Programming retinal Müller glial cells for supporting neuronal survival in retinal degeneration

遗传性视网膜营养不良症（IRD）是一种罕见的视网膜疾病，它引起光感受器变性，从而导致患者视力严重下降。驱动IRD的致病突变非常复杂，目前已知的基因超过200个。由于这些突变中只有一小部分会影响视锥细胞感光细胞，而另一方面，视锥细胞是日光视觉中使用的主要感光细胞类型，因此一个重要的治疗概念是保留视锥细胞的功能，而不依赖于针对任何潜在突变开发矫正性基因疗法。目前已经发现了许多有效的抗光感受器变性的神经保护因子，其中大多数是由视网膜主要的神经胶质细胞视网膜<s:1>神经胶质细胞（RMG）提供的，用于终身保持光感受器的功能。然而，在IRD条件下，RMG提供的内在支持不足，我们假设增强RMG的神经保护特性将延长IRD中光感受器的存活和视网膜功能。我们的目标是通过CRISPR/(d) cas9驱动RMG重编程为神经保护表型，同时通过干扰胶质细胞/免疫反应来抑制不良胶质细胞反应来实现这一目标。我们将重点关注四种不同的神经营养因子，即CNTF和GDNF，它们都代表了IRD中最有效的神经保护因子。此外，我们将包括CXCL10，一种新的RMG衍生的神经营养因子，我们之前证实了它对光受体有直接的促生存作用。最后，我们将探索RdCVF，一种杆状细胞衍生的神经营养因子，被发现直接支持锥体存活，但不是RMG自然表达的。我们将使用的模型是从猪身上分离出的原始RMG以及器官型视网膜外植体，这将能够在完整的组织背景下靶向RMG。该项目的最终目标是通过同时激活促存活蛋白表达和抑制胶质/炎症反应，在完整的组织环境中开发促存活表型的RMG。这将提供一种全新的、不依赖突变的治疗策略，可能克服以前神经保护治疗的局限性，最终防止疾病进展，从而在对个体患者进行矫正基因治疗或基因添加治疗之前保护脆弱的光感受器。它也可以被设想为适用于那些已经失去所有杆状光感受器的患者，因此矫正基因治疗的进展太大，但仍然有剩余的锥驱动视力，可以保留。此外，该疗法也可应用于IRD以外的其他视网膜退行性疾病，如青光眼、年龄相关性黄斑变性和糖尿病视网膜病变。

Inherited retinal dystrophies (IRD) are rare disorders of the retina that cause degeneration of photoreceptors and thus result in severe decline of visual ability for the affected patients. The causative mutations driving IRD are extremely complex covering so far more than 200 known genes. Since only a smaller fraction of those mutations affect cone photoreceptors, while on the other hand, cones are the main photoreceptor cell type used in daylight vision, an important therapeutic concept is to preserve cone function independent of developing corrective gene therapies for any underlying mutation. Many neuroprotective factors effective against photoreceptor degeneration have been identified and most of them are normally provided by retinal Müller glial cells (RMG), the main glial cell of the retina, for preserving photoreceptor function throughout life. However, under IRD conditions, the intrinsic support provided by RMG is insufficient and we hypothesize that boosting the neuroprotective properties of RMG will prolong photoreceptors survival and retinal function in IRD.We aim to achieve this by CRISPR/(d)CAS9-driven re-programming of RMG into a neuroprotective phenotype and simultaneously suppressing adverse glial responses by interfering with gliotic/immune responses. We will focus on four different neurotrophic factors, namely, CNTF and GDNF, which both represent the as yet most effective neuroprotective factors in IRD. Further we will include CXCL10, a novel RMG-derived neurotrophic factor that we previously validated for a direct pro-survival effect on photoreceptors and finally, we will explore RdCVF, a rod-derived neurotrophic factor that was found to directly support cone survival, but is not naturally expressed by RMG. The models we will be using are primary RMG isolated from pigs as well as organotypic retinal explants, which will enable to target RMG in the intact tissue context. The final goal of this project is development of a pro-survival phenotype of RMG within the intact tissue context by simultaneous activation of pro-survival protein expression and inhibition of gliotic/inflammatory reactions.This will provide a completely novel and mutation-independent therapeutic strategy that might overcome previous limitations of neuroprotective therapy and eventually prevent disease progression and thus preserve fragile photoreceptors before corrective gene therapy or gene addition therapy becomes available for the individual patient. It also could be envisioned to be applicable to patients, which have already lost all rod photoreceptors and hence are too far progressed for corrective gene therapy, but still have residual cone-driven vision, which could be preserved. Further, this therapy may also be applicable beyond IRD to other degenerative diseases of the retina, such as glaucoma, age-related macular degeneration and diabetic retinopathy.