Department of Chemical Engineering, The University of California at Berkeley, Berkeley, California 94720-3190, USA.
Mol Ther. 2009 Dec;17(12):2096-102. doi: 10.1038/mt.2009.181. Epub 2009 Aug 11.
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.
腺相关病毒基因治疗在治疗视网膜疾病方面显示出巨大的潜力，目前正在进行三项有前途的临床试验。当腺相关病毒 (AAV) 血清型通过视网膜下途径给药时，可以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负面影响，从而改变微环境并影响视网膜神经元的存活。玻璃体内给药可以避免这个问题，但是只有 AAV2 可以从玻璃体内感染视网膜细胞，而且转导仅限于视网膜内层。因此，我们试图研究并减少玻璃体内转导的障碍。我们对几种 AAV 血清型衣壳进行了荧光标记，并在玻璃体内注射后观察它们在视网膜中的分布。AAV2、8 和 9 在玻璃体视网膜交界处积聚。AAV1 和 5 没有积聚，表明在内界膜 (ILM) 上缺乏适当的受体。重要的是，用非特异性蛋白酶轻度消化 ILM 可以显著增强玻璃体内多种视网膜细胞类型的转导，其中 AAV5 在所有视网膜层中都介导了特别显著的表达。这种蛋白酶处理对视网膜功能没有影响，如视网膜电图 (ERG) 和视皮层细胞群体反应所示。这些发现可能有助于避免与目前临床基因治疗中必需的视网膜下注射相关的限制、风险和损伤。
