 治疗通过HGF根除角膜瘢痕的机制消除体内预先存在的角膜瘢痕 通过肌成纤维细胞和BMP 7中的选择性凋亡形成瘢痕防止新伤口愈合的重新出现 由于通过平衡促纤维化Smads和（B）HGF+ BMP 7而导致已建立的肌成纤维细胞死亡 递送到基质中不会损害角膜透明性所需的特征性胶原原纤维形成， 维持正常的角膜稳态，并且在体内对眼睛是安全的。3)测试2个新假设（a） 基质成纤维细胞向肌成纤维细胞的分化通过α-平滑肌肌动蛋白（TGFβ1下游 靶基因），并由辅阻遏物（5 '-TG-3'-相互作用因子（TGIF）1、TGIF 2和 SnoN）和共激活剂（CBP和p300），和（B）转分化过程可以被 在兔模型中体内有效地过表达鉴定的共阻遏物或沉默共激活物。 成功完成拟议的研究填补了关键的知识空白，并显着提高我们的 了解调节角膜伤口愈合的分子机制， 纳米医学方法治疗角膜失明，并显著推进了纤维化研究领域。

Corneal scarring from ocular trauma is a major cause of blindness in veterans, active military personnel and civilians affecting 1.5 million Americans each year. Presently, over 167,000 Veterans are legally blind, 1.5 million suffer significantly compromised vision, and >7000 veterans are becoming blind each year. The annual cost for treating blindness in America is $139 billion. Current therapies offer short-term relief, cause multiple side effects and often fails to treat preexisting corneal scars for which corneal transplant surgery is a standard of care. With current funding we have successfully identified potentially the first non-surgical therapy to cure pre-existing corneal scars in vivo, uncovered epigenetic mechanisms regulating corneal fibrosis, established novel single and 2-gene therapies to block myofibroblast formation, characterized novel TGFβ DNA-binding proteins, developed a canine in vivo corneal fibrosis model, and identified many newer molecular targets, genes, and drugs to control corneal fibrosis or thinning as evident from 22 publications. Our central hypothesis is that nanoparticle-mediated gene therapy offers long-term relief from corneal blindness without significant side effects and long-term goal remains the development of innovative bench- to-bedside translational nanomedicine approaches to cure pre- and post-corneal scarring in vivo. This project tests 5 novel hypotheses formulated into 3 independent aims: 1) Tests the hypothesis that PEI2- GNPs-mediated Id3 gene (Inhibitor of differentiation) delivery into rabbit stroma will limit transdifferentiation of stromal fibroblast to myofibroblast (a key mechanism causing undesirable wound healing and fibrosis) in the cornea in vivo by disrupting the binding of E-proteins to the promoter of α-smooth muscle actin (TGFβ downstream target gene). 2) Tests 2 hypotheses that (a) PEI2-GNPs-delivered localized HGF+BMP7 gene therapy eliminates preexisting corneal scarring in vivo via mechanisms in which HGF eradicates corneal scarring by selective apoptosis in myofibroblasts and BMP7 prevents re-emergence of new wound healing due to death of established myofibroblasts by counterbalancing profibrotic Smads and (b) HGF+BMP7 delivery into stroma does not compromise characteristic collagen fibrillogenesis required for corneal clarity, maintains normal corneal homeostasis, and is safe to the eye in vivo. 3) Tests 2 novel hypotheses (a) stromal fibroblast differentiation to myofibroblast proceeds via α-smooth muscle actin (TGFβ1 downstream target gene) and is tightly controlled by the co-repressor (5'-TG-3'-interacting factor (TGIF)1, TGIF2, and SnoN) and co-activator (CBP and p300), and (b) the transdifferentiation process can be arrested by the over-expression of identified co-repressor or silencing of co-activator efficiently in vivo in a rabbit model. Successful completion of the proposed research fills critical knowledge gaps and significantly improve our understanding of molecular mechanisms regulating corneal wound healing, lead development of novel nanomedicine approaches for corneal blindness and significantly advances the fibrosis research field.