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免权，但视网膜细胞表达补体系统(CS)的组成部分，补体系统是天然免疫系统的一条途径。由脂褐素积聚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)的一个特征。这种年轻人的遗传性视网膜营养不良(IRD)是由ATP结合盒A亚家族成员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)主要在视网膜色素上皮、小胶质细胞和血管细胞中表达，而唯一的阳性调节因子备解素(CFP)主要在Müller细胞、小胶质细胞和神经元中表达。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CS活性再平衡可能是一种有前景的、基因无关的IRD治疗策略，如具有复杂或未知遗传学的STGD1(约25%的STGD和约40%-50%的IRD患者)。事实上，最近CS过多也与疾病进展有关，例如在视网膜色素变性中。针对CS关键因素重复玻璃体内注射重组蛋白和治疗性抗体的治疗潜力的临床试验已经得出了支持我们的假设的积极结果。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(截断的CFH)或阻断CFP的抗体片段来调节视网膜CS。外源蛋白的表达可以应激高度特化的视网膜神经元，如光感受器。因此，我们认为Müler细胞是基因添加方法的理想靶点，因为它们(I)具有固有的高分泌活性，(Ii)它们与每种视网膜细胞类型接触巨大，因此，它们分泌的蛋白质到达每个视网膜亚室，(Iii)由于它们独特的形态，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，可以积极地塑造视网膜补体的稳态。在生成腺相关病毒载体来驱动我们的治疗蛋白的Müller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，从而阻止疾病的进展，并对Müler细胞生理产生积极的影响。在细胞类型特定的水平上深入表征STGD1中补体调节工具的作用，识别补体因子的局部来源及其调节，具有很高的创新性，可能会导致对目前已经在临床试验中测试的治疗方法的重新思考。最终，特别是那些不适合基因纠正策略的STGD(和IRD)患者将从我们希望开发的治疗工具中受益。

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.