Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

眼外伤造成的角膜疤痕是退伍军人、现役军人和 平民每年影响150万美国人。目前，超过167,000名退伍军人合法失明，1.5 每年有数百万人视力严重受损，7000名退伍军人失明。这个 在美国，每年治疗失明的费用为1390亿美元。目前的治疗方法提供了短期缓解，原因 多个副作用，往往无法治疗角膜移植手术治疗的先前存在的角膜疤痕 这是护理的标准。以目前的资金，我们已经成功地确定了第一个潜在的非手术 体内治愈先前存在的角膜疤痕的治疗，揭示调节角膜的表观遗传机制 纤维化，建立了阻止肌成纤维细胞形成的新的单基因和双基因疗法，其特征是新的 转化生长因子βdna结合蛋白，建立了犬活体角膜纤维化模型，并鉴定了许多新的 控制角膜纤维化或变薄的分子靶点、基因和药物，从22篇文献中可见一斑。 我们的中心假设是纳米颗粒介导的基因治疗可以提供长期的角膜缓解作用。 盲目无明显副作用和长远目标仍是发展创新板凳-- 床边转译纳米药物治疗活体角膜前和后瘢痕形成的方法。这 项目测试了5个形成为3个独立目标的新假设：1)测试假设PEI2- GNP介导的ID3基因导入兔基质将限制转分化 从基质成纤维细胞到肌成纤维细胞(导致不良伤口愈合和纤维化的关键机制)在 通过破坏E蛋白与转化生长因子-平滑肌肌动蛋白启动子(α-β)的结合而体内角膜 下游靶基因)。2)检验2个假设：(A)PEI2-GNPs传递定位的HGF+BMP7基因 治疗通过HGF根除角膜的机制消除体内原有的角膜疤痕 肌成纤维细胞和BMP7选择性凋亡的瘢痕形成防止新创面愈合的再次出现 由于已建立的肌成纤维细胞通过平衡促纤维化的Smads和(B)HGF+BMP7而死亡 向基质中输送不会影响角膜透明所需的特有的胶原纤维形成， 维持正常的角膜动态平衡，在体内对眼睛是安全的。3)检验2个新假说(A) 基质成纤维细胞通过α-平滑肌肌动蛋白(β-1下游)向肌成纤维细胞分化 靶基因)，并受共抑制物(5‘-TG-3’-相互作用因子(TGIF)1、TGIF2和 SnoN)和共激活子(CBP和p300)，以及(B)转分化过程可被 在兔模型中高效地过表达已识别的共抑制因子或沉默共激活因子。 成功完成拟议的研究填补了关键的知识空白，并显著提高了我们的 了解调控角膜伤口愈合的分子机制，引领新的发展 纳米药物治疗角膜失明，极大地推动了纤维化研究领域的发展。

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.