Oxidative Damage and Cone Cell Death in RP

描述(申请人提供)：视网膜色素变性(RP)是一组疾病，数百种不同的突变中的一种会导致视杆细胞死亡，导致夜盲，然后是视锥细胞逐渐死亡，导致视野狭窄和最终失明。视杆细胞的丢失导致外视网膜氧气水平升高，在当前的授权期内，我们证明了这伴随着视锥细胞的进行性氧化损伤，导致功能降低和细胞死亡。使用转基因方法，我们证明了在RP小鼠模型中过表达内源性抗氧化防御系统的组件可以减少氧化损伤，促进视锥功能和存活，但前提是线粒体或胞浆中存在超氧化物歧化酶(SOD)和过氧化氢解毒酶的共表达。这表明，在RP的环境下，胞质和线粒体中都存在过量的ROS，同时也表明为了利用ROS解毒酶来治疗RP，需要多基因转移。我们开始将我们的发现转化为基于基因的RP治疗，方法是确定一种病毒载体/启动子组合，在小鼠或猪视网膜下或玻璃体内注射后转导光感受器，并开始在小鼠和猪的RP模型中进行治疗性转基因研究。在下一个赠款期间，我们使用定量技术来评估多基因转移对单个组分转基因表达的影响。我们将继续我们目前在小鼠和猪的RP模型上的实验，以回答通过多基因转移靶向线粒体或胞浆是否提供了更大的锥体挽救。最后，我们将确定同时针对线粒体和胞质间隔是否比单独针对其中任何一个提供更好的结果。这项提议的潜在影响很大，因为它将 提供有关多基因转移的重要新信息，这将使基因治疗领域的所有研究人员感兴趣，最重要的是，将提供可能导致临床试验的RP大型动物模型的关键疗效数据。

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.