Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Rehovot, Israel.
Center for Retinal and Macular Degenerations (CRMD), Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Exp Eye Res. 2018 Nov;176:227-234. doi: 10.1016/j.exer.2018.08.021. Epub 2018 Aug 29.
Recombinant adeno associated viruses (AAV) are the most commonly used vectors in animal model studies of gene therapy for retinal diseases. The ability of a vector to localize and remain in the target tissue, and in this manner to avoid off-target effects beyond the site of delivery, is critical to the efficacy and safety of the treatment. The in vivo imaging system (IVIS) is a non-invasive imaging tool used for detection and quantification of bioluminescence activity in rodents. Our aim was to investigate whether IVIS can detect localization and biodistribution of AAV5 vector in mice following subretinal (SR) and intravitreal (IVT) injections. AAV5 carrying firefly luciferase DNA under control of the ubiquitous cytomegalovirus (CMV) promoter was injected unilaterally IVT or SR (in the central or peripheral retina) of forty-one mice. Luciferase activity was tracked for up to 60 weeks in the longest surviving animals, using repeated (up to 12 times) IVIS bioluminescence imaging. Luciferase presence was also confirmed immunohistochemically (IHC) and by PCR in representative animals. In the SR group, IVIS readings demonstrated luciferase activity in all (32/32) eyes, and luciferase presence was confirmed by IHC (4/4 eyes) and PCR (12/12 eyes). In the IVT group, IVIS readings demonstrated luciferase activity in 7/9 eyes, and luciferase presence was confirmed by PCR in 5/5 eyes and by IHC (2/2 eyes). In two SR-injected animals (one each from the central and peripheral injection sites), PCR detected luciferase presence in the ipsilateral optic nerves, a finding that was not detected by IVIS or IHC. Our results show that when evaluating SR delivery, IVIS has a sensitivity and specificity of 100% compared with the gold standard PCR. When evaluating IVT delivery, IVIS has a sensitivity of 78% and specificity of 100%. These finding confirm the ability of IVIS to detect in-vivo localized expression of AAV following SR delivery in the retina up to 60 weeks post-treatment, using repeated imaging for longitudinal evaluation, without fading of the biological signal, thereby replacing the need for post mortem processing in order to confirm vector expression. However, IVIS is probably not sensitive enough, compared with genome detection, to demonstrate biodistribution to the optic nerve, as it could not detect luciferase activity in ipsilateral optic nerves following SR delivery in mice.
重组腺相关病毒（AAV）是用于视网膜疾病基因治疗的动物模型研究中最常用的载体。载体在目标组织中的定位和保持能力，以及避免输送部位以外的脱靶效应，对于治疗的疗效和安全性至关重要。活体成像系统（IVIS）是一种非侵入性成像工具，用于检测和定量分析啮齿动物的生物发光活性。我们的目的是研究 IVIS 是否可以检测到 AAV5 载体在小鼠的视网膜下（SR）和玻璃体内（IVT）注射后的定位和分布。携带萤火虫荧光素酶 DNA 的 AAV5 由普遍存在的巨细胞病毒（CMV）启动子控制，单侧 IVT 或 SR（在中央或周边视网膜）注射到 41 只小鼠中。使用重复（多达 12 次）IVIS 生物发光成像，在最长时间存活的动物中最多可跟踪 60 周的荧光素酶活性。在代表性动物中，通过免疫组织化学（IHC）和 PCR 也证实了荧光素酶的存在。在 SR 组中，IVIS 读数显示 32/32 只眼均有荧光素酶活性，并且 IHC（4/4 只眼）和 PCR（12/12 只眼）证实了荧光素酶的存在。在 IVT 组中，7/9 只眼的 IVIS 读数显示有荧光素酶活性，PCR 证实了 5/5 只眼和 IHC（2/2 只眼）中的荧光素酶存在。在 2 只 SR 注射的动物（分别来自中央和周边注射部位）中，PCR 检测到同侧视神经中存在荧光素酶，而 IVIS 或 IHC 未检测到该信号。我们的结果表明，与金标准 PCR 相比，当评估 SR 给药时，IVIS 的灵敏度和特异性均为 100%。当评估 IVT 给药时，IVIS 的灵敏度为 78%，特异性为 100%。这些发现证实了 IVIS 在视网膜 SR 给药后长达 60 周的时间内能够重复成像进行纵向评估，以检测体内 AAV 的局部表达，而无需生物信号衰减，从而取代了为了确认载体表达而进行的死后处理的需要。然而，与基因组检测相比，IVIS 可能不够敏感，无法检测到 SR 给药后同侧视神经中的荧光素酶活性，因此无法检测到小鼠 SR 给药后的视神经中的荧光素酶活性。
