Lee Steven Hyun Seung, Chang HeeSoon, Kim Hee Jong, Choi Jun-Sub, Kim Jin, Kim Ji Hyun, Woo Ha-Na, Nah Seung Kwan, Jung Sang Joon, Lee Joo Yong, Park Keerang, Park Tae Kwann, Lee Heuiran
Department of Microbiology, College of Medicine, University of Ulsan, Seoul 05505, Korea.
Bio-Medical Institute of Technology, College of Medicine, University of Ulsan, Seoul 05505, Korea.
Mol Ther Methods Clin Dev. 2019 Jul 3;14:171-179. doi: 10.1016/j.omtm.2019.06.004. eCollection 2019 Sep 13.
Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3' UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.
脉络膜新生血管（CNV）是年龄相关性黄斑变性（AMD）湿性亚型的决定性特征，AMD是一个在全球范围内迅速加剧的健康问题。此前，我们已经证明了使用通过重组腺相关病毒（rAAV）递送的短发夹RNA（shRNA）进行基因治疗对抗CNV的潜力，该方法通过同时抑制两种mTOR复合物，以一种全新的方式消除哺乳动物雷帕霉素靶蛋白（mTOR）活性。基因治疗的靶点和用途均代表了一种针对AMD的新型治疗方式。在此，之前研究中用作报告基因的异种绿色荧光蛋白（GFP）基因从病毒载体中移除，以进一步开发用于临床试验的治疗方法。取而代之的是，使用源自人泛素蛋白连接酶E3A（UBE3A）基因3'非翻译区（UTR）的填充DNA，以确保病毒基因组大小达到最优，从而实现高效的rAAV组装。通过眼底荧光血管造影术测定，在激光诱导的小鼠模型中，含有填充DNA的病毒载体rAAV2-shmTOR-SD在减少CNV方面与编码shRNA和GFP表达盒的载体效果相当。使用抗CD31的免疫组织化学证实了这些结果，而TUNEL检测表明rAAV2-shmTOR-SD也具有抗凋亡特性。rAAV2-shmTOR-SD所展现的特性证明了其作为治疗湿性AMD的人类基因疗法的潜力。
