Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous
Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous
复制标题
DOI：
10.1038/mt.2009.181
复制
发表时间：
2009-12-01
影响因子：
12.4
通讯作者：
Flannery, John G.
中科院分区：
文献类型：
--
作者：
Dalkara, Deniz;Kolstad, Kathleen D.;Flannery, John G.
腺相关病毒基因疗法在治疗视网膜疾病方面显示出巨大的希望，有三个有希望的临床试验正在进行中。许多腺相关病毒（AAV）血清型可以在视网膜下注射时感染视网膜的各种细胞，但伴随这种注射的视网膜脱离会引起对视网膜神经元微环境和存活产生负面影响的变化。玻璃体内给药可以避免这个问题，但只有AAV2可以从玻璃体感染视网膜细胞，并且转导仅限于视网膜内。因此，我们试图研究并减少玻璃体转导的障碍。我们荧光标记了几种AAV血清型衣壳，并观察了它们在玻璃体内注射后的视网膜分布。AAV2、8和9聚集在玻璃体视网膜交界处。AAV1和aav5没有积累，表明在内限制膜（ILM）缺乏合适的受体。重要的是，用非特异性蛋白酶对ILM进行温和消化，可以大大增强玻璃体中多种视网膜细胞类型的转导，其中AAV5介导了所有视网膜层中特别显著的表达。通过视网膜电图（ERG）和视觉皮层细胞群反应显示，这种蛋白酶治疗对视网膜功能没有影响。这些发现可能有助于避免目前临床基因治疗所必需的视网膜下注射的局限性、风险和损伤。
Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy.