基于OPTN（E50K）突变致NTG致病机制的干细胞治疗研究

正常眼压青光眼（NTG）发病隐匿，视功能损害严重，其治疗是青光眼领域的难题。OPTN基因是NTG相关致病基因。申请者前期研究证实OPTN（E50K）突变影响神经营养因子表达及分泌是导致RGCs死亡的机制之一。另外，正常OPTN蛋白作为自噬接合体，在细胞自噬过程起关键作用；而自噬功能异常则会引起机体干细胞干性和旁分泌作用下降，进一步阻碍其对受损RGCs的修复。本项目基于OPTN（E50K）突变致RGCs损伤多重机制和骨髓优质干细胞高效归巢修复受损组织的特性，寻找NTG治疗的新方法和新思路。我们拟在体外对骨髓干细胞中自我更新能力占优势的Sca-1+干细胞亚群进行分选富集，对CRISPR-Cas9技术构建的OPTN（E50K）突变NTG小鼠进行骨髓重建，观察Sca-1+亚群干细胞对NTG致RGCs损伤的修复和对视功能的改善，探讨其作用机制，为NTG等视神经退行性疾病的干细胞治疗提供新策略。

Normal tension glaucoma (NTG), a common cause of vision loss and blindness that occurs without grossly abnormal intraocular pressure. RGCs degeneration under normal pressure remains poorly understood, which now is a dilemma in the glaucoma field. The cell death is irreversible and the retinal ganglion cells have very limited ability to regenerate after injury. Mutations in optineurin (OPTN) are linked to the pathology of NTG. Emerging evidence indicated that OPTN(E50K) mutation is involved in the damage of transportation and expression for neurotrophic factors, resulting in RGCs death of mutant. Nevertheless, the role played by the pathogenic autophagy that underlies the abnormal stemness and paracrine effect of stem cell degeneration, associated with defective capacity of restoration for damaged RGCs. According to the multiple pathophysiological mechanisms underlying the E50K mutation-mediated glaucomatous RGC degeneration and the possibility of the pluripotent bone marrow stem cell based regeneration of retinal cells and repair of neurodegenerative diseases, we tried to develop a new strategy of stem cell therapy for NTG. A novel mouse model was established based on a mutation by CRISPR-Cas9 in mouse optineurin associated with hereditary, normal tension glaucoma, which improved the design and phenotype of currently available optineurin mice. Further, we propose to isolate the bone marrow Sca-1+ subset of stem cells using magnetic activated cell sorting and identify their neuroprotective effect on organotypical retinal explant of the mutants and their homing efficiency in bone marrow reconstituted chimeras in vivo in conjunction with the model of E50K mutation-mediated normal tension glaucoma. We hypothesize that the bone marrow-derived Sca-1+ subset of stem cells exerts beneficial paracrine actions on the injured retina of E50K mutation by releasing biologically active factors, regulating pathogenic autophagy, and all these may play an important role in bone marrow Sca-1+ subset of stem cell-mediated retinal neuroprotection. Our study is to evaluate the translational potential of bone marrow derived subpopulation of stem cells for the treatment of NTG, alongside targeting possible novel therapeutical factors through stem cell-independent method. It will provide a more efficient stem cell sub-type for the treatment ocular diseases and glaucoma in future. We believe that the current proposal will provide a new strategy of stem cell therapy for NTG and identify the most efficient therapy for cell application in ophthalmological treatment.