Tropisms of AAV for subretinal delivery to the neonatal mouse retina and its application for in vivo rescue of developmental photoreceptor disorders.
Tropisms of AAV for subretinal delivery to the neonatal mouse retina and its application for in vivo rescue of developmental photoreceptor disorders.
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DOI：
10.1371/journal.pone.0054146
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Furukawa T
中科院分区：
文献类型：
--
作者：
Watanabe S;Sanuki R;Ueno S;Koyasu T;Hasegawa T;Furukawa T
腺相关病毒（AAV）被公认为用于体内基因转移到哺乳动物视网膜中的载体。该病毒不仅有望用于视网膜疾病的基因治疗，而且还有望用于视网膜基因的体内功能分析。先前的报道表明，AAV可以感染发育中的小鼠视网膜中的各种细胞类型。然而，AAV在发育中的视网膜中的向性尚未被详细研究。我们将AAV-CAG-mCherry的七种AAV血清型（AAV 2/1、2/2、2/5、2/8、2/9、2/10和2/11）视网膜下递送到P0小鼠视网膜中，并通过其在视网膜细胞中的感染程度定量评估每种血清型的嗜性。将AAV视网膜下注射到出生后第0天（P0）的小鼠视网膜中后，用不同的AAV有效地转导各种视网膜细胞类型。用AAV 2/5有效地转导光感受器细胞。除了双极和Müller神经胶质细胞外，视网膜细胞用AAV 2/9有效转导。用AAV 2/1、AAV 2/2、AAV 2/8、AAV 2/9和AAV 2/10有效地转导水平细胞和/或神经节细胞。为了证实AAV介导的基因转移到P0小鼠视网膜中的有用性，我们对锥-杆同源框基因敲除（Crx KO）小鼠进行了AAV介导的拯救，该小鼠表现出外节形成缺陷、平坦视网膜电图（ERG）反应和感光细胞变性。我们将在Crx 2kb启动子控制下表达Crx的AAV注射到新生儿Crx KO视网膜中。我们发现AAV介导的Crx表达显著降低了Crx KO视网膜的异常。在目前的研究中，我们报告了合适的AAV向性递送到发育中的小鼠视网膜中。在光感受器细胞中使用AAV 2/5，我们证明了基因替代由Crx缺失引起的视网膜光感受器发育障碍和随后变性的可能性。
Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail. We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina. In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.
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