Pawlyk B S, Bulgakov O V, Sun X, Adamian M, Shu X, Smith A J, Berson E L, Ali R R, Khani S, Wright A F, Sandberg M A, Li T
Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA, USA.
National Eye Institute, Bethesda, MA, USA.
Gene Ther. 2016 Feb;23(2):196-204. doi: 10.1038/gt.2015.93. Epub 2015 Sep 8.
The X-linked RP3 gene codes for the ciliary protein RPGR and accounts for over 10% of inherited retinal degenerations. The critical RPGR-ORF15 splice variant contains a highly repetitive purine-rich linker region that renders it unstable and difficult to adapt for gene therapy. To test the hypothesis that the precise length of the linker region is not critical for function, we evaluated whether adeno-associated virus-mediated replacement gene therapy with a human ORF15 variant containing in-frame shortening of the linker region could reconstitute RPGR function in vivo. We delivered human RPGR-ORF15 replacement genes with deletion of most (314 codons, 'short form') or 1/3 (126 codons, 'long form') of the linker region to Rpgr null mice. Human RPGR-ORF15 expression was detected post treatment with both forms of ORF15 transgenes. However, only the long form correctly localized to the connecting cilia and led to significant functional and morphological rescue of rods and cones. Thus the highly repetitive region of RPGR is functionally important but that moderate shortening of its length, which confers the advantage of added stability, preserves its function. These findings provide a theoretical basis for optimizing replacement gene design in clinical trials for X-linked RP3.
X连锁的RP3基因编码睫状蛋白RPGR，在遗传性视网膜变性中占比超过10%。关键的RPGR-ORF15剪接变体包含一个高度重复的富含嘌呤的连接区，这使其不稳定且难以应用于基因治疗。为了验证连接区的精确长度对功能并非至关重要这一假设，我们评估了用一种连接区发生读码框内缩短的人ORF15变体进行腺相关病毒介导的替代基因治疗是否能在体内重建RPGR功能。我们将缺失大部分（314个密码子，“短形式”）或1/3（126个密码子，“长形式”）连接区的人RPGR-ORF15替代基因导入Rpgr基因敲除小鼠体内。两种形式的ORF15转基因在治疗后均检测到了人RPGR-ORF15的表达。然而，只有长形式正确定位于连接纤毛，并导致视杆和视锥细胞在功能和形态上得到显著挽救。因此，RPGR的高度重复区域在功能上很重要，但适度缩短其长度（这赋予了增加稳定性的优势）仍能保留其功能。这些发现为优化X连锁RP3临床试验中的替代基因设计提供了理论基础。
