Targeting Müller cells for complement modulating gene addition therapy in a mouse model for Stargardt disease type 1

虽然视网膜具有免疫特权，但视网膜细胞表达补体系统（CS）的成分，这是先天免疫系统的一条途径。由脂褐素积累引起的视网膜补体激活和神经破坏性副炎症是Stargardt病1型（STGD1）的一个标志。这种年轻人的遗传性视网膜营养不良（IRD）是由ATP结合盒亚家族A成员4 （ABCA4）基因突变引起的。我们发现，主要的抑制性补体调节因子补体因子H （CFH）主要表达在视网膜色素上皮、小胶质细胞和血管细胞中，而唯一已知的阳性调节因子properdin （CFP）主要表达在突触细胞、小胶质细胞和神经元中。在ABCA4-/- STGD1小鼠模型中，我们发现与对照组相比，补体因子在各种视网膜细胞群中的表达增加。细胞类型特异性的CS活性再平衡可能是一种有希望的、与基因无关的IRD治疗策略，如具有复杂或未知遗传的STGD1（约25%的STGD和约40-50%的所有IRD患者）。事实上，最近超调的CS也与疾病进展有关，例如视网膜色素变性。研究玻璃体内反复注射重组蛋白和针对CS关键因子的治疗性抗体的治疗潜力的临床试验已经给出了支持我们假设的积极结果。我们现在的目标是开发一种长期有效的基因添加疗法，通过表达mini-FH（截断CFH）或阻断CFP的抗体片段来调节视网膜CS。外源蛋白的表达可能会对高度特化的视网膜神经元（如光感受器）造成压力。因此，我们认为m<s:1> ller细胞是基因添加方法的理想目标，因为它们(i)具有高分泌活性，（ii）与每种视网膜细胞类型接触很大，因此，它们分泌的蛋白质可以到达每个视网膜亚室，（iii）鉴于它们独特的形态，它们可以通过相对安全的玻璃体内注射来解决，（iv）它们本身是CS激活剂的主要来源，因此，能积极塑造视网膜补体稳态。在生成腺相关病毒载体驱动我们的治疗性蛋白在m<s:1> ller细胞特异性表达后，我们将对ABCA4-/-小鼠进行治疗，以确定这是否可以抑制补体活性，从而阻止疾病进展并积极影响m<s:1> ller细胞生理学。在细胞类型特异性水平上对补体调节工具在STGD1中的作用进行深入表征，识别补体因子的局部来源及其调控，这是高度创新的，可能会导致对已经在临床试验中测试的当前治疗方法的重新思考。最后，特别是那些不适合基因校正策略的STGD（和IRD）患者将受益于我们希望开发的治疗工具。

Although the retina is immune privileged, retinal cells express components of the complement system (CS), a pathway of the innate immune system. Retinal complement activation and neuro-destructive parainflammation induced by lipofuscin accumulation is a hallmark in Stargardt disease type 1 (STGD1). This inherited retinal dystrophy (IRD) of young adults is caused by mutations in the ATP binding cassette subfamily A member 4 (ABCA4) gene. We showed that the main inhibitory complement regulator, complement factor H (CFH), was mainly expressed in retinal pigment epithelium, microglia and vascular cells, while the only known positive regulator, properdin (CFP), was primarily detected in Müller cells, microglia and neurons. In the ABCA4-/- STGD1 mouse model, we found an increased expression of complement factors in various retinal cell populations compared to controls. Cell type-specific rebalancing of CS activity could be a promising, gene-independent therapeutic strategy for IRD such as STGD1 with complicated or unknown genetics (about 25% STGD and ~40-50% of all IRD patients). Indeed, overshooting CS has recently also been associated with disease progression, e.g., in retinitis pigmentosa. Clinical trials investigating the therapeutic potential of repetitive intravitreal injections of recombinant proteins and therapeutic antibodies targeting key factors of the CS already gave positive results supporting our hypothesis. We now aim to develop a long-term efficient gene addition therapy modulating the retinal CS by expression of mini-FH (truncated CFH) or an antibody fragment blocking CFP. Expression of exogenous protein could stress the highly specialized retinal neurons like photoreceptors. Accordingly, we consider Müller cells as ideal targets for a gene addition approach as they (i) have an intrinsically high secretory activity, (ii) are huge contacting every retinal cell type and, thus, proteins secreted by them reach every retinal subcompartment, (iii) given their distinct morphology they can be addressed by comparably safe intravitreal injections and (iv) they themselves are a major source of activators of the CS and, thus, could actively shape the retinal complement homeostasis. After generating adeno-associated viral vectors driving Müller cell-specific expression of our therapeutic proteins, we will treat ABCA4-/- mice to determine whether this can dampen complement activity, thereby halt disease progression and positively affect Müller cell physiology.The in-depth characterization of effects of complement modulatory tools in STGD1 at cell type-specific level identifying the local source of complement factors and regulation thereof is highly innovative and probably will lead to a rethinking of current therapeutic approaches already tested in clinical trials. In the end, especially those STGD (and IRD) patients not suited for gene correction strategies will benefit from the therapeutic tools we hope to develop.