The Human Rhodopsin Kinase Promoter in an AAV5 Vector Confers Rod- and Cone-Specific Expression in the Primate Retina
The Human Rhodopsin Kinase Promoter in an AAV5 Vector Confers Rod- and Cone-Specific Expression in the Primate Retina
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DOI：
10.1089/hum.2012.125
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发表时间：
2012-10-01
影响因子：
4.2
通讯作者：
Gamlin, Paul D.
中科院分区：
文献类型：
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作者：
Boye, Shannon E.;Alexander, John J.;Gamlin, Paul D.
腺相关病毒(AAV)已被证明是治疗视网膜疾病的有效基因载体。正在进行的使用2型AAV载体在视网膜色素上皮表达RPE65的临床试验已被证明是安全和有效的。虽然许多视网膜疾病动物模型的概念验证研究表明，将基因转移到神经视网膜也将是有效的，但以光感受器为靶向的AAV载体尚未用于临床，主要是因为尚未证明一种有效但唯一地针对所有灵长类光感受器的载体。在这里，我们评估了含有人视紫红质激酶(HGRK1)启动子的血清5型AAV载体在非人类灵长类动物(NHP)视网膜下传递时，其靶向视杆和视锥感受器转基因表达的能力。体内荧光眼底成像证实，AAV5-hGRK1介导的绿色荧光蛋白(GFP)表达仅限于治疗眼的注射泡。光学相干断层扫描(OCT)显示注射后无大体病理改变。在接受简单视网膜下注射(即没有出血)的受试者的注射后血清样本中，未检测到针对AAV5的中和抗体。视网膜切片的免疫组织化学证实hGRK1在NHP视网膜的视杆细胞和视锥细胞中都是活跃的，并且是特异的。生物分布研究显示，载体基因组向周围组织的传播最小。这些结果表明，AAV5-hGRK1是一种安全有效的AAV血清型/启动子组合，可在临床上将治疗性转基因表达蛋白靶向杆状和锥体。
Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.