Gene therapy restores vision and delays degeneration in the CNGB1-/- mouse model of retinitis pigmentosa
Gene therapy restores vision and delays degeneration in the CNGB1-/- mouse model of retinitis pigmentosa
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DOI：
10.1093/hmg/dds290
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发表时间：
2012-10-15
影响因子：
3.5
通讯作者：
Michalakis, Stylianos
中科院分区：
文献类型：
--
作者：
Koch, Susanne;Sothilingam, Vithiyanjali;Michalakis, Stylianos
视网膜色素变性是一组遗传性异质性、严重的视网膜疾病，常导致法定失明。已经发现，杆状环核苷酸门控(CNG)通道CNGB1a亚单位的突变会导致患者出现RP。在这里，我们通过重组腺相关病毒(AAV)载体在CNGB1基因敲除(CNGB1(/))小鼠模型中展示了基因治疗作为一种潜在的治疗RP的有效性。为了实现相对较大的CNGB1a基因的有效包装和杆特异性表达(约4kb)，我们使用了带有短杆特异性启动子和短调控元件的AAV表达盒。将治疗性AAVs注射到2周龄CNGB1(/)小鼠的视网膜下腔后，我们通过分析(I)CNG通道的表达和定位、(Ii)视网膜功能和形态以及(Iii)视觉引导行为来评估视觉系统的恢复。我们发现，处理不仅导致全长CNGB1a的表达，而且还恢复了先前降解的杆状CNG通道CNGA1亚单位的正常水平。这两种蛋白共同定位于视杆外节，并在CNGB1(/)视网膜治疗区域内形成规则的CNG通道复合体，导致显著的形态保留和视网膜变性的延缓。在视网膜电信号分析中，我们还观察到视杆驱动光反应的恢复。最后，在棒依赖视觉引导行为测试中，经CNGB1(/)治疗的小鼠的表现明显好于未经治疗的小鼠。综上所述，这项工作为通过AAV介导的基因置换治疗杆状通道病相关RP提供了一个概念验证。
Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1(/)) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (approximate to 4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1(/) mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1(/) retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1(/) mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.