Semple-Rowland Susan L, Coggin William E, Geesey Mero, Eccles Kristofer S, Abraham Leah, Pachigar Krunal, Ludlow Rachel, Khani Shahrokh C, Smith W Clay
Department of Neuroscience, University of Florida McKnight Brain Institute, 100 Newell Dr., Rm L1-100 Box 100244, Gainesville, FL 32610-0244, USA.
Mol Vis. 2010 May 27;16:916-34.
Growing evidence suggests that successful treatment of many inherited photoreceptor diseases will require multi-protein therapies that not only correct the genetic defects linked to these diseases but also slow or halt the related degenerative phenotypes. To be effective, it is likely that therapeutic protein expression will need to be targeted to specific cell types. The purpose of this study was to develop dual-promoter lentiviral vectors that target expression of two proteins to retinal cones and rods, rods only, or Müller cells.
Dual-promoter lentivectors were constructed using the following promoters: Xenopus opsin promoter (XOPS)1.3, murine opsin promoter (MOPS), interphotoreceptor retinoid binding protein promoter (IRBP156), rhodopsin kinase (RK), neural retina leucine zipper (NRLL), vimentin (VIM), cluster differentiation (CD44), and glial fibrillary acidic protein (GFAP). Vectors were packaged and injected into the neural tubes of chicken embryos. The activities of the promoters alone, in duplicate, or when paired with a different promoter were analyzed in transduced, fully-developed retinas, using direct fluorescent and immunofluorescent microscopy.
IRBP156, NRLL, and RK were active in cones and rods while XOPS1.3 was active only in rods. Of the glial promoters, only GFAP activity was restricted to Müller cells; both VIM and CD44 were active in Müller and neural cells. Dual-promoter vectors carrying IRBP156 and RK or XOPS1.3 and MOPS, in the order listed, exhibited robust expression of both reporter transgenes in cones and rods or rods only, respectively. Expression of the upstream transgene was much lower than the downstream transgene in dual-promoter vectors constructed using two copies of either RK or IRBP156. Analyses of the expression of a dual-promoter vector carrying CD44 and VIM in the order listed showed that the activity of the VIM promoter was more restricted to glial cells when paired with the CD44 promoter, while the activity of the CD44 promoter was inhibited to the extent that no CD44-driven reporter protein was detected in transduced cells.
We have identified two dual-promoter vectors, one that targets cones and rods and one that targets rods alone. Both vectors reliably express the two proteins encoded by the transgenes they carry. When two well matched promoters are not available, we found that it is possible to target expression of two proteins to single cells using dual-promoter vectors carrying two copies of the same promoter. These vectors should be useful in studies of retina when co-delivery of a reporter protein with an experimental protein is desired or when expression of two exogenous proteins in targeted cells is required.
越来越多的证据表明，成功治疗许多遗传性光感受器疾病需要多种蛋白质疗法，这些疗法不仅要纠正与这些疾病相关的基因缺陷，还要减缓或阻止相关的退行性表型。为了有效，治疗性蛋白质表达可能需要靶向特定的细胞类型。本研究的目的是开发双启动子慢病毒载体，将两种蛋白质的表达靶向视网膜视锥细胞和视杆细胞、仅视杆细胞或穆勒细胞。
使用以下启动子构建双启动子慢病毒载体：非洲爪蟾视蛋白启动子（XOPS）1.3、小鼠视蛋白启动子（MOPS）、光感受器间类视黄醇结合蛋白启动子（IRBP156）、视紫红质激酶（RK）、神经视网膜亮氨酸拉链（NRLL）、波形蛋白（VIM）、分化簇（CD44）和胶质纤维酸性蛋白（GFAP）。将载体包装并注射到鸡胚的神经管中。使用直接荧光和免疫荧光显微镜在转导的、完全发育的视网膜中分析单独的启动子、重复的启动子或与不同启动子配对时的启动子活性。
IRBP156、NRLL和RK在视锥细胞和视杆细胞中具有活性，而XOPS1.3仅在视杆细胞中具有活性。在胶质细胞启动子中，只有GFAP活性局限于穆勒细胞；VIM和CD44在穆勒细胞和神经细胞中均具有活性。按所列顺序携带IRBP156和RK或XOPS1.3和MOPS的双启动子载体分别在视锥细胞和视杆细胞或仅视杆细胞中表现出两种报告转基因的强劲表达。在使用两个拷贝的RK或IRBP156构建的双启动子载体中，上游转基因的表达远低于下游转基因。对按所列顺序携带CD44和VIM的双启动子载体的表达分析表明，当与CD44启动子配对时，VIM启动子的活性更局限于胶质细胞，而CD44启动子的活性受到抑制，以至于在转导细胞中未检测到CD44驱动的报告蛋白。
我们鉴定出两种双启动子载体，一种靶向视锥细胞和视杆细胞，另一种仅靶向视杆细胞。两种载体都能可靠地表达它们携带的转基因所编码的两种蛋白质。当没有两个匹配良好的启动子时，我们发现使用携带同一启动子两个拷贝的双启动子载体将两种蛋白质的表达靶向单个细胞是可行的。当需要将报告蛋白与实验蛋白共同递送或需要在靶向细胞中表达两种外源蛋白时，这些载体在视网膜研究中应该会很有用。
