Oxidative Damage and Cone Cell Death in RP

描述（由申请人提供）：色素性视网膜炎（RP）是一组疾病，其中数百种不同的突变之一导致视杆细胞死亡，导致夜盲症，随后视锥细胞逐渐死亡，导致视野狭窄，最终失明。视杆细胞的丧失导致视网膜外氧水平升高，在目前的资助期内，我们证明这伴随着视锥细胞的进行性氧化损伤，导致功能下降和细胞死亡。通过转基因方法，我们证明了内源性抗氧化防御系统成分在小鼠RP模型中过表达可以减少氧化损伤，促进锥体功能和存活，但前提是线粒体或细胞质中有超氧化物歧化酶（SOD）和h2o2解毒酶的共同表达。这表明在RP的情况下，细胞质和线粒体中都存在过多的活性氧（ROS），也表明为了利用ROS解毒酶治疗RP，需要多基因转移。我们开始将我们的发现转化为一种基于基因的RP治疗方法，通过鉴定一种病毒载体/启动子组合，在小鼠或猪的视网膜下或玻璃体内注射后转导光感受器，我们开始在小鼠和猪的RP模型中进行治疗性转基因研究。在下一个拨款期间，我们将使用定量技术来评估多基因转移对单个成分转基因表达的影响。我们将继续在小鼠和猪RP模型中进行目前的实验，以回答多基因转移靶向线粒体或细胞质是否能提供更大的锥体拯救。最后，我们将确定同时靶向线粒体和细胞质室是否比单独靶向任一室提供更好的结果。这项提议的潜在影响很大，因为它将

DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) is a group of diseases in which one of hundreds of different mutations leads to death of rods resulting in night blindness followed by gradual death of cones causing constriction of visual fields and eventual blindness. The loss of rods results in elevated levels of oxygen in the outer retina and during the current grant period, we demonstrated that this is accompanied by progressive oxidative damage in cones causing reduced function and cell death. Using a transgenic approach, we demonstrated that over-expression of components of the endogenous antioxidant defense system in mouse models of RP can reduce oxidative damage and promote cone function and survival, but only if there is co- expression of a superoxide dismutase (SOD) and a H2O2-detoxifying enzyme in mitochondria or in the cytosol. This suggests that in the setting of RP, there are excessive reactive oxygen species (ROS) in both the cytosol and mitochondria and also indicates that in order to utilize ROS-detoxifying enzymes to treat RP, multi- gene transfer will be needed. We began translating our findings into a gene-based therapy for RP by identifying a viral vector/promoter combination that transduces photoreceptors after subretinal or intravitreous injections in mice or pigs and we initiated studies with therapeutic transgenes in mouse and pig models of RP. During the next grant period we use quantitative techniques to assess the effect of multi- gene transfer on the expression of individual component transgenes. We will continue our current experiments in both mouse and pig models of RP that will answer whether targeting the mitochondria or cytosol with multi-gene transfer provides greater cone rescue. Finally we will determine whether targeting both the mitochondrial and cytosolic compartments provides better outcomes than targeting either alone. The potential impact of this proposal is high because it will provide important new information regarding multi-gene transfer that will interest all investigator in the gene therapy field, and most importantly will provide critical efficacy data in a large animl model of RP that could lead to clinical trials. PUBLIC HEALTH RELEVANCE: This project seeks to develop a gene-based treatment to prevent cone cell death in patients with retinitis pigmentosa (RP) by augmenting the endogenous antioxidant defense system. RP is a group of diseases in which hundreds of different mutations lead to rod cell death followed by progressive oxidative damage to cones and eventual blindness. This treatment which targets cones will apply to all patients with RP regardless of the underlying mutation and therefore could remedy a large unmet medical need providing major public health impact.